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The essential role of histone H2A deubiquitinase MYSM1 in natural killer cell maturation and HSC homeostasis

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The essential role of histone H2A deubiquitinase MYSM1 in natural killer cell maturation and HSC homeostasis

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Content THE ESSENTIAL ROLE OF HISTONE H2A DEUBIQUITINASE MYSMI IN
NATURAL KILLER CELL MATURATION AND HSC HOMEOSTASIS

by
Vijayalakshmi Nandakumar

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETIC, MOLECULAR AND CELLULAR BIOLOGY)

December 2013

Copyright 2013 Vijayalakshmi Nandakumar
ii

Dedication
I dedicate this work to my family: my parents, Dr. and Mrs. Nandakumar, my husband,
Mr. Harshavardhan Bhaskaran and my brother, Mr. Vivek Nandakumar for their
unconditional love, support and confidence in me through these years. They have been a
great source of energy, inspiration and motivation for my life. Without their constant
encouragement, it would not have been possible for me to pursue my PhD.

iii

Acknowledgements
I hereby take the opportunity to thank all those people who have helped me during the
course of my PhD.

First and foremost, I would like to thank my advisor Dr. Si Yi Chen for his guidance and
support during the entire time span of my PhD. Dr. Chen has given me the opportunity to
carry out cutting-edge research in Immunology. My research in his lab has not only
enabled me to expand my scientific knowledge, but has also helped me to hone my
technical and presentation skills. Through his mentorship, I have learnt to appreciate the
relevance of basic immunology research in a disease setting. I would also like to thank
Dr. Xue Huang for her valuable advice and guidance during the course of my PhD.

Next, I would like to thank all my co-workers in the lab (past and current) who have
made the entire experience of PhD not only rewarding but enjoyable. I would like to
extend my special gratitude to Dr. Tao Wang who have made direct contribution to some
of the experiments described in this thesis. My special thanks to Dr. Xiaoxia Jiang for her
valuable advice and technical suggestions.

I would like to thank my committee members: Dr. Omid Akbari, Dr. Andre. J. Ouellette,
Dr. Weiming Yuan and Dr. Keigo Machida for being extremely supportive. I thank them
for their guidance and for helping me analyze scientific questions from different
dimensions.
iv

Finally, I would like to thank my family and friends both in India and in US, for
encouraging and supporting me throughout this process. Most importantly, I would like
to thank my parents and my husband who have been instrumental in motivating me to
achieve my academic goals. They have always taught and helped me strive harder and
face any challenges with optimism. None of this would have been possible without their
support.

v

Table of Contents

Dedications ......................................................................................................................... ii
Acknowledgements ............................................................................................................ iii
List of Tables ..................................................................................................................... ix
List of Figures ......................................................................................................................x
Abstract ............................................................................................................................. xii
Chapter 1. Epigenetic regulation of Hematopoiesis ............................................................1
1.1 Hematopoiesis ............................................................................................................1
1.2 Epigenetics .................................................................................................................2
1.3 Histone H2A Ubiquitination/Deubiquitination ..........................................................2
1.4 MYSM1, a novel Histone H2A Deubiquitinase .........................................................4
1.5 Creation of MYSM1-KO first floxed mice ................................................................5
1.6 Rationale and Objectives of this thesis ......................................................................6
1.7 Significance of this study ...........................................................................................9
Chapter 2. Epigenetic control of NK cell maturation by MYSM1 ....................................11
2.1 Introduction ..............................................................................................................11
2.2 Materials and Methods .............................................................................................13
2.2.1 Animals..............................................................................................................13
2.2.2 Cell preparation .................................................................................................13
2.2.3 Flow cytometry and Cell sorting .......................................................................14
2.2.4 Lentivirus and retrovirus production and transduction .....................................15
2.2.5 In vitro generation of NK cells on OP9 culture .................................................16
2.2.6 Transplantation assays .......................................................................................17
2.3 Results ......................................................................................................................18
2.3.1 Reduction in the frequency and number of NK cells in MYSM1-/- mice ........18
2.3.2 Defective NK cell maturation but normal NK lineage commitment in MYSM1-
/- mice .........................................................................................................................20
2.3.3 MYSM1-/- NK cells are phenotypically immature ...........................................25
2.3.4 MYSM1 is intrinsically required for NK cell maturation in vivo .....................29
2.3.5 MYSM1 is intrinsically required for NK cell maturation in vitro.....................32
vi

2.4 Conclusion ................................................................................................................35
Chapter 3. Epigenetic control of Id2 transcription by MYSM1 during NK cell
development .......................................................................................................................37
3.1 Introduction ..............................................................................................................37
3.2 Materials and Methods .............................................................................................40
3.2.1 Animals..............................................................................................................40
3.2.2 Cell preparation .................................................................................................40
3.2.3 Flow cytometry and Cell sorting .......................................................................40
3.2.4 Chromatin Immunoprecipitation .......................................................................40
3.2.5 Protein Immunoprecipitation and Immunoblotting ...........................................42
3.2.6 Quantitative RT-PCR ........................................................................................43
3.2.7 Statistics .............................................................................................................43
3.3 Results ......................................................................................................................43
3.3.1 MYSM1 is required for Id2 transcription ..........................................................43
3.3.2 MYSM1 associates with the ID2 locus -/- .......................................................47
3.3.3 MYSM1 interacts with NFIL3 and this interaction is critical for their
recruitment to the ID2 locus .......................................................................................51
3.3.4 Id2 locus of the MYSM1-/- NK cells is poised in its repressed state................53
3.4 Conclusion ................................................................................................................57
Chapter 4. The essential role of MYSM1 in the maintenance, self-renewal and
differentiation of hematopoietic stem cells ........................................................................59
4.1 Introduction ..............................................................................................................59
4.2 Materials and Methods .............................................................................................61
4.2.1 Animals..............................................................................................................61
4.2.2 Cell preparation .................................................................................................62
4.2.3 Flow cytometry and cell sorting ........................................................................62
4.2.4 Cell-proliferation and cell-cycle studies ............................................................63
4.2.5 Colony assays ....................................................................................................63
4.2.5 Treatment with 5-FU .........................................................................................64
4.2.6 Pyronin and Hoechst staining ............................................................................64
4.2.7 Intracytoplasmic staining...................................................................................65
vii

4.3 Results ......................................................................................................................65
4.3.1 MYSM1 controls HSC differentiation during its transition to MPPs ...............65
4.3.2 Loss of MYSM1 drives HSCs from quiescence to rapid cycling ....................69
4.3.3 Loss of MYSM1 results in failed recovery of HSC pool ..................................70
4.3.4 MYSM1 deficiency causes increased apoptosis in HSCs .................................72
4.3.4 MYSM1-deficient HSCs display intrinsic functional defects and impaired
engraftment .................................................................................................................74
4.4 Conclusion ................................................................................................................76
Chapter 5. The control of HSC homeostasis by MYSM1-mediated epigenetic regulation79
5.1 Introduction ..............................................................................................................79
5.2 Materials and Methods .............................................................................................82
5.2.1 Animals..............................................................................................................82
5.2.2 Cell preparation .................................................................................................82
5.2.3 Flow cytometry and cell sorting ........................................................................82
5.2.4 Immunoblot analysis and immunoprecipitation ................................................82
5.2.5 RNA extraction and real-time PCR ...................................................................83
5.2.6 Chromatin immunoprecipitaion.........................................................................83
5.2.7 Luciferase reporter assays .................................................................................84
5.2.8 PCR array ..........................................................................................................84
5.3 Results ......................................................................................................................85
5.3.1 Gfi1 gene expression is regulated by MYSM1 in HSC ....................................85
5.3.2 Gfi1 is a direct transcriptional target of MYSM1 and is regulated by MYSM1
and its coordinated action with Gata2 and Runx1 .....................................................87
5.3.3 Gfi1 partially restores the function of MYSM1-/- HSCs ..................................90
5.3.4 MYSM1 orchestrates histone modifications at the Gfi1 locus ..........................94
5.4 Conclusion ................................................................................................................95
Chapter 6. Overall Summary and Future Perspectives ......................................................98
Bibliography ....................................................................................................................107
Appendices .......................................................................................................................120
Appendix A: Supplementary information for Chapter 2 ..........................................120
Appendix B: Supplementary information for Chapter 3 ..........................................126
viii

Appendix C: Supplementary information for Chapter 4 ..........................................130
Appendix D: Supplementary information for Chapter 5 ..........................................134

ix

List of Tables

Table B.1 Primers used for q-RT PCR of NK cell development-related factors .............128
Table B.2 Primers used for chIP analysis of Id2 locus ....................................................129
Table D.1 List of genes changed more than 2 fold in the Mouse RT
2
Profiler
TM
PCR array
..........................................................................................................................................134
Table D.2 Mouse hematopoiesis RT
2
Profiler
TM
PCR array data ...................................134
Table D.3 Primers used for q-RT PCR of HSC development-related factors .................136

x

List of Figures

Figure 1.1 Flow chart depicting the process of hematopoiesis ...........................................1
Figure 1.2 Schematic representation of creation of MYSM1-KO first floxed mice ...........6
Figure 2.1 Reduction in the frequency and number of NK cells in MYSM1-/- mice .......19
Figure 2.2 Defective NK cell maturation but normal NK lineage commitment in
MYSM1-/- mice .................................................................................................................21
Figure 2.3 MYSM1-/- NK cells are phenotypically immature ..........................................27
Figure 2.4 MYSM1 is intrinsically required for NK cell maturation ................................30
Figure 3.1 Schematic depicting the molecular regulation of NK cell development ..........39
Figure 3.2 MYSM1 is required for ID2 transcription .......................................................45
Figure 3.3 MYSM1 associates with the Id2 locus .............................................................49
Figure 3.4 Id2 locus of the MYSM1-/- NK cells is poised in its repressed state...............55
Figure 4.1 Schematic depicting the stages of cell differentiation from stem cells. ...........60
Figure 4.2 MYSM1 deficiency results in a reduction of HSC and its progenitors ............68
Figure 4.3 Loss of MYSM1 drives HSC from quiescence to rapid proliferation ..............69
Figure 4.4 Exhaustion of Hematopoietic stem cell in the absence of MYSM1 .................71
Figure 4.5 MYSM1 deficiency leads to increased HSC apoptosis ...................................73
Figure 4.6 Cell-intrinsic defects of MYSM1-deficient HSC .............................................75
Figure 5.1 Requirements of Transcription Factors in Hematopoiesis ...............................81
Figure 5.2 Differential Gene expression change between wild-type and MYSM1-/- LSK
cells ....................................................................................................................................86
Figure 5.3 Gfi1 is a target of MYSM1 ...............................................................................89
Figure 5.4. Forced expression of Gfi1 partially restores the function of MYSM1-
deficient HSC. ....................................................................................................................92
Figure 5.5 MYSM1 Orchestrates Histone Modifications at the Gfi1 Locus ....................95
Figure A.1 Confirmation of the reduction in MYSM1 mRNA levels in MYSM1-/- NK
cells and T cell frequencies and numbers in WT vs MYSM1-/- mice .............................120
Figure A.2 Thymic NK cells were not defective in MYSM1-/- mice .............................121
Figure A.3 Flt3
-
IL-7R
+
CLPs were increased in MYSM1-/- mice ..................................121

xi

Figure A.4 Transition to immature Lin-CD122+NK1.1+NKp46- NK cells is unaffected in
MYSM1-/- mice ...............................................................................................................122
Figure A.5. Additional analysis for the cell-intrinsic role of MYSM1 in NK cell
maturation in vitro and in vivo .........................................................................................123
Figure A.6 Sorting gating scheme and validation of purity of the sorted cells used in our
experiments .....................................................................................................................124
Figure B.1 qRT-PCR analyses of NK cell development transcription factors in sorted WT
and MYSM1-/- mature NK cells (CD122
+
Lin
-
NK1.1
+
DX5
+
) ..........................................126
Figure B.2 qRT-PCR analyses of ID2 in sorted WT NK cell and NK cell progenitor
subsets from bone marrow ...............................................................................................126
Figure B.3 Enrichment of known NK cell specific interactions in the chromatin fractions
prepared for chIP assays ..................................................................................................127
Figure C.1 Reduction in the absolute numbers of hematopoietic lineage cells in MYSM1-
/- mice ..............................................................................................................................130
Figure C.2 Characterization of MYSM1 in HSC and its compartments .........................131
Figure C.3 Loss of quiescence in MYSM1-deficient HSCs ............................................131
Figure C.4 MYSM1-deficient HSCs display defective engraftment ...............................132

xii

Abstract
Histone modifications play critical roles in regulating hematopoietic cell development
and differentiation. The importance of several histone H2A ubiquitinases and
deubiquitinases (ubiquitin-specific proteases) in transcription regulation have been
demonstrated recently. In our recent study, we found that Myb-like, SWIRM, and MPN
domains-containing protein 1 (MYSM1), a histone H2A deubiquitinase (2A-DUB), is
required for early B cell development by de-repressing EBF1 transcription (Jiang et al.,
2011).
So far, very little is known about the epigenetic control of NK cell development. In the
first half of the thesis, we present our finding that NK cell development is severely
impaired in mice deficient (-/-) in the histone H2A deubiquitinase MYSM1 (Nandakumar
et al., 2013). We demonstrated that MYSM1 is required for NK cell maturation but not
for NK lineage specification and commitment. We also found that MYSM1 intrinsically
controls this NK cell maturation. Mechanistic studies revealed that the expression of
transcription factor Id2, a critical factor for NK cell development, is impaired in
MYSM1-/- NK cells. MYSM1 was found to interact with NFIL3 (E4BP4), a critical
factor for mouse NK cell development and the recruitment of NFIL3 to the Id2 locus is
dependent on MYSM1. Further, we observed that MYSM1 is involved in maintaining an
active chromatin at the ID2 locus to promote NK cell development. Hence this study, for
the first time, uncovers the critical epigenetic regulation of NK cell development by
histone H2A deubiquitinase, MYSM1 through the transcriptional control of transcription
factors important for NK cell development.
xiii

Epigenetic histone modifications also play critical roles in the control of self-renewal and
differentiation of hematopoietic stem cells (HSC). MYSM1 is recently identified for its
critical role in HSC functional maintenance (Nijnik et al., 2011). In the study presented in
the second half of this thesis, in addition to confirming this function of MYSM1, we
provide more evidence for how MYSM1 controls HSC homeostasis using a MYSM1
deficient (-/-) mice (Wang Tao & Nandakumar et al., 2013). MYSM1 deletion drove
HSCs from quiescence into rapid cycling, elevated their apoptotic rate, resulting in an
exhaustion of the stem cell pool. This lead to an impaired self-renewal and lineage
reconstituting abilities in the MYSM1-deficient mice. Our study identified Gfi1 as one of
the candidate genes responsible for the hematopoietic stem cell defect in MYSM1-
deficient mice. Mechanistic studies revealed that MYSM1 modulates histone
modifications and directs key transcriptional factors-recruitment, such as Gata2 and
Runx1, to Gfi1 locus in HSC. We found that MYSM1 directly associates with the Gfi1
enhancer element and promotes its transcription through Gata2 and Runx1
transactivation. Thus, our study not only elaborates on the initial reports of MYSM1's
association with HSC homeostasis but also delineates a possible epigenetic mechanism
through which MYSM1 carries out this function in the hematopoietic stem cells.
1

Chapter 1. Epigenetic regulation of Hematopoiesis
1.1 Hematopoesis
In adults, hematopoietic stem cells (HSCs) from bone marrow develop into all classes of
blood cells through progressive loss of differentiation potentials for other cell lineages.
HSCs differentiate into multipotent progenitors (MPPs), which further differentiate into
lymphoid and myeloid lineages in a symmetrical fashion. This is the first step of
irreversible lineage commitment from HSCs during early hematopoietic ontogeny (A. Y.
Lai & Kondo, 2008). The lymphoid lineage includes T, B, and natural killer (NK) cells,
and the myeloid lineage comprises of erythrocytes, megakaryocytes, granulocytes, and
monocyte/macrophage. Lymphoid and myeloid lineages develop from the lineage-
restricted progenitors: Common lymphoid progenitors (CLPs) and Common myeloid
progenitors (CMPs), both derived from MPPs.
Figure 1.1 Flow chart depicting the process of hematopoiesis
The blood stem cell (HSC) is the parent cell that gives rise to all the hematopoetic
lineages. Adapted from (A. Y. Lai & Kondo, 2008).

2

1.2 Epigenetics
Cell development involves epigenetics; the underlying genome of a stem cell and its
differentiated progenitors and mature cells remains identical and it’s the epigenetic
regulation that helps in different lineage commitment and development (Goldberg, Allis,
& Bernstein, 2007). Nucleosomes, the basic units of chromatin, are composed of genomic
DNA wrapped around the histone octamer, which is comprised of four core histones
(H2A, H2B, H3, and H4) (Luger, Mader, Richmond, Sargent, & Richmond, 1997). A
mammalian cell packs 1.7 meters of genomic DNA into a 5 micrometer nucleus in the
chromatin (Ho & Crabtree, 2010). Thus, most chromatin is organized in highly compact
structures (Ho & Crabtree, 2010). At least three epigenetic processes work in concert to
control the assembly and status of chromatin: DNA methylation (Jones & Baylin, 2002;
Suzuki & Bird, 2008), histone modifications (Kouzarides, 2007), and ATP-dependent
chromatin remodeling (Ho & Crabtree, 2010). Histones are subject to more than 100
post-translational modifications, including methylation, acetylation, phosphorylation,
sumoylation, and ubiquitination (Kouzarides, 2007). The different combinations of these
histone modifications, termed the “histone code” (Jenuwein & Allis, 2001; Strahl &
Allis, 2000), determine chromatin structure and states, thus defining not only distinct
transcription patterns, but also cellular identity and functional states (Kouzarides, 2007;
B. Li, Carey, & Workman, 2007).

1.3 Histone H2A Ubiquitination/deubiquitination

Protein mono- or poly-ubiquitination plays a critical role in a variety of cellular
processes, including protein degradation, cell cycle, protein trafficking, signal
3

transduction, and transcriptional regulation (Komander, Clague, & Urbe, 2009). H2A
monoubiquitination at the conserved residue lysine (K) 119 was discovered in 1975
(Goldknopf et al., 1975), and has since been found to be a relatively abundant
modification, comprising between 5% to 15% of the total H2A (Komander et al., 2009).
Polyubiquitination of a protein is usually marked for its degradation; however,
monoubiquitination of histone 2A represents a non-degradative, epigenetic signal.
Among the four core histones, H2A at K119 and H2B at K120 were found to be mono-
ubiquitinated (Komander et al., 2009). So far, at least two histone H2A ubiquitinases
(ubiquitin E3 ligase) have been identified. The H2A ubiquitinase Ring1B/Ring 2 was
identified as the core component of the Polycomb repressive complex 1 (PRC1) (L.
Wang et al., 2004). Biochemical and functional analysis of the PRC1 complex has
revealed Ring1B to be the catalytic subunit, which can be greatly stimulated by Bmi1 and
Ring1a (Cao, Tsukada, & Zhang, 2005; de Napoles et al., 2004; H. Wang et al., 2004).
Another H2A ubiquitinase, 2A-HUB/hRUL138, was recently found to suppress
chemokine genes in N-CoR/HDAC1/3 corepressor complex (Zhou et al., 2008).
Recently, an increasing number of histone H2A deubiquitinases, including USP16/Ubp-
M, 2A-DUB, USP21, USP22, and PR-DUB/calypso, have been identified (Joo et al.,
2007; Malleshaiah, Shahrezaei, Swain, & Michnick, 2010; Nakagawa et al., 2008; Zhao
et al., 2008; Zhu et al., 2007). In addition, several histone ubiquitinases and
deubiquitinases of histone H2B have also been discovered (Zhao et al., 2008; Zhu et al.,
2007) (Zhou, Wang, & Rosenfeld, 2009). Ubp-M plays an important role in regulating
the mitotic phase of the cell cycle and HOX gene expression (Cai, Babbitt, & Marchesi,
4

1999; Joo et al., 2007). USP22, one component of the GCNs HAT-containing
TFTC/STAGA complex, is able to deubiquitinate histone H2A and H2B in vitro and is
required for the transcription activation of the androgen receptor (AR) (Zhao et al., 2008).

1.4 MYSM1, a novel Histone H2A Deubiquitinase
Zhu et al. recently identified Myb-like, SWIRM, and MPN domains-containing protein 1
(MYSM1) as a histone H2A deubiquitinase (2A-DUB) and that its H2A deubiquitination
activity is required for the activation of several target genes in prostate cancer cells (Zhu
et al., 2007). The JAMM/MPN (JAB1/MPN/Mov34 metalloenzymes) domain possesses
an intrinsic metalloprotease activity that hydrolyzes the isopeptide bonds of ubiquitin
chains. The SANT (switching-defective protein 3, adaptor 2, nuclear receptor co-
repressor, and transcription factor IIIB domain) has a strong similarity to the DNA-
binding domain of Myb-related proteins and is a motif capable of binding to DNA and
histones, which exists in many transcription regulators (Boyer, Latek, & Peterson, 2004).
The SWIRM domain is named for its presence in the proteins Swi3, Rsc8, and Moira,
which are homologous members in the SWI/SNF-family of ATP-dependent chromatin
remodeling complexes, and favors interactions with linker DNA and/or N-terminal tails
of histone H3 (Qian et al., 2005). Yoneyama et al. (2007) demonstrated that the SWIRM
domain of MYSM1, initially cloned from a brain cDNA library (Nagase, Nakayama,
Nakajima, Kikuno, & Ohara, 2001), formed a helix-turn-helix fold with 5 alpha helices
and that its SANT domain bound DNA in a manner similar to that of the Myb DNA-
binding domain (M Yoneyama et al., 2007). Zhu et al. demonstrated that MYSM1 was a
5

component of a complex that included the histone acetyltransferase PCAF in human
embryonic kidney cells (Zhu et al., 2007). Although the mechanisms by which histone
H2A ubiquitinases regulate gene transcription are unclear, it was proposed that MYSM1
forms a complex with PCAF to regulate transcription initiation and elongation by a
stepwise coordination of histone acetylation, H2A deubiquitination, and linker histone H1
disassociation from the nucleosome (Zhou et al., 2009; Zhu et al., 2007).

1.5 Creation of MYSM1-KO first floxed mice
MYSM1-deficient (-/-) mice (MYSM1-KO first floxed mice) is generated through a “KO
first” strategy. This strategy was based on inserting a cassette (tm1a allele) into an intron
of an intact target gene that produces a knockout at the RNA processing level. A splice
acceptor (SA) in the cassette captures the RNA transcript and an efficient
polyadenylation termination signal truncates the transcript (prematurely), preventing the
gene from being transcribed into mRNA downstream of the cassette site as described by
Skarnes et al (Skarnes et al., 2011). Subsequent crossing with Cre transgenic mice and
Cre expression results in the deletion of the floxed MYSM1 exon 3. The MYSM1 KO-
first floxed sperm in the C57BL/6J background (MYSM1_A04; MYSM1tm1a
(ΔMP)Wtsi MGI#: 2444584) were provided by the KOMP Repository at UC Davis. In
vitro fertilization, microinjection, chimera production, and the generation of MYSM1
KO-first floxed mouse founders were carried out at the USC Transgenic/KO Mouse Core
Facility.

6

Figure 1.2 Schematic representation of creation of MYSM1-KO first floxed mice.
Diagram of the MYSM1 targeted vector and crossbreeding with Cre mice to delete the
floxed MYSM1 exon. pA: polyA; FRT: Flippase recognition target site; SA: splice
acceptor. Adapted from (X. X. Jiang et al., 2011)

1.6 Rationale and Objectives of this thesis
Hematopoetic stem cells (HSCs) and their progenies have distinct chromatin states
(epigenomes) established through epigenetic modifications, which allows for the
expression and pre-assembly of key transcription factors at lineage-specific promoters in
HSCs, progenitors, and differentiated cells (Weishaupt, Sigvardsson, & Attema, 2010)
Recently, increasing numbers of histone modifiers have been found to be critical in
hematopoiesis and leukemogenesis (Rice, Hormaeche, & Licht, 2007). Despite the early
discovery, the biological role of histone ubiquitination remains the least understood
among various histone modifications. This study was designed to understand the
physiological function of MYSM1, contributing to the basic biological understanding of
reversible histone ubiquitination in general, since the physiological functions of the entire
group of newly identified histone deubiquitinases are unknown (Zhou et al., 2009).
7

Recently, we generated MYSM1-deficient (KO or -/-) mice and Jiang et al from our lab
(X. X. Jiang et al., 2011) found that MYSM1 is essential for early B cell development by
de-repressing the transcription of EBF1 and other transcription factors. Moreover, we
found that MYSM1 interacts with E2A, a transcriptional activator of EBF1, and
BRM/BRG1, a chromatin remodeler SWI/SNF component, for targeting the EBF1 locus.
In addition, MYSM1 orchestrates H2A deubiquitination, as well as other histone
modifications at the EBF1 locus. Thus, our recent study reveals a critical role of MYSM1
in early B cell development by de-repressing transcription of EBF1 and other factors in B
cell progenitors (X.-X. Jiang et al., 2011).

Furthermore, we found that MYSM1 deficiency severely impaired the maturation of NK
cells, however early stages of NK cell development were not significantly compromised.
These important, unexpected preliminary findings formed the basis of this thesis and we
further aimed to mechanistically investigate the epigenetic regulation of maturation of
NK cells by MYSM1. In the first half of the thesis, we show how MYSM1 is an essential
and intrinsic requirement for NK cell development. Concurrently, another group (A.
Nijnik et al., 2012) reported the role of MYSM1 in BM hematopoiesis and function with
a specific defect at the early stage of stem cell differentiation. Although MYSM1
deficiency does not affect commitment to NK cell lineage we were curious to identify the
specific development defect at the early stages of HSC differentiation. This formed the
basis for the second half of this thesis, where we sought to identify more precisely the
defective subset within the HSC compartment of MYSM1-/- mice since this was not
8

detailed out by Nijnik et al. Eventually, we also attempted to examine the role of
MYSM1 in HSC maintenance, self-renewal, and function in a detailed manner, which is
presented in the latter half of this thesis.

Several studies on a limited set of genes and at a genome-wide level indicate that uH2A
is generally linked to transcription repression (Cao et al., 2005; Nakagawa et al., 2008).
The H2A ubiquitinases Ring1B and 2A-HUB/hRUL138 are core components of the
Polycomb repressive complex 1 and other transcription-repressive complexes (Fang,
Chen, Chadwick, Li, & Zhang, 2004). Recently, an increasing number of histone H2A
DUBs, including USP16/Ubp-M, 2A-DUB, USP21, USP22 and PR-DUB/calypso, have
been identified (Cao et al., 2005). Members in our lab have shown that MYSM1 have
different effects on different cell lineages, as exhibited by a marked decrease in B cells,
NK cells, an increase in granulocytes and unaltered levels of T cells in the MYSM1-
deficient mice (X. X. Jiang et al., 2011; Nandakumar, Chou, Zang, Huang, & Chen,
2013; Wang Tao & Nandakumar et al., 2013). Our results also suggest that MYSM1 may
have different functions in different cells owing to its selective ability to deubiquitinate
specific chromatin locations, thus regulating the transcription of distinct sets of genes.

In the second half of our analyses, we attempted to find a possible mechanism for the
observed defective NK cell maturation and HSC biology in MYSM1-deficient mice. We
tested the hypothesis that MYSM1 controls the transcription of genes associated with NK
cell development and HSC homeostasis by selectively deubiquitinating uH2A at specific
9

chromatin locations of such genes. Based on comprehensive analysis of differential gene
expression in the presence and absence of MYSM1 and through genetic complementation
studies conducted on MYSM1-deficient cells, we identified Id2 and Gfi-1 as candidate
targets of MYSM1 during NK cell maturation and HSC development respectively.
Hence, in the last section of each half, we demonstrate the underlying mechanism by
which MYSM1 controls Id2 and Gfi-1 gene transcription during these hematopoietic cell
developments.

1.7 Significance of this study
This study is significant and novel for several reasons. Over the past 10 years, major
advance have been made in our understanding of the transcriptional factors that promote
specification and commitment during B and T lymphocyte development (Nutt SL, 2007
(Singh H, 2005). In contrast molecular mechanisms controlling NK cell development are
not well understood. A thorough understanding of this mechanism is necessary because
impaired regulation of NK cell development can lead to immune deficiency,
autoimmunity and NK cell malignancies that are difficult to diagnose and treat (Di Santo,
2006). Not only that, current efforts on understanding NK cell development have been
largely focused on transcription factors, receptors, signal transducers, and cytokines
involved in this process. This study aimed to investigate the regulation of NK cell
development through an epigenetic mechanism, using the MYSM1-KO first floxed mice
in combination with other molecular biological techniques.

10

To date, the physiological roles of the entire group of newly identified histone
deubiquitinases are unknown and the biological role of histone ubiquitination remains the
least understood among various histone modifications. This study attempted to uncover
the physiological functions of the 2A-DUB/MYSM1, as well as contributes to the
fundamental understanding of the reversible histone ubiquitination in general. Therefore,
this study is one of the first one to mechanistically investigate the transcription control of
genes in NK cell and HSC precursors by MYSM1. Furthermore, understanding the
epigenetic regulation of these hematopoietic cell development by MYSM1 in detail may
provide new targets or strategies for the prevention and treatment of autoimmune,
infectious diseases and other hematological malignancies (Stuart H. Orkin & Leonard I.
Zon, 2008).

11

Chapter 2. Epigenetic control of NK cell maturation by MYSM1
2.1 Introduction
Natural killer (NK) cells are lymphocytes that play critical role in adaptive and innate
immune responses (Nandakumar et al., 2013). They can recognize the virus-infected and
cancerous cells through their multiple surface-expressed activatory and inhibitory
receptors and lyse them through a cytotoxic effect (Smyth, Hayakawa, Takeda, & Yagita,
2002). Natural killing occurs through the release of granzyme- and perforin- containing
cytoplasmic granules through a metabolically active process. Not only their response is
rapid in the innate immune system, but also produces a distinct set of cytokines such as
IFN- γ, TNF-α, IL-10, 1L-5, and 1L-13 or chemokines such as MIP-1α and –β and
RANTES, which can further elicit an adaptive immune response (Yokoyama & Kim,
2006). Together, these functional activities of NK cells help to eliminate the susceptible
targets through multiple ways and help to amplify the inflammatory response (Di Santo,
2006; Nandakumar et al., 2013).

NK cells develop from the common lymphoid progenitors (CLPs) just like B cells and T
cells. The primary site of NK cell development is bone marrow although some evidence
shows the presence of immature NK cells in the liver and thymus suggesting that NK
cells may also develop at these sites (Yokoyama & Kim, 2006). NK cell development in
the bone marrow (BM) is primarily defined by the step-wise expression of CD122 (IL-2
and IL-15 receptor-β chain), NK1.1 (activating NK receptor), and DX5 (integrin α2) (S
Kim et al., 2002; Rosmaraki et al., 2001). CD122
+
NK1.1
-
DX5
-
Lin
-
cells are originally
12

described as NK progenitors (NKPs); but it’s been recently shown that this population
also exhibits a T-cell potential in a notch-dependent manner both in vivo and in vitro
(Nozad Charoudeh et al., 2010). For convenience, CD122
+
NK1.1
-
DX5
-
Lin
-
cells are still
referred to as ‘NK progenitors’ in this study. Based on a refined analysis of markers
expressed on these progenitors (CD27, IL-7R, and CD244), NKPS enriched for NK cell
potential known as rNKPs (refined NKPs) and an intermediate stage between NKPs and
CLPs known as preNKPs has recently been identified (Fathman et al., 2011). Acquisition
of NK1.1 occurs at the immature NK (iNK) cell stage, during when multiple NK
receptors including NKp46, a preferential marker expressed in NK cells and conserved in
mammals, begin to express (Narni-Mancinelli et al., 2011; Vosshenrich et al., 2005). This
stage when the NKp46 onsets has been marked for its irreversible engagement into NK
cell lineage since NK1.1
+
NKp46
-
cells can still give rise to both NK and T cells in
contrary to the NK1.1
+
NKp46
+
cells (Narni-Mancinelli et al., 2011; Nozad Charoudeh et
al., 2010). Following this, cells transition into mature NK cells (mNK) on sequential
acquirement of DX5, CD11B and KLRG1 expression and on down-regulation of c-KIT,
CD27 and CD51 expression (S Kim et al., 2002; Rosmaraki et al., 2001).

In our recent study, we found that MYSM1 plays a critical role in early B cell
Commitment and development (X.-X. Jiang et al., 2011; Anastasia Nijnik et al., 2012). In
this study, we reveal an important and intrinsic role of MYSM1 in the maturation of NK
cells, but not for the NK lineage specification and early development (Nandakumar et al.,
2013).
13

2.2 Materials and Methods
2.2.1 Animals.
MYSM1-deficient (-/-) mice (MYSM1-KO first floxed mice) is generated through a “KO
first” strategy as described in 1.5. CD45.1 mice were purchased from Jackson
Laboratory. All of these mice were of C57BL/6J background (Nandakumar et al., 2013).
All animal breeding and experiments were approved and performed in accordance with
the University of Southern California Institutional Animal Care and Use Committee.

2.2.2 Cell preparation
Single cell suspensions were obtained by either flushing the femoral bone marrow cells
with PBS or by passing the spleen, lymph node (pooled cervical, inguinal and axillary)
and thymus through 70-μm cell strainers (BD) followed by treatment with red blood cell
(RBC) lysis buffer (Sigma-Aldrich)(Nandakumar et al., 2013). To obtain single lung cell
suspensions, lungs were perfused with 20 ml PBS through the right ventricle, minced
using iridectomy scissors, and digested with collagenase III and DNase I, as described in
(Vermaelen, Carro-Muino, Lambrecht, & Pauwels, 2001). For making cell suspension
from peripheral blood, it was diluted with PBS (1:1) and poured onto the top of Ficoll
solution without getting mixed. It was then centrifuged for 20 min at 1,600 rpm after
which the layer of white blood cells that separates out as a white ring was harvested and
washed twice with PBS. Following single cell suspension-preparations, all the samples
were subjected to red blood cell lysis using the lysis buffer (Sigma-Aldrich) after which
the samples were ready for subsequent analysis.
14

2.2.3 Flow cytometry and cell sorting
Single-cell suspensions of bone marrow (BM), thymus, spleens and peripheral blood
were prepared and were first stained for 20 min at 4° C with CD16/CD32 Fc-blocking
antibody (2.4G2), in flow cytometry buffer (PBS with 1% FBS), followed by incubation
with a ‘cocktail’ of antibodies conjugated to different fluorophores(Nandakumar et al.,
2013). For intracellular staining, BD intracellular staining was used following
manufacturer’s instructions. The following anti-mouse antibodies conjugated with
different flurochromes were from BD Biosciences or eBioscience or BioLegend: NK1.1
(PK136), CD3e (145-2C11), CD49b (DX5), CD122 (TM-β1), B220 (RA3-6B2), 2B4
(2B4), NKG2D (CX5), NKG2A/C/E (20d5), KLRG1 (CF1 Ly49D (4E5), Ly49G2 (Cwy-
3), Ly49C/I/H (5E6), NKp46 (29A1.4), CD11b (M1/70), CD45.1 (A20), CD45.2 (104),
CD4 (L3T4), CD8a (53-6.7), Sca1 (anti-Ly6A; D7), CD117 (c-Kit; 2B8), CD127 (anti-
IL-7Rα; A7R34), CD19 (ID3), CD11b (M1/70), CD27 (LG.7F9), CD244 (2B4), Flt3
(A2F10), rat IgG2a k isotype (R35-95), rat IgG2b k isotype (A95-1), rat IgG1 k isotype
(R3-34), rat IgG1 λ isotype (A110-1), hamster IgG1 k isotype (A19-3), and mouse IgG2a
k isotype (G155-178), Lin cocktail (with CD3e 145-2C11, Ly-6G/Ly-6C RB6-8C5,
CD45R/B220 RA3-6B2, TER-119/Erythroid cells Ter-119 and CD11B M1/70). 7-
Amino-actinomycin D (BD) was used to exclude dead cells from the analysis. Data was
collected on FACSCanto II (BD) and analyzed using FlowJo software (Tree Star, Inc.).

For sorting the NK progenitors and mature NK cells, BM cells (for NKPs) and/or spleen
cells (for mNKS) were first enriched for CD122 cells by incubation with anti-CD122
15

biotinylated antibody followed by anti-biotin Micro beads (Miltenyi Biotec), and then
positive selection was done on 25 LS MACS column (Miltenyi Biotec). The enriched
CD122
+
cells were next stained with directly conjugated antibodies against lineage
markers: anti-CD19, Gr-1, Mac-1, CD3, CD4, CD8, and Ter119 as well as anti-NK1.1
and DX5 or anti-NK1.1 and sorted by flow cytometry using FACS Aria III as indicated
in Fig.A.6 (Nandakumar et al., 2013)

For sorting KLS, CLPs, rNKP and pre NKP subsets, lineage positive cells were first
depleted by incubation with Lin

cocktail and anti-NK1.1 and anti-NKP46 biotinylated
antibodies followed by anti-biotin Micro beads (Miltenyi Biotec) and then negative
selection was done on 25 LD MACS column (Miltenyi Biotec). Following depletion,
cells were stained with different surface markers and sorted by flow cytometry using
FACS Aria III as follows: from bone marrow, KLS (Lin
−
Sca1
+
c-Kit
+
), CLP cells (Lin
-
c-
KIT
-
CD27
+
CD244
+
IL-7R
+
Flt3
+
CD122
+
), pre NKPs (Lin
-
c-KIT
-
CD27
+
CD244
+
IL-
7R
+
Flt3
-
CD122
-
) and rNKPs (Lin
-
c-KIT
-
CD27
+
CD244
+
IL-7R
+
Flt3
-
CD122
+
). All sorts for
indicated cell phenotypes were performed on BD FACSAria (BD Biosciences) with
purities greater than 98% (Nandakumar et al., 2013).

2.2.4 Lentivirus and retrovirus production and transduction.
Recombinant lentiviral vectors were produced as described in our previous publications
(Nandakumar et al., 2013; R. Schroers, Davis, C. M., Wagner, H-J., and Chen, S-Y. &
2002; R. Schroers et al., 2000). Lentiviruses were produced by co-transfection of 293T
16

cells with packaging plasmids (VSVg, Rev, and Gag/Pol) and lentiviral constructs
encoding MYSM1 (LV- MYSM1), or eGFP alone (LV-GFP) (X.-X. Jiang et al., 2011;
Nandakumar et al., 2013) for 60 to 72 hours. MSCV-IRES-GFP (RV-GFP) and MSCV-
IRES-Id2 (RV-Id2) were kindly provided by CA Klug from the University of Alabama-
Birmingham. Retroviruses were produced by transfection of these retroviral plasmids into
the Phoenix packaging cells and retroviral containing supernatants were harvested 60 to
72 hours later. For transduction, 50,000 NK progenitors (sorted from 5-6 mice) were
cultured overnight in Opti MEM plus Gluta Max 1 time medium (Invitrogen) containing
10% fetal bovine serum (FBS; Sigma-Aldrich), 1% penicillin/streptomycin (Sigma-
Aldrich), 1%10
-2
M 2-mercaptoethanol (Sigma-Aldrich) supplemented with cytokines
with final concentrations: interleukin-7 (IL-7, 20 ng/mL), KIT ligand (KL, 25 ng/mL)
and IL-2 (50 ng/mL) (all cytokines purchased from PeproTech). Lentiviral or retroviral
supernatants were applied to culture dishes pretreated with RetroNectin (TaKaRa) and
centrifuged at 3,000 rpm for 90 minutes and then incubated at 37
o
C in the presence of
polybrene (4 µg/ml) for an additional 24 hours. Cells were then washed and resuspended
in fresh media and transduced cells were used for subsequent in vitro differentiation
assays (Nandakumar et al., 2013).

2.2.5 In vitro generation of NK cells on OP9 cultures.
This experiment was performed as described in(Nozad Charoudeh et al., 2010).
Briefly,10,000 fresh or transduced NK progenitors or KLS (Lin
−
Sca1
+
c-Kit
+
) cells were
plated on previously established, approximately 80% confluent stroma cell monolayers in
17

24-well plates in 1.5 mL of Opti MEM plus Gluta Max 1 time medium (Invitrogen)
containing 10% fetal bovine serum (FBS; Sigma-Aldrich), 1% penicillin/streptomycin
(Sigma-Aldrich), 1%10
-2
M 2-mercaptoethanol (Sigma-Aldrich) supplemented with
cytokines with final concentrations: interleukin-7 (IL-7, 20 ng/mL), KIT ligand (KL, 25
ng/mL) for first week only and Fms-like tyrosinekinase-3 ligand (FL, 25 ng/mL), IL-15
(25 ng/mL), and IL-2(50 ng/mL) (all cytokines purchased from PeproTech). Cell viability
was tested by using Trypan blue dye when cells were plated onto stroma and at the end of
the experiment. Cells were cultured at 37°C for 14 days and then harvested for assessing
the NK cell outgrowth (Nandakumar et al., 2013).

2.2.6 Transplantation assays.
This experiment was performed as described in (Nozad Charoudeh et al., 2010). Briefly,
sublethally-irradiated (350 cGy) 10- to 12-week-old wild type C57BL/6 (CD45.1)
recipient mice were transplanted with 1500 NKPs sorted from 10- to 12-week-old WT
and MYSM1-/- CD45.2 donors mice or with 1500 WT and MYSM1-/- CD45.2 NKPs
mixed with 3000 CD45.1 WT NKPs through retro-orbital injection. At 21 days after
transplantation, recipient mice were analyzed for NK cell donor-derived reconstitution in
the spleen by FACS. The spleen cells were stained with antibodies against CD45.2 and
CD45.1 (to distinguish donor and recipient) (Nandakumar et al., 2013)

18

2.3 Results
2.3.1 Reduction in the frequency and number of NK cells in MYSM1-/- mice
When systemically examining hematopoiesis of MYSM1-deficient (-/-) mice (MYSM1-
KO first floxed mice), besides the severe reduction in peripheral B cells in MYSM1-/-
mice reported in our recent study (X.-X. Jiang et al., 2011; Anastasia Nijnik et al., 2012),
we found a drastic reduction in both the percentages and cell numbers of NK1.1
+
CD3
-

NK cells in various lymphoid tissues of MYSM1-/- mice, compared to its wild-type
littermates (Figs. 2.1A, 2.1B & 2.1D). Jiang et al reported the reduction in the protein and
mRNA levels of MYSM1 in various tissues and cell lineages such as B-cells, T cells and
HSCs (X.-X. Jiang et al., 2011). We confirmed the reduction in MYSM1 transcript levels
by about 8-10 times in the NK cells of MYSM1-/- mice compared to its wild-type controls
using real-time PCR (Fig. A.1, A). In the bone marrow, the primary site of NK cell
development, there was 3 folds reduction and the peripheral NK cells examined in the
spleen, blood, lymph node (pooled inguinal, axillary and cervical), liver and lung showed
a reduction of ~ 3-9 folds in the percentages of NK cells (Figs. 2.1A-B)(Nandakumar et
al., 2013).

In contrast, NK1.1
+
CD3
+
NKT cells did not display any significant differences in their
frequencies in the MYSM1-/- mice compared to its wild-type littermates (Figs. 2.1A,
2.1C & 2.1E). We examined T cells and found that in contrast to NK cell frequencies, the
frequencies of CD4
+
T cells were not significantly altered and that of the CD8
+
T cells
was slightly decreased in spleen but were comparable in the peripheral blood of MYSM1-
19

/- mice (Figs. A.1, B-C). We examined thymus and found that there were some
alterations in the thymic T cell development with the reduction of DN2 frequencies (Fig.
A.1, D) (Nandakumar et al., 2013).
Figure 2.1 Reduction in the frequency and number of NK cells in MYSM1-/- mice
A. Representative flow cytometric profiles of NK1.1+CD3- NK cells or NK1.1+CD3+
NKT cells cells in the femurs and tibias of bone marrow (BM), spleen, peripheral blood
(PBL), lymph node (LN), Liver and Lung of wild-type (WT) and MYSM1-/- mice.
Numbers indicate percentages of cells in each quadrant. B-C. Quantification of the
average percentages of NK and NKT cells in the indicated organs of WT and MYSM1-/-
mice.D-E. Average of total cell numbers of the NK and NKT cells in the indicated organs
Data shown are mean + SEM of n>6 mice per group and are representative of at least 3
independent experiments.*, P < 0.05; **, P < 0.01; ***, P < 0.001. (Adapted from
(Nandakumar et al., 2013)

20

However, the frequencies of double positive CD4CD8 thymocytes were only slightly
reduced and that of the single positive CD4 and CD8 thymocytes were roughly similar
between WT and MYSM1-/- mice (Figs. A.1, A-B). We noticed that the frequencies of
Lin
-
CD122
+
DX5
+
IL-7R
+
thymic NK cells were also not significantly compromised in
MYSM1-/- mice (Figs. A.2, A-B)(Nandakumar et al., 2013).

The absolute cell numbers of NK1.1
+
CD3
+
NKT cells, CD4
+
T cells and CD8
+
T cells
(Figs. 2.1E & A.1, E) were significantly reduced in MYSM1-/- mice compared to its wild-
type littermates due to a severe reduction in total cell numbers of the MYSM1-/- mice (X.-
X. Jiang et al., 2011; Anastasia Nijnik et al., 2012). Nevertheless, in contrast to the
numbers of NKT and T lymphocytes (Figs. 2.1E & A.1, E), the drastic reduction in
NK1.1
+
CD3
-
NK cell numbers in the various lymphoid tissues (Fig. 2.1D)(Nandakumar
et al., 2013) is contributed by both reduction in frequencies and total cell numbers of the
MYSM1-/- mice compared to its wild-type littermates. Together, these data suggest that,
in addition to B cell lineage, MYSM1 is necessary for the generation of NK cells, but not
NKT and T lymphocytes.

2.3.2 Defective NK cell maturation but normal NK lineage commitment in MYSM1-/-
mice

To dissect in detail the effect of MYSM1 deficiency on NK cell development, we
assessed the surface expression of CD122, NK1.1 and DX5 on hematopoietic cells to
gate out precisely the different stages of NK cell development. The three major
developmental stages of NK cells are defined as NK progenitor (NKP; Lin
-
21

CD122
+
NK1.1
-
DX5
-
), immature NK (iNK; Lin
-
CD122
+
NK1.1
+
DX5
-
) cell and mature
NK (mNK; Lin
-
CD122
+
NK1.1
+
DX5
+
) cell (Di Santo, 2006; S. Kim, 2002). The
frequencies of mNK cells were drastically reduced (~5 to 6 folds), whereas the
frequencies of NKPs and iNK cells were increased both in bone marrow and peripheral
tissues of MYSM1-/- mice (Figs. 2.2A-B). The absolute cell numbers were increased at
the NKP stage, not much affected at the iNK cell stage, but severely reduced at the mNK
cell stage of development in the MYSM1-/- bone marrow in comparison to its wild-type
controls (Figs. 2.2C). There was a 17 and 29 folds reduction in the cell numbers of Lin
-
CD122
+
NK1.1
+
DX5
+
mature NK cells in the bone marrow and spleen respectively owing
to a severe reduction in the frequencies of mNK cells in MYSM1-/- mice compared to its
wild-type littermates (Figs. 2.2B & C) (Nandakumar et al., 2013).
Figure 2.2 Defective NK cell maturation but normal NK lineage commitment in
MYSM1-/- mice.
A. Representative flow cytometric analyses of WT and MYSM1-/- mice using NK1.1 and
DX5 to identify NKP (NK1.1
-
DX5
-
), iNK (NK1.1
+
DX5
-
), and mNK (NK1.1
+
DX5
+
) cells
(lower panel) in CD122
+
CD3
-
B220
-
lymphocytes (upper panel) in the femurs and tibias
of bone marrow, spleen and peripheral blood of WT and MYSM1-/- mice. Numbers
indicate percentages of cells in each quadrant. NKP - NK progenitors; iNK - immature
NK; mNK - mature NK. B. Quantification of the average percentages of NKP, iNK and
mNK cells in the bone marrow and spleen (smNK) C. Average of total cell numbers of
NKP, iNK and mNK cells in the bone marrow and spleen (smNK) of WT and MYSM1-/-
mice. Data shown are mean + SEM of n>6 mice per group and are representative of at
least 3 independent experiments *, P < 0.05; **, P < 0.01; ***, P < 0.001. D.
Representative flow cytometric analyses of WT and MYSM1-/- bone marrow using Flt3
and CD122 to identify CLP (FLT3
+
CD122
_
), pre-NKP (FLT3
-
CD122
-
), and rNKP
(FLT3
-
CD122
+
) in Lin
-
c-KIT
-
CD27
+
CD244
+
IL7R
+
cells. CLP - Common lymphoid
progenitor. Numbers indicate percentages of cells in each quadrant. E. Quantification of
the average percentages of CLPs, pre-NKPs and rNKPs cells in the bone marrow. F.
Average of total cell numbers of CLPs, pre-NKPs and rNKPs in the bone marrow of WT
and MYSM1-/- mice. Data shown are mean + SEM of n=5 mice per group; *, P < 0.05;
**, P < 0.01; ***, P < 0.001. G. MYSM1 mRNA expression at each NK developmental

22

Figure 2.2 continued

stage and in the progenitors of NK cells: Lin
-
c-KIT
-
CD27
+
CD244
+
IL-7R
+
Flt3
-
CD122
-

preNKPs, Lin
-
CD122
+
NK1.1
-
DX5
-
NKPs, Lin
-
c-KIT
-
CD27
+
CD244
+
IL-7R
+
Flt3
-
CD122
+
rNKPs, Lin
-
CD122
+
NK1.1
+
DX5
-
iNK cells, Lin
-
CD122
+
NK1.1
+
DX5
+
mNK cells, Lin
-
c-
KIT
-
CD27
+
CD244
+
IL-7R
+
Flt3
+
CD122
-
CLPs and Lin
-
cKit
+
Sca1
+
KLS cells. All cells
were sorted from pooled femurs and tibias of wild-type bone marrow. MYSM1
expression was normalized to GAPDH; Data are representative of five independent
experiments (Nandakumar et al., 2013)

23

Previously, impairment in the development of B-lymphocytes at early stages of their
differentiation has been demonstrated in the MYSM1-/- mice (X.-X. Jiang et al., 2011;
Anastasia Nijnik et al., 2012). In order to test if MYSM1 regulates NK fate commitment
from its lymphoid progenitors similar to B-cells, we evaluated the NK precursor
compartment of wild-type and MYSM1-/- mice. Lin
-
IL-7R
+
cKit
int
Sca1
+
cells, commonly
defined as common lymphoid progenitors (CLP) can be fractionated into Flt3
+
and Flt3-
CLPs. Flt3
+
CLPS were severely affected in the MYSM1-/- mice (Figs.A.3, A-A.3,
C)(Nandakumar et al., 2013) due to loss of Flt3
+
KLS (Lin
-
c-KIT
+
Sca1
+
) subsets, as also
observed and reported by Nijnik et al (Anastasia Nijnik et al., 2012). However, to
precisely identify the CLPS that can differentiate into NKPs, Fathman et al recently
developed a strategy to distinguish CLPs, preNKPs and rNKPs (refined NKPs) based on
their refined analysis of markers that are expressed on these subsets (Fathman et al.,
2011). CD27 and CD244 are cell surface markers expressed on CLPs and maintained on
mature NK cells (Fathman et al., 2011). Additionally, on commitment to NK lineage and
with loss of B, T, and DC lineage differentiation potentials, Flt3
+
CLPs lose Flt3
expression and acquire CD122 expression sequentially (Fathman et al., 2011). Within the
Lin
-
CD27
+
CD244
+
IL-7R
+
subset, differential expression of Flt3 and CD122 and the NK
lineage bias exhibited by these cells in functional assays distinguished three progenitor
subsets: Flt3
+
CD122
-
(CLPs), Flt3
-
CD122
-
(preNKPs) and Flt3
-
CD122
+
(rNKPs). Based
on our analysis using the above markers, we found a severe reduction in the frequencies
and total cell numbers of CLPs defined as Lin
-
c-KIT
-
CD27
+
CD244
+
IL-7R
+
Flt3
+
CD122
-

cells; in contrast the frequencies of pre-NKPS defined as Lin
-
c-KIT
-
CD27
+
CD244
+
IL-
24

7R
+
Flt3
-
CD122
-
cells and that of rNKPs defined as Lin
-
c-KIT
-
CD27
+
CD244
+
IL-7R
+
Flt3
-
CD122
+
cells were significantly increased (3 folds) in the bone marrow of MYSM1-/-
mice compared to its wild-type controls (Figs. 2.2D-F)(Nandakumar et al., 2013). The
absolute cell numbers of pre-NKPs and rNKPs were similar between the MYSM1-/- and
wild-type mice (Fig. 2.2E).

Collectively, our data show that the reduction of Flt3
+
CLPS
in the MYSM1-/- mice, did not compromise the generation of Flt3
-
CLPs, comprising of
pre-NKPs and rNKPs (Figs. 2.2D-F & Figs. A.3, A-A3, C)(Nandakumar et al., 2013).

Real-time PCR analyses of sorted WT KLS cells, CLPs, pre NKPs, rNKPs, NKPs, iNK
cells and mNK cells showed higher mRNA levels of MYSM1 at rNKP, iNK and mNK
cells than that expressed by their progenitors (Fig. 2.2G). The sharp increase in the
mRNA levels at the rNKP stage suggests the dependence of later stages of NK cell
development on this onset of MYSM1 and is consistent with the block observed in NK
cell maturation rather in its lineage commitment in the MYSM1-/- mice (Fig.
2.2G)(Nandakumar et al., 2013).

Together, these data demonstrate a block in the maturation of NK cells during NK cell
development in MYSM1-/- mice, while the development of NKP and immature NK cells
from their lymphoid precursors were not compromised. Thus, different from its critical
role in early B cell commitment, MYSM1 is required for the maturation of NK cells, but
not for NK lineage commitment or its transition to iNK cells(Nandakumar et al., 2013).

25

2.3.3 MYSM1-/- NK cells are phenotypically immature
The reduction observed in the mNK cell subsets in the bone marrow and peripheral
organs but not of that of iNK cells (Figs. 2.2A-C)(Nandakumar et al., 2013), raised the
question if the NK cells that egress from the MYSM1-/- bone marrow to the peripheral
organs are mainly from the iNK cell subsets. To test this, we assessed the expression of a
panel of well-characterized mature and immature cell surface antigens on wild-type and
MYSM1-/- NK cells.

The most mature NK cells express the markers DX5, CD11B, KLRG1 but not the
immature markers c-KIT, CD27 and CD51 (Hayakawa & Smyth, 2006; Huntington et al.,
2007; S. Kim, 2002; Robbins, Tessmer, Mikayama, & Brossay, 2004). Flow cytometric
analysis of a panel of NK developmental markers in the MYSM1-/- and wild-type splenic
NK1.1
+
CD3
-
B220
-
NK cells (Figs 2.3A-B) revealed that MYSM1-/- NK cells exhibited
lower levels of expression of mature markers such as DX5, CD11B and KLRGI but the
immature markers displayed increased (CD51) or unaltered (c-KIT) levels compared to
that in the wild-type cells (Figs 2.3A-B). However, CD27 expression was decreased in
the MYSM1-/- NK cells compared to that in the wild-type cells (Figs 2.3A-B)
(Nandakumar et al., 2013).

Hayakawa et al (Hayakawa & Smyth, 2006) proposed that NK cell maturation can be
sub-divided into four stages based on the expression levels of CD27 and CD11B such as
DN CD27
-
CD11B
-
, CD27
+
CD11B
-
, DP CD27
+
CD11B
+
and CD27
-
CD11B
+
in the order
26

of development(Hayakawa & Smyth, 2006). In the MYSM1-/- bone marrow and
peripheral organs, there was a significant decrease in the frequencies of CD27
-
CD11B
+
(~
4-8 folds) and CD27
+
CD11B
+
(~18-48 folds) NK cell subsets and an increase in the
frequencies of CD27
-
CD11B
-
(~ 8-13 folds) NK cell subsets compared to that in the wild-
type tissues (Figs. 2.3C-D)(Nandakumar et al., 2013). CD27
+
CD11B
-
NK cell subsets
were decreased as well in the MYSM1-/- mice (except in the blood), but less severely than
the DP and CD27
-
CD11B
+
subsets (Figs. 2.3C-D). Our analyses revealed that MYSM1-/-
NK cells were predominantly composed of the DN subsets in comparison to the wild-
type NK cells that were mainly DP and CD27
-
CD11B
+
NK cells (Figs. 2.3C-D). Narni-
Mancinelli E et al (Narni-Mancinelli et al., 2011) suggested that engagement of cells into
NK cell lineage occurs after the acquisition of NK1.1 and before the expression of
NKp46. Our analysis of NK1.1 vs NKp46 on CD122
+
B220
-
CD3
-
BM lymphocytes
showed that NK1.1
+
NKp46
+
cells were reduced by only ~1.5 folds in the MYSM1-/- mice
(Fig. A.4, A). However, mature NK cell subsets gated from NK1.1
+
NKp46
+
lymphocytes
such as: NKp46
+
DX5
+
or CD27
+
CD11B
+
or CD27
-
CD11B
+
NK cell subsets were
severely affected in their development in the MYSM1-/- mice. We also compared CD11B
versus KLRGI (most mature NK cell marker) expression and found that MYSM1-/- NK
cell compartment was predominated by CD11B
-
KLRG1
-
NK cells and were severely
compromised in their mature NK cell pool (CD11B
+
KLRG1
-
,

CD11B
+
KLRG1
+
,CD11B
-
KLRG1
+
) compared to that of the wild-type controls (Fig. 2.3E)(Nandakumar et al.,
2013). Together, these data add more to our finding by showing that MYSM1 is required
for the maturation of NK cells specifically, during the onset of NKP46 and during its
27

transition to mature NK cells, but not for NK lineage commitment or in its transition to
immature Lin-CD122+NK1.1+NKp46- NK cells.

Activatory receptors on NK cells are necessary for target recognition and induction of
NK-cell mediated cytolysis (Moretta et al., 2001). MYSM1-/- mice exhibited lower levels
of expression of activatory receptors 2B4, Ly49D, NKp46, LY49H, and CD11b than did
WT controls (Figs. 2.3A-B). However the expression of NKG2D, an activatory receptor,
remained unaltered in the MYSM1-/- NK cells compared to that in the wild-type cells
(Figs. 2.3A-B)(Nandakumar et al., 2013).
Figure 2. 3 MYSM1-/- NK cells are phenotypically immature
A. Representative flow cytometric profiles of the 4 stage NK cell development: Double
negative (DN) CD27
-
CD11B
-
, CD27
+
CD11B
-
, double positive (DP) CD27
+
CD11B
+
and
CD27
-
CD11B
+
expression on NK1.1
+
B220
-
CD3
-
NK cells in the femurs and tibias of
bone marrow, spleen and peripheral blood of WT and MYSM1-/- mice. Numbers indicate
percentages of cells in each quadrant. B. Quantification of the average percentages in NK
cells (NK1.1
+
B220
-
CD3
-
) being DN, CD27
+
CD11B
-
, DP and CD27
-
CD11B
+
populations
in the indicated organs. C. Representative flow cytometric plots of KLRG1

vs CD11B

expression on NK1.1
+
B220
-
CD3
-
lymphocytes in the spleens of WT and MYSM1-/- mice.
Numbers indicate percentages of cells in each quadrant. Data shown are mean + SEM of
n=5 mice per group and are representative of at least 3 independent experiments; *, P <
0.05; **, P < 0.01; ***, P < 0.001. D. Representative flow cytometric histograms of NK
developmental markers gated on splenic NK1.1
+
B220
-
CD3
-
NK cells of WT and
MYSM1-/- mice. Numbers near the gate indicate percentages of cells within the positive
gate. Numbers against ‘*’ indicate relative median fluorescence intensities (MFIs) of
specific markers above the auto fluorescence of the corresponding isotype controls in the
WT and MYSM1-/- mice. E. Quantification of the average percentages of the
developmental markers in the splenic NK cells (NK1.1
+
B220
-
CD3
-
) being decreased
(top), unaltered (middle) or increased (bottom) in the MYSM1-/- mice vs wild-type
controls. Data shown are mean + SEM of n=3 mice per group and are representative of at
least 3 independent experiments; *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Nandakumar
et al., 2013)

28

Figure 2.3 continued

For complete NK cell maturation, NK cell education through recognition of self-MHC
class I molecule by their inhibitory receptors (LY49C/I, LY49G2, NKG2A/C/E) is
necessary and this process occurs during the transition from iNK to mNK cell stage. We
analyzed the expression of these inhibitory receptors and found that there was a
significant decrease in the levels of Ly49G2 and LY49C/I levels in the MYSM1-/- NK
29

cells compared to that in the wild-type cells, however, the NKG2A/C/E levels remained
unaltered (Figs. 2.3A-B)(Nandakumar et al., 2013).

Together, the severe decrease in the levels of many mature developmental markers
including the activatory and inhibitory receptors demonstrates the predominance of an
immature nature in the MYSM1-deficient NK cells.

2.3.4 MYSM1 is intrinsically required for NK cell maturation in vivo
Our data demonstrates a defect in the MYSM1-/- mNK development but not in the NK
lineage commitment or in their transition to immature NK cells (Figs. 2.2A-F). To
investigate whether the defective NK cell maturation in vivo comes from the
microenvironment or from a phenomenon intrinsic to NK progenitors, we performed
transplantation assays using NKPs as recently described(Nozad Charoudeh et al., 2010).
In order to account for the bias that may arise from the reduced MYSM1-/- HSC
progenitors in comparison to that of the wild-types (Anastasia Nijnik et al., 2012), we did
not transplant bone marrow cells. We transplanted equal numbers of NKPs instead to
establish a more direct and accurate comparison between the wild-type and MYSM1-/-
NK cell maturation from their committed NK progenitors. Lin
-
CD122
+
NK1.1
-
DX5
-
NKPS sorted to high purity (Fig. A.6)(Nandakumar et al., 2013) from CD45.2 bone
marrows of wild-type and MYSM1-/- mice were injected intravenously into sub-lethally
irradiated CD45.1 WT B6.SJL-PtprcPep3/BoyJ recipients. Flow cytometric analysis of
spleen 21 days post transplantation, showed similar frequencies and numbers of total
30

CD45.2 chimerism in the recipient mouse reconstituted with wild-type or MYSM1-/-
donor cells (Figs 2.4A-B). Also, donor-derived reconstitution of T cells was similar in the
recipient mouse reconstituted with wild-type or MYSM1-/- donor cells and as expected
transplanted NKPs did not contribute to detectable B-cell reconstitution (Figs 2.4A).
CD45.2 immature NK (B220
-
CD3
-
NK1.1
+
DX5
-
) chimerism was evident in the recipient
mice which received WT donor cells and increased in the recipient mice which received
MYSM1-/- donor cells (Figs 2.4A), however the reconstitution of mature NK cells (B220
-
CD3
-
NK1.1
+
DX5
+
)

occurred only with the wild-type donor cells and was severely
defective in the recipient mice that received MYSM1-/- donor cells (Figs 2.4A-B),
indicating an intrinsic role of MYSM1 in NK cell maturation (Nandakumar et al., 2013).
We also used a mixed-chimera model, in which we transplanted equal numbers of WT
CD45.1 and WT or MYSM1-/- CD45.2 NKPs into sub-lethally irradiated CD45.1 WT
recipients (Fig. A.5, A). We were unable to compare the NK-cell reconstitution ability of
CD45.1 and CD45.2 cells side by side due to the presence of host CD45.1 cells in the
sub-lethally irradiated recipient mice. Nevertheless, these data that WT CD45.2 NKPs
contributed to a normal NK cell reconstitution in contrast to MYSM1-/- CD45.2 NKPs
that showed a defective NK cell maturation in the recipient mice was consistent with that
of the NKP chimera study, re-affirming the conclusion that MYSM1 is an intrinsic factor
for NK-cell maturation in vivo(Nandakumar et al., 2013).
Figure 2. 4 MYSM1 is intrinsically required for NK cell maturation
A-B. Sublethally-irradiated 10- to 12- week-old WT B6.SJL-PtprcPep3/BoyJ recipient
mice were transplanted with 1500 bone marrow Lin
-
CD122
+
NK1.1
-
DX5
-
NKPs sorted
from 9- to 12-week-old CD45.2 WT and MYSM1-/- mice. Donor-derived lineage

31

Figure 2.4 continued

reconstitution was evaluated in the spleen 21days after transplantation by FACS. A.
Representative FACS plots of donor-derived CD45.2 NK (NK1.1
+
DX5
+
CD3
-
B220
-
)
reconstitution in the spleen. The specific gates are indicated in the text above the plot and
by arrows B. Percent and absolute cell numbers of donor-derived total (CD45.2) cells and
donor derived NK cells (NK1.1
+
DX5
+
CD3
-
B220
-
) from the spleens of mice transplanted
with WT and MYSM1-/- NKPs. NKPs were injected into 5 individual mice per group in
each experiment. Data shown are representative of 1 of 2 experiments and are mean +
SEM of n>5 mice per group in each experiment *, P < 0.05; **, P < 0.01; ***, P < 0.001.
C-D. In vitro NK differentiation assays. FACs-sorted 10,000 Lin
-
CD122
+
NK1.1
-
DX5
-
NKPs from 12-week-old WT and MYSM1-/- mice were cultured on OP9 stroma with
cytokines: KL, IL-7 (first week only), FL, IL-2, and IL-15 for 14 days, cells were
harvested and evaluated for NK cell outgrowth by FACS. 7AAD was used to exclude
dead cells. C. Shown are representative flow cytometric profiles of NK cells
(NK1.1
+
DX5
+
CD3
-
) generated from WT and KO NKPs and numbers indicate
percentages of cells in each quadrant. D. Mean proportions of NK1.1
+
DX5
+
CD3
-
NK
cells generated in vitro per input WT and MYSM1-/- NKPs. Data shown are mean + SEM
of three independent experiments; *, P < 0.05; **, P < 0.01; ***, P < 0.001. E-F. In vitro
MYSM1 rescue assays. FACs-sorted WT and MYSM1-/- CD122
+
Lin
-
NK1.1
-
DX5
-
NKPs
from the bone marrow were transduced with a recombinant lentiviral vector LV-MYSM1
or a control lentiviral vector LV-GFP and 10,000 transduced cells were transferred to
OP9 cell co-culture and subjected to NK cell generation as explained above for 14 days,
cells were harvested and assessed for NK cell outgrowth by flow cytometric analysis.
7AAD was used to exclude dead cells. E. Shown are representative flow cytometric
profiles of NK cells (NK1.1
+
DX5
+
CD3
-
) generated from WT NKPs, MYSM1-/- NKPs,
WT NKPs over-expressed with MYSM1 or MYSM1-/- NKPs rescued with MYSM1 and
numbers indicate percentages of cells in each quadrant. F. Mean proportions of
NK1.1
+
DX5
+
CD3
-
NK cells generated in vitro per input respective NKPs as indicated.
Data shown are mean + SEM three independent experiments; *, P < 0.05; **, P < 0.01;
***, P < 0.001 (Nandakumar et al., 2013)

32

Figure 2.4 continued

2.3.5 MYSM1 is intrinsically required for NK cell maturation in vitro

To further confirm the intrinsic role of MYSM1 in NK cell maturation, we performed in
vitro NK cell generation assays, once again from NKPs (Fig. A.6)(Nandakumar et al.,
2013), and not from hematopoietic progenitors to exclude possibilities of the defective
KLS (Lin
-
c-KIT
+
Sca1
+
) differentiation at earlier stages of hematopoiesis (Anastasia
Nijnik et al., 2012) impacting on mNK cell generation in the MYSM1-deficient mice. We
made use of the protocol developed by Nozad Charoudeh et al (Nozad Charoudeh et al.,
2010) in which as few as 1-10 NKPs co-cultured on OP9 stroma cell lines for 14 days in
the presence of KIT ligand(KL), Fms-like tyrosinekinase-3 ligand (FL), IL-7 (first week
33

only), IL-2, and IL-15 gave rise to a large number of NK cell clones. In consistent with
the in vivo results, NKPs did not produce any detectable B220
+
B-cells (Fig 2.4C). But,
there was a severe defect in the development of MYSM1-/- Lin
-
CD122
+
NK1.1
-
DX5
-
NKPs into NK1.1
+
DX5
+
mNK cells in comparison to WT NKPs that differentiated
normally into mNK cells in the presence of IL-15 (Figs 2.4C-D)(Nandakumar et al.,
2013).

Since our data showed no defect in the MYSM1-/- NK lineage commitment in vivo (Figs.
2.4A-F), we speculated to see similar trend to that of NK cell generation from MYSM1-/-
NKPS even when MYSM1-/- KLS cells were used as inputs; our data proved the same
(Figs. A.5, B - A.5, C). The fact that not only KLS but also NKP-derived NK cells
suffered a maturation defect reconfirms our previous finding that the defective KLS
differentiation at the early stages of hematopoiesis (Anastasia Nijnik et al., 2012) did not
contribute to the impairment in the development of mature NK cells in the MYSM1-/-
mice (Figs. 2.4A-F). Together, these results recapitulated the in vivo phenotype of
MYSM1-/- mice.

To further test the direct relationship between MYSM1 and NK cell development, we
extended the in vitro system and carried out a rescue assay of MYSM1-/- NKPs using a
recombinant lentiviral vector (LV) expressing MYSM1 (X.-X. Jiang et al., 2011). NKPS
sorted to high purity (Fig. A.6) were transduced with LV-MYSM1 and the transduced
cells were subjected to NK cell differentiation in vitro. Figs. 2.4E-F show that forced
34

expression of MYSM1 in vitro rescued the defective ability of MYSM1-/- NKPs in
generating NK1.1
+
DX5
+
mNK cells. In contrast, transduction with control LV-GFP failed
to rescue the developmental defect of MYSM1-/- NKPs (Figs. 2.4E-F). Furthermore,
ectopic expression of MYSM1 in wild-type NKPs increased the proportion of mature NK
cells produced (Figs. 2.4E-F), showing that MYSM1 over-expression can boost NK cell
development in vitro. Collectively, these data demonstrate the dependence of NK cell
maturation on MYSM1(Nandakumar et al., 2013).

IL-15 is a critical factor for NK cell development and homeostasis and both IL-15-/- or
IL-15R-/- mice were defective in their production of mature NK cells (Kennedy et al.,
2000). We were curious to know how MYSM1 responds to IL-15 and hence checked the
outgrowth of NK cells from WT NKPs over-expressed with MYSM1 in the absence of
IL-15. Interestingly, over-expression of MYSM1 in WT NKPS significantly increased the
generation of NK cells even in the absence of exogenous IL-15 (Figs. A.5, D - A.5, E).
Not only that, upon IL-15 stimulation, the mRNA levels of MYSM1 was significantly
increased both in the WT NK cells derived in vitro from NKPs over-expressed with
MYSM1 (Fig. A.5, F) and in the exvivo cultured freshly sorted Lin
-
CD3
-
CD122
+
NK1.1
+
NK Cells (Fig. A.5, G). These data suggest that MYSM1 functions downstream IL-15
during NK cell development.Thus both our in vivo and in vitro data indicate that NK
maturation requires cell-intrinsic MYSM1 expression(Nandakumar et al., 2013).

35

2.4 Conclusion
Natural killer cells are a subset of lymphocytes that kill virus-infected and tumor cells
and signal adaptive immune system to kill the pathogens through the production of
several inflammatory cytokines. Unlike T and B lymphocytes, the transcription factors
essential for the development of NK cells are not well-understood. Although many
transcription factors have been identified at the later stages of NK cell development, no
transcription factors have been identified at the initial stages for the emergence of NK
cell progenitors from their multipotent precursors.

The epigenetic changes act as a control machinery to modulate the expression of the
transcription factors and its access to the different gene regulatory regions involved in the
development of different cell lineages. The ubiquitination of histone H2A (uH2A) is one
of the sparsely studied epigenetic modification; however recent studies have identified
the role of several histone H2A ubiquitinases and deubiquitinases, specifically in the
lymphocytes.

In our study, we identified a novel histone H2A deubiquitinase, MYSM1 and created its
knockout mice to determine the function of this gene. In our recent study, we found that
the histone H2A deubiquitinase MYSM1, a Myb-like, SWIRM, and MPN domain-
containing protein is essential for early B cell development by derepressing the
transcription of EBF1 in B cell progenitors (X. X. Jiang et al., 2011). Later on in this
study, we found that MYSM1’s deletion lead to an impaired development of NK cells as
36

well. In this study, we found that NK cell development is severely impaired in MYSM1-
deficient (-/-) mice, while the development of NKT and CD4+ and CD8+ T lymphocytes
are not apparently compromised in MYSM1-deficient mice. We demonstrated a block in
the transition from immature NK cells to mature NK cells in various lymphoid tissues of
MYSM1-/- mice, while the development of NKP and immature NK cells from their
lymphoid precursors are not compromised. These data indicate an essential role of
MYSM1 in the maturation of NK cells, but not the lineage specification. We
unequivocally demonstrated that MYSM1 has an intrinsic role in NK cell maturation. In
conclusion, this study is one of the first studies to explore the epigenetic regulation
during NK cell development.

37

Chapter 3. Epigenetic control of Id2 transcription by MYSM1 during
NK cell Development

3.1 Introduction
Natural killer (NK) cells are part of innate immune system and provide rapid response
against cancer and infection. Many transcriptions factors play critical roles in the
regulation of gene transcription involved in immune cell fate and differentiation
(Luevano, Madrigal, & Saudemont, 2012). The use of Knockout mice has facilitated the
identification of transcription factors essential for NK cell lineage commitment,
maturation or of tissue-specific NK cells (Luevano et al., 2012). NK cell development
occurs in different stages, CD34+ hematopoietic stem cells differentiate into common
lymphoid progenitors that can give rise to NK cell precursors that then develop into
immature NK cells. Finally, the maturation of immature NK cells into fully functional
NK cells involves the acquisition of activating and inhibitory receptors that regulate NK
cell effector functions (Lanier, 2008).

Transcription factors such as ID2 and ID3 have been shown to control the development
of mature NK cells from their precursors while GATA-3, T-bet, Eomes and IRF2 are
known to be involved in generating functional NK cells that can exit bone marrow and
enter peripheral tissues to perform their function (Boos, Ramirez, & Kee, 2008; M. Boos,
Y. Yokota, G. Eberl, & B. Kee, 2007). However, unlike T and B lymphocytes, the
molecular mechanisms that regulate the transcription of these key transcription factors
during NK cell development remain poorly defined (Nandakumar et al., 2013).
38

ID2 proteins are important class of proteins essential for NK cell development and
maturation since the late 1990s. Mice deficient for ID2 have been shown to lack LNs,
Peyer’s patches as well as to have reduced number of mature NK cells in the spleen
(Luevano et al., 2012; Yokota et al., 1999). Also, when ID3 was over expressed in CD34
progenitors, T cell development was inhibited and NK cell differentiation was favored
(Yokota et al., 1999). Although the role of ID2 in the regulation of NK cell development
was further confirmed (M. Boos et al., 2007), they reported that ID2 seems to be essential
only at the later stage in differentiation. ID2–/– mice have reduced numbers of mature
NK cells in the periphery while the numbers of immature NK cells and NK cell
precursors remained unaffected, emphasizing the importance of ID2 in NK cell
maturation (M. Boos et al., 2007). The phenotype observed in ID2–/– animals was linked
to the capacity of ID2 to inhibit E2A proteins (Luevano et al., 2012). NFIL3-/- mice had
impaired development of immature and mature NK cells but all other hematopoietic cell
lineages developed normally highlighting that NFIL3(E4bp4) is essential for the
progression from NK cell precursors to immature NK cells and then from immature NK
cells to mature NK cells(Gascoyne et al., 2009; Luevano et al., 2012). Eomes is another
transcription factor essential for NK cell maturation and in the maintenance of functional
NK cells (Gordon et al., 2012; Luevano et al., 2012). Ets-1 is an essential transcription
factor for the differentiation of NK cell progenitors, specifically to induce the expression
of other TFs involved in the regulation of NK cell development such as T-bet and ID2
(Luevano et al., 2012; Ramirez et al., 2012).

39

Figure 3. 1 Schematic depicting the molecular regulation of NK cell development.
Transcription factors involved in murine NK cell development and maturation. Adapted
from (Luevano et al., 2012)

Natural killer cell development in humans and mice has been explored for several
decades. Yet, understanding the pathways and regulation of events that control NK cell
development remains a challenge(Luevano et al., 2012). In this previous chapter, we
reveal an important and intrinsic role of MYSM1 in the maturation of NK cells, but not
for the NK lineage specification and early development. In this chapter, we further
delineate the underlying mechanism by which MYSM1 controls target gene transcription
and NK cell maturation.
40

3.2 Materials and Methods
3.2.1 Animals.
MYSM1-deficient (-/-) mice (MYSM1-KO first floxed mice) is generated through a “KO
first” strategy as described in 1.5.

3.2.2 Cell preparation
Single cell suspensions were obtained as described in 2.2.2

3.2.3 Flow cytometry and Cell sorting
These methods are described in 2.2.3

3.2.4 Chromatin Immunoprecipitation
Chromatin was immunoprecipitated according to the manufacturer’s instruction (#9002,
Cell Signaling)(Nandakumar et al., 2013). Facs-sorted Lin
-
CD122
+
NK1.1
+
NK cells from
pooled bone marrow and spleens cells were used for all our chIP assays. The purity of the
sorted cells was confirmed by reanalysis (> 98% pure). Due to a drastic reduction in NK
cell numbers in the MYSM1-/- mice, 6-7 MYSM1-/- mice were used to get ~ 1 million
cells and not less than 1 million WT or MYSM1-/- cells were used for each pull-down. At
a time 8X10
6
WT and MYSM1-/- cells were sorted separately, crosslinked with 1%
(vol/vol) formaldehyde at room temperature for 10 min, incubated with glycine for 5 min
at room temperature, washed with ice-cold PBS and the cross-linked cell pellets were
stored in batches at -80⁰C for subsequent immunoprecipitations. The chromatin
41

preparations were verified for enrichment of known NK cell specific interactions
(RUNX3 binding to NKp46 promoter) through standard chIP assays as described below
and this was done in every newly prepared chromatin fractions. The frozen pellets from
16X10
6
cells were thawed and then sequentially washed in ice-cold buffer A and buffer
B, followed by digestion with MNase. Nuclear pellet was suspended in ChIP buffer,
chromatin sheared by sonication (the Branson Sonifier 450) to an average size of about
400 - 600 base pairs. After centrifugation at 10,000 rpm for 10 minutes, sheared
chromatin was diluted in ChIP buffer and precleared by addition of protein G plus
agarose beads (for IgG purification; santacruz sc-2003) or protein L plus agarose beads
(for IgM purification; santacruz, sc-2336) for 1 h at 4° C. The beads were discarded and
the supernatant was then incubated with one of these antibodies (ab-Abcam; sc-
Santacruz),: H3K4me3 (ab-1012), H3K4me2 (ab-7766), H3K27me3 (ab-6002), NFIL3
(F-1, sc-74414), ubH2A (E6C5, 05-678; from Millipore), anti-MYSM1(X.-X. Jiang et al.,
2011), RNA POL II s5p (H14, MMS-134R; from Covance), RNA POL II s2p (H5,
MMS-129R; from Covance) and control anti-IgG (cell-signalling) or anti-mouse IgM
(12-488, Millipore) at 4° C overnight. At the next day, protein G or protein L plus
agarose beads were added and incubated for 2h at 4° C. Beads were harvested by
centrifugation followed by 3 low salt washes and one high salt wash. Beads were then
eluted with ChIP elution buffer. The elutes and input were then treated with proteinase K
and RNase A and heated at 65° C for 2h to reverse the formaldehyde cross-links. DNA
fragments were purified using columns(Nandakumar et al., 2013).
42

For sequential two-step ChIP experiments (Zhou et al., 2008), complexes from initial
anti-MYSM1 ChIP were eluted and diluted 15 folds in ChIP buffer and re-
immunoprecipitated with anti-NFIL3 and anti-IgG (cell-signalling). Eluted DNA and
sheared input material was analyzed by real-time PCR using primers shown in
supplementary Table 2. The percent immunoprecipitated DNA (for the protein of interest
along the ID2 locus) of the total input DNA is calculated. The binding of the protein of
interest was determined by comparing the percent inputs of protein of interest with that of
the negative control IgG or IgM for significant enrichment (Nandakumar et al., 2013).

3.2.5 Protein immunoprecipitation and immunoblotting.
Total lysates were prepared from HEK 293T cells transiently transfected with pCMV
Nfil3, pCMV Tox, pCMV ETS-1, pCMV Eomes and pCMV-FLAG-MYSM1 or pCMV-
FLAG expression plasmids or lysates were prepared from FACS sorted NK1.1
+
CD3
-

splenic and bone marrow NK cells (20X10
6
) of WT mice using the cytoplasmic/Nuclear
complex CoIP kit according to the manufacturer’s instructions (Active Motif)
(Nandakumar et al., 2013). Briefly, lysates were incubated with anti-Flag (F1804, Sigma)
or anti-MYSM1 (ab107542) or control IgG antibodies at 4° C overnight, followed by
incubation with protein A/G agarose beads for additional 2h. After washing three times
with washing buffer, immunoprecipitates were boiled in SDS sample buffer, resolved by
4-12% Bis-Tris NuPAGE gel (Invitrogen), transferred to PVDF membrane and probed
with one of these antibodies, anti-NFIL3 (Abcam, ab93785), anti-Flag (C-17, sc-835),
Anti-ETS-1 (sc-55581), anti-TOX (sc-374137), Anti-EOMES (sc-98555) and anti-Actin
43

(sc-1616). 1-10% input was used to detect the total protein and anti-actin is used as a
control to verify equal starting amounts of protein for immunoprecipitation. For
expression vectors, full length cDNA sequences were purchased from Origene
(Nandakumar et al., 2013).

3.2.6 Quantitative RT-PCR.
Total RNA from isolated cells was purified with RNeasy Minikit (Qiagen) according to
the manufacturer’s instructions (X.-X. Jiang et al., 2011; Nandakumar et al., 2013). The
iScript™ Select cDNA Synthesis Kit (BIO-RAD) was used for reverse transcription
using oligi-dT primers or random hexamers. A SYBR Green PCR kit (BIO-RAD) was
used for quantitative real-time PCR and results were quantified with an ICycler IQ (BIO-
RAD). Sequences of primer pairs are listed in the Supplementary Table 2.

3.2.7 Statistics.
Statistics was calculated with the prism software (Graphpad prism). To test the
significant differences between two groups, a Student’s t test with a two-tailed p value
was used. Statistical significance was reached at p<0.05

3.3 Results
3.3.1 MYSM1 is required for Id2 transcription
Given that MYSM1, a histone H2A deubiquitinase functions as a transcriptional regulator
(X.-X. Jiang et al., 2011; Zhou et al., 2008), we set out to assess whether expression of
44

any genes having implication in NK cell development (Boos et al., 2008) were altered in
MYSM1-/- mature NK cells and their progenitors by qRT-PCR assays. We detected a
marked reduction in the mRNA levels of Id2 (~6 folds) accompanied with an increase in
the mRNA levels of E2A in the sorted MYSM1-/- mNK (CD122
+
Lin
-
NK1.1
+
DX5
+
) and
NKP (CD122
+
Lin
-
NK1.1
-
DX5
-
) cells compared to that of the WT cells. mRNA levels of
TBET and GATA3 were also decreased in the MYSM1-/- mNK cells compared to that of
wild-type but less severely than Id2 (Figs. 3.2A-B & Fig. B.1)(Nandakumar et al., 2013).

ID2 and ID3 are both expressed in NKPs but Id2 is the predominant transcription factor
in mature NK cells (M. D. Boos, Y. Yokota, G. Eberl, & B. L. Kee, 2007; Ikawa,
Fujimoto, Kawamoto, Katsura, & Yokota, 2001). In the absence of Id2, NKP and iNK
cell development is not affected likely due to the functional compensation of ID3 protein,
highly expressed during these stages of NK cell development. Similar to MYSM1, NK
cells fail to mature in the absence of Id2 (M. D. Boos et al., 2007). An earlier onset of
expression for MYSM1 mRNA (peaks in rNKPs) as compared to Id2 (peaks in iNK) is
consistent with the hypothesis that Id2 transcription is dependent on MYSM1 during NK
cell development (Figs. 2.2G & Fig. B.2). These data combined with the significant
reduction in the mRNA levels of Id2 in the MYSM1-/- NK cells (Figs. 3.2A-
B)(Nandakumar et al., 2013) indicates that MYSM1 is required for Id2 transcription.

Subsequent to this, we sought to test if MYSM1 controlled Id2 transcription during NK
cell development. To do this, we transduced MYSM1-/- NKPs with LV-MYSM1 or
45

control LV-GFP and transferred them to an OP9 co-culture system in the presence of NK
cell conditioned media. After 14 days, Id2 mRNA levels of the in vitro generated and
post-sorted NK1.1
+
cells were examined by qRT-PCR. Fig. 3.2C shows that forced
expression of MYSM1, but not that of GFP control, significantly increased the mRNA
levels of Id2 in the MYSM1-/- NK cells generated in vitro, indicating that Id2 is directly
or indirectly transcriptionally-regulated by MYSM1.

Next, we further tested if the defective expression of Id2 in MYSM1-/- NK cells is indeed
one of the possible causes for the observed defective NK maturation. To do so, we
transduced WT and MYSM1-/- NKPs with a retrovirus (RV) expressing Id2 protein and
examined the NK cell generation in vitro for two weeks using similar assays as explained
above. Forced Id2 expression rescued the NK cell maturation defect in the MYSM1-/-
NKPs (Figs. 3.2D&E).

Collectively, these data demonstrate that MYSM1 is required for Id2 transcription and
that the defective Id2 transcription in MYSM1-/- NK cells is likely one possible cause for
their defective NK cell maturation (Nandakumar et al., 2013).
Figure 3. 2 MYSM1 is required for ID2 transcription
A-B. Real-time PCR analyses of a panel of NK development transcription factors in
sorted A. NKPs (CD122
+
Lin
-
NK1.1
-
DX5
-
), B. mNKs (CD122
+
Lin
-
NK1.1
+
DX5
+
) from
the femurs and tibias of bone marrow pooled from 10-15 MYSM1-/- and WT mice. Data
are mean + SEM of triplicate determinations from one of two or more independent
experiments *, P < 0.05; **, P < 0.01; ***, P < 0.001. C. ID2 mRNA levels of in vitro-
derived and post-sorted NK1.1
+
NK cells from WT or MYSM1-/- NKPs transduced with
or without LV-MYSM1 and control LV-GFP vectors. mRNA expressions were
normalized to GAPDH and values are presented as relative expression with that of the
mRNA levels of NK cells derived from LV-GFP transduced-WT NKPs (values set to 1).
46

Figure 3.2 continued
Data are mean + SEM of triplicate determinations from one of two independent
experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. D-E. Forced expression of ID2
rescued defective NK cell maturation. FACs-sorted WT and MYSM1-/- CD122
+
Lin
-
NK1.1
-
DX5
-
NKPs from the bone marrow were transduced with a recombinant retroviral
vector RV-Id2 or a control retroviral vector RV-GFP and 10,000 transduced cells were
transferred to OP9 cell co-culture and subjected to NK cell generation as explained above
for 14 days, cells were harvested and assessed for NK cell outgrowth by flow cytometric
analysis. 7AAD was used to exclude dead cells. E. Shown are representative flow
cytometric profiles of NK cells (NK1.1
+
DX5
+
CD3
-
) generated from MYSM1-/- NKPs or
MYSM1-/- NKPs transd(Nandakumar et al., 2013)uced with ID2 and numbers indicate
percentages of cells in each quadrant. F. Mean proportions of NK1.1
+
DX5
+
CD3
-
NK
cells generated in vitro per input respective NKPs as indicated. Data shown are mean +
SEM three independent experiments; *, P < 0.05; **, P < 0.01; ***, P <
0.001.(Nandakumar et al., 2013)

47

Figure 3.2 continued

3.3.2 MYSM1 associates with the Id2 locus.
To further gain insight into the molecular mechanisms controlling Id2 transcription, we
first set to examine whether there is an association of MYSM1 protein with the Id2 locus
using chIP assays(Nandakumar et al., 2013).

For all our chIP assays, we used Lin
-
CD3
-
CD122
+
NK1.1
+
NK cells (comprising of
mature and immature NK) sorted from the bone marrow and spleens cells of WT or
MYSM1-/- mice to get sufficient number of cells (Fig A.6). Since Id2’s expression is
required at the later stages of NK cell development and homeostasis (M. D. Boos et al.,
2007), Id2’s transcriptional regulation should be evident in the mature splenic and bone
marrow NK cells and consistent with this notion of ours, Ramirez et al recently
demonstrated the ETS-1-mediated Id2 regulation using sorted splenic mNK cells
(Ramirez et al., 2012). A panel of PCR primer pairs spanning −1Kb of the promoter
region of Id2 encompassing different transcription factor binding sites and +1Kb of the
Id2 locus encompassing the initiator sequence (INR) and the first coding exon of Id2 was
constructed (Fig. 3.2A)(Nandakumar et al., 2013). As a negative control, we chose an
48

intergenic region (IGR), that is not known to contain any regulatory sequences, ~ 3.8kb
3’ Id2 locus (Zhang et al., 2012). Although sorted to 98-99% purity (Fig A.6), we
validated our chromatin preparations by checking for the enrichment of known
interactions in NK cell specific-genes. To this end, we made use of two controls: RUNX3
is known to bind the proximal Nkp46 promoter in a NK cell-specific manner (C. B. Lai &
Mager, 2012). Although, NKp46 expression is reduced by ~ 1.5 folds in MYSM1-/- NK
cells, there was a substantial level of residual NKp46 present (Fig. 2.3D). We speculated
to see an intact RUNX3-binding of the proximal Nkp46 promoter since RUNX3
expression was unaffected by MYSM1 deletion (Fig B.1) and we confirmed this using
chIP assay (Fig B.3). RUNX3 can also bind to Cd122 promoter (C. B. Lai & Mager,
2012; Ohno et al., 2008), whose expression is unaltered in MYSM1-/- mice (Fig 2.3A);
we used this as another control to validate our chromatin preparation (Fig B.3). Anti-IgG
was used as a negative control for the antibody of interest used in the
immunoprecipitations, unless otherwise indicated(Nandakumar et al., 2013).

Chromatin from the sorted cells were immunoprecipitated with either anti-MYSM1 or
anti-IgG and the precipitated DNA was tested for the enrichment of Id2 gene. In
comparison to the IgG control, there was a significant enrichment of Id2 gene using
primers pairs 5 (comprising C/EBP binding site) and less significantly using primer pairs
4 (comprising E-BOX binding site) and no enrichment was observed in the intergenic
region (Fig. 3.3B), suggesting a direct association of MYSM1 with the Id2 locus in NK
cells. This further adds to our previous finding that MYSM1 regulates Id2 transcription
49

and indeed through a direct association. However, at this point we were clueless on how
MYSM1 selectively gets targeted to the Id2 regulatory elements in NK cells, whether
through its direct DNA binding activity mediated via its N-terminal SANT domain
(Misao Yoneyama et al., 2007) or with the help of other DNA-binding proteins and
regulators of Id2 gene(Nandakumar et al., 2013).
Figure 3. 3 MYSM1 associates with the Id2 locus.
A. Schematic diagram of the Id2 locus: promoter encompassing different transcription
factor binding sites (small circles), transcription initation site (INR), fist exonic region
(black) and an intergenic region (IGR) 3’ Id2 gene. The positions of the primers used for
chIP assays are indicated by numbers.B. ChIP assays of sorted WT NK cells (Lin-CD3-
CD122+NK1.1+) comprising of mature and immature NK cells from the pooled bone
marrow and spleen cells using anti-MYSM1 antibody. The precipitated DNA was
analyzed by quantitative PCR using the indicated primers along Id2 locus. The relative
amount of immunoprecipitated DNA is presented as a percentage of input DNA. The
binding of MYSM1 was determined by comparing the chIP fractions with that of control
IgG. The IGR region served as a negative control. Data are mean + SEM of triplicate
determinations from one of three independent experiments *, P < 0.05; **, P < 0.01; ***,
P < 0.001. C-F. MYSM1 interacts with NFIL3 and this interaction is critical for their
recruitment to the ID2 locus. C. Co-immunoprecipitation assays: Cell lysates from
HEK293T cells co-transfected with pCMV-Nfil3 and pCMV-FLAG-MYSM1 or pCMV-
FLAG expression vectors were precipitated with an anti-Flag antibody or anti-IgG
(negative control), and precipitated proteins were analyzed by western blot by probing
with anti-NFIL3 antibody. 10% percent of the input was loaded. D. Cell lysates of 2 X
107 WT NK1.1+CD3- cells were incubated with anti-MYSM1 antibody and anti-IgG
(negative control) antibody and immunoprecipitated proteins were analyzed by western
blot by probing with anti-NFIL3 antibody. 1% of the input was loaded. Actin is used as a
control to confirm equal starting amounts of protein for immunoprecipitation. Data are
representative of two (D) or three independent (C) experiments. E. ChIP assays of sorted
WT and MYSM1-/- NK cells (Lin-CD3-CD122+NK1.1+) using anti-NFIL3 and anti-IgG
antibodies. The precipitated DNA was analyzed by quantitative PCR using the indicated
primers along Id2 locus. The relative amount of immunoprecipitated DNA is presented as
a percentage of input DNA. The binding of NFIL3 was determined by comparing the
chIP fractions with control IgG and was further compared with the WT
immunoprecipitated chromatin fractions. The IGR region served as a negative control.
Data are mean + SEM of triplicate determinations from one of three independent
experiments *, P < 0.05; **, P < 0.01; ***, P < 0.001. F. Sequential ChIP assays of sorted
WT NK cells (Lin-CD3-CD122+NK1.1+) using anti-MYSM1, anti-NFIL3 and the
corresponding negative control antibodies (IgG). The precipitated chromatin from the
first ChIP with anti-MYSM1 was re-precipitated with antibody against 2nd protein
50

Figure 3.3 continued

NFIL3 and IgG control. The precipitated DNA was analyzed by quantitative PCR using
the indicated primers along Id2 locus. The relative amount of immunoprecipitated DNA
is presented as a percentage of input DNA. The binding of MYSM1 and NFIL3 was
determined by comparing the chIP fractions with the corresponding 1st chIP or 2nd chIP
control IgGs. The IGR region served as a negative control. Data are mean + SEM of
triplicate determinations from one of two independent experiments *, P < 0.05; **, P <
0.01; ***, P < 0.001(Nandakumar et al., 2013).

51

3.3.3 MYSM1 interacts with NFIL3 and this interaction is critical for their recruitment
to the Id2 locus

To further gain insight on how MYSM1 activates Id2 transcription, we examined whether
MYSM1 is required for the recruitment of regulators of Id2 gene transcription. We
surveyed to know if MYSM1 specifically facilitates the recruitment of any NK cell
development-related transcription factors such as NFIL3, TOX, KLF4, ETS-1, EOMES
(Luevano et al., 2012) through co-immunoprecipitation assays with over-expressed
proteins in 293-T cells. Interestingly, NFIL3 protein, essential for NK cell maturation
through its activation of Id2 transcription (Gascoyne et al., 2009) readily co-
immunoprecipitated with MYSM1 when over-expressed in 293-T cells that were
transfected with pCMV-MYSM1-FLAG but not in the cells that were transfected with the
pCMV-FLAG (Fig. 3.3C). None of the other proteins co-immunoprecipitated with
MYSM1, at least in our experimental settings. Also, a strong association of endogenous
MYSM1 and endogenous NFIL3 was detected in the NK1.1
+
CD3
-
NK cells pooled from
WT spleens and bone marrow (Fig. 3.3D) (Nandakumar et al., 2013).

Next we tested whether MYSM1’s interaction with NFIL3 is required for the recruitment
of these proteins to the Id2 locus through chIP assays of WT and MYSM1-/- Lin
-
CD3
-
CD122
+
NK1.1
+
NK cells. We found that NFIL3 indeed associated with the promoter
region of Id2 locus in WT NK cells at the C/EBP binding site (primer 5) (Fig. 3.3E). Id2
promoter encompasses two C/EBP binding sites: CbE2 (−81 to −73) and CbE3 (−73 to
−65) (Karaya et al., 2005) and our finding was consistent with the earlier reports that
suggested the consensus C/EBP binding sites (ATTGC/GCAAT) as one of the high-
52

affinity binding sites for bZIP factors such as NFIL3 (E4BP4) (Haas, Cantwell, Johnson,
& Burch, 1995; F. Li, Liu, Jo, & Curry, 2011). However, the association of transcription
factor NFIL3 with the Id2 locus was lost in the absence of MYSM1 and in the intergenic
region (Fig. 3.3E). This indicates an important role of MYSM1 in the recruitment of
NFIL3 to the Id2 locus in NK cells (Nandakumar et al., 2013).

To further confirm the co-recruitment and co-localization of MYSM1 and NFIL3 at the
Id2 locus, we performed sequential two-step chIP assays with WT Lin
-
CD3
-
CD122
+
NK1.1
+
NK cells. To do this, chromatin was first immunoprecipitated with anti-
MYSM1 antibody, followed by a second immunoprecipitation with anti-NFIL3. MYSM1
and NFIL3 were found to be co-localized at the promoter region of the Id2 locus in the
WT NK cells (Fig. 3.3F). Co-localization was enriched at the C/EBP binding site again
(primer 5); this provides explanation at least partially for how MYSM1 may get targeted
to the Id2 locus, possibly through its interaction with the DNA binding NFIL3 protein.
The association of these factors at the Id2 locus was not detectable in the intergenic
region (Fig. 3.3F)(Nandakumar et al., 2013).

MYSM1 also has its own DNA binding activity (Misao Yoneyama et al., 2007) and its
possible that its own or its concerted action with the DNA-binding NFIL3 protein may
help bring it closer to the DNA regulatory elements of Id2 locus. However, the fact that
NFIL3’s association is lost in absence of MYSM1 shows that MYSM1 and its interaction
with NFIL3 are critical for stable association of NFIL3 at the Id2 locus during NK cell
53

development. Collectively these results may imply the existence of some kind of a
concerted molecular mechanism in which NFIL3 may help in targeting MYSM1 to Id2
promoter and MYSM1 in turn enhances NFIL3 binding by inducing localized alterations
in the nucleosomic structures of the Id2 locus(Nandakumar et al., 2013).

3.3.4 Id2 locus of the MYSM1-/- NK cells is poised in its repressed state
Cell development is controlled by activation of a specific genetic program, distinctive of
a specific stage of cell-development and repression of the genetic program corresponding
to the previous cellular state. This process is regulated at the transcriptional level, to a
large extent by transcription factors that can dictate the epigenetic signature of specific
locus (Shilatifard, 2006). MYSM1 was previously reported to regulate target gene
transcription by deubiquitinating histone K119, coordinating with other histone
modifications and by subsequently recruiting transcription factors to the target locus (X.-
X. Jiang et al., 2011; Zhou et al., 2008). We examined if the chromatin context of the Id2
locus was altered in MYSM1-/- NK cells, consistent with its reduced transcript levels, by
performing chIP assays. We determined the chromatin state of the locus in
MYSM1/NFIL3 - C/EBP binding site, transcription start site, first-coding exon of the Id2
locus and in the negative control intergenic region 3’ of Id2 locus (Fig. 3.4A)
(Nandakumar et al., 2013).

As expected, the chromatin of WT NK cells was enhanced with active marks (H3K4me3,
H3K4me2) and showed a loss of ubH2A (H2AK119Ub1) and undetectable levels of
54

repressive marks (H3K27me3) at the Id2 locus (Fig. 3.4A). However, the chromatin of
MYSM1-/- NK cells showed a gain of repressive ubH2A (H2AK119Ub1) and H3K27me3
marks. Interestingly in comparison to the wild-types, MYSM1-/- NK cells displayed
similar levels of H3K4me2 modification (Fig. 3.4A), often associated with ‘poised’
chromatin similar to H3K4me3-H3K27me3 ‘bivalent’ chromatins (Bernstein et al., 2006;
K. Orford et al., 2008). No enrichment of any of the histone modifications were observed
in the negative control intergenic region (Fig. 3.4A) (Nandakumar et al., 2013).

Previous studies reported that loss of histone H2A deubiquitination in B-cell precursors
lead to an increased binding of the H2A monoubiquitinase, Bmi-1(X.-X. Jiang et al.,
2011; Oguro et al., 2010). To test if H2A mono-ubiquitinated/deubiquitinated Id2 locus
in NK cells, exhibits similar trend to that observed by others in B cell precursors, we
checked the binding of Bmi-1 and Ring1b to Id2 locus in the WT and MYSM1-deficient
NK cells. Consistent with the increased H2A marks, Bmi-1’s and Ring1b’s association
was enhanced in the MYSM1-/- NK cells compared to that of WT NK cells (Fig. 3.4B).
One of the downstream consequences of H2A monoubiquitination is RNA polymerase
(pol) II pausing (Stock et al., 2007) and hence to follow up and confirm if the Id2 locus of
MYSM1-/- NK cells is indeed only poised and not inactivated, we further checked the
binding of RNA polymerase to the Id2 promoter at its different phosphorylated states.
Enrichment of ser 5p within 8 Kb of the transcription start site but absence of ser 2p
(present throughout the coding regions of active genes) is an hallmark of ‘poised’ or
‘paused’ chromatin (Brookes & Pombo, 2009). We checked the levels of these two
55

differentially modified polymerases at the Id2 locus and found that RNA pol II ser 5p
was enriched in the promoter and coding region of WT and MYSM1-/- NK cells but RNA
pol II ser 2p was enriched only in the promoter and coding regions of WT NK cells (Fig.
3.4C). This combined with the histone modification profiles of the chromatin clearly
indicates that Id2 gene of MYSM1-deficient NK cells are poised in its repressed state
while that of the WT NK cells are enriched with active chromatin marks contributing to
its active Id2 gene transcription.

Together, our data indicates that Id2 gene is poised in its repressed state for future
activation and that MYSM1-mediated epigenetic alterations may suspend its chromatin
from a poised to that of an activated state for subsequent induction of NK cell
development (Nandakumar et al., 2013).
Figure 3. 4 Id2 locus of the MYSM1-/- NK cells is poised in its repressed state
A-C. ChIP assays of sorted WT and MYSM1-/- NK cells (Lin-CD3-CD122+NK1.1+)
using A. antibodies against histone modifications: anti-H3K27me3, anti-H3K4me3, anti-
H3K4me2, anti-H2AK119ub1 and their corresponding negative control antibodies (anti-
IgG or anti-IgM). B. antibodies against histone H2A monoubiquitinases: anti-Bmi-1,
anti-Ring1b and anti-IgG C. antibodies against RNA polymerases: anti-RNA pol II s2p
(H5; with an elongation pol complex), anti-RNA POL II s5p (H14; with a paused pol
complex) and anti-IgM. The precipitated DNA was analyzed by quantitative PCR using
the indicated primers along Id2 locus. The relative amount of immunoprecipitated DNA
is presented as a percentage of input DNA. The binding of proteins was determined by
comparing the chIP fractions with control IgG and was further compared with the WT
immunoprecipitated chromatin fractions. The IGR region served as a negative control.
Data are mean + SEM of triplicate determinations from one of three independent
experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001(Nandakumar et al., 2013).

56

Figure 3.4 continued

57

3.4 Conclusion
Epigenetics refers to irreversible changes not in the genomic sequence but outside the
genome and may impact gene expression, cellular phenotype, and function. Examples of
such modifications are acetylation, methylation or phosphorylation of the N-terminal tails
of histone proteins. Natural killer (NK) cells are part of innate immune system and they
exhibit their effector functions through a repertoire of germline–encoded receptors that
do not undergo somatic recombination (Cichocki, Miller, Anderson, & Bryceson, 2013;
Lanier, 2008). Peripheral NK cells are quite heterogeneous with regards to development
and function and a hierarchy of upstream elements control the induction of various
effector functions (Björkström et al., 2010). Despite several years of investigation of NK
cell development, the precise mechanisms underlying the development, maturation and
maintenance of functional NK cells remains only partially understood (Cichocki et al.,
2013).

This study unequivocally proves the intrinsic requirement of MYSM1 for NK-cell
maturation through epigenetic regulation of an important NK cell transcription factor,
ID2. This study is one of the first studies to explore the epigenetic regulation during NK
cell development. Mechanistic studies revealed that the expression of the transcription
factor ID2 is impaired in MYSM1-/- NK cells and NKP cells. Our data further reveals
that MYSM1 associates with the ID2 locus and that it coordinates with other histone
modifiers and transcription factors to promote the transcription of ID2 gene(Nandakumar
et al., 2013). Further mechanistic studies demonstrated that MYSM1 interacts with
58

NFIL3/E4BP4 protein, a key transcription factor of NK development and function, and
that its interaction with NFIL3 is required for ID2 transcription. Hence, this study unfolds
the regulatory events of the previously known key transcription factors of NK cell
development such as NFIL3 and ID2 through an epigenetic mechanism (Nandakumar et
al., 2013).

This study through the employment of ChIP and sequential ChIP-techniques have
assessed the histone modifications and DNA-binding proteins exclusively in the NK
cells. The invent of high-resolution, genome-wide analyses may provide a deeper
understanding of NK cell differentiation and their execution of effector function
(Cichocki et al., 2013). Understanding genomic association of proteins along with global
expression of genes through analyses such as microarrays or RNA-seq may be useful to
produce a genome-wide view of the chromatin state and the associated transcriptional
regulation within a defined population of cells (Cichocki et al., 2013). T cell and B cell
differentiation and chromatin states of genes essential for this process have been widely
studied using these technologies (Cichocki et al., 2013). The limited numbers makes the
application of high-throughput technologies to primary NK cells difficult(Cichocki et al.,
2013), but improvements in reagents and techniques may overcome these obstacles.

59

Chapter 4. The Essential Role of MYSM1 in the Maintenance, Self-
renewal and Differentiation of Hematopoietic Stem Cells

4.1 Introduction
HSC are responsible for giving rise to all lineages of blood cells. To sustain blood cell
production throughout the lifetime of an individual, a steady state condition is established
during postnatal life, in which HSC self-renewal and differentiation are carefully
regulated to maintain the HSC pool (Abramovich & Humphries, 2005; K. W. Orford &
Scadden, 2008; S. H. Orkin & L. I. Zon, 2008; Wang Tao & Nandakumar et al., 2013).
Hematopoietic development is regulated by a dynamic balance between HSC self-
renewal and differentiation to mature effector cells. HSCs self-renewal and
differentiation must be in proper balance since lesser self-renewal or more differentiation
may endanger the endurance of hematopoiesis throughout life; in contrary excessive self-
renewal and/or aberrant differentiation may lead to leukemogenesis(Tothova & Gilliland,
2007).

In 1991, Ogawa et al. reported that BM cells can be divided into c-kit
+
cells that do not
express Lin (Mac-1, Gr-1, Ter119, and B220) markers, and c-kit
-
cells that do not include
hematopoietic progenitor cells (Ogawa et al., 1991). From then on, Lin
-
Sca-1
+
c-
kit
+
(LSK ) cells has been used as the population in which HSCs are highly concentrated
(Okada et al., 1992; Osawa et al., 1996). HSC can be functionally classified as either
long-term (LT-HSC) or short-term (ST-HSC) according to their capacity to give rise to
life-long or transient hematopoiesis (J. L. Christensen & I. L. Weissman, 2001; Thal et
al., 2009). Osawa et al. showed that CD34
+
LSK cells are capable of only short-term
60

multilineage differentiation while CD34
-/Lo
LSK cells are capable of long-term
multilineage reconstitution capacity and can differentiate into CD34
+
LSK cells (Osawa
et al., 1996). LSK fraction also can be divided into two populations by expression level of
Flk-2. While LT-HSC are enriched in the Flk-2
-
LSK fraction, the Flk-2
+
LSK cells are
mainly ST-HSC (Julie L. Christensen & Irving L. Weissman, 2001).

In recent years, Kiel et al. demonstrated that murine HSCs can be distinguished by their
expression of SLAM family markers (CD150, CD244 and CD48). They reported that one
out of every 4.8 (21%) of CD150
+
CD48
-
cells from young adult murine BM gave long-
term multilineage reconstitution (Kiel et al., 2005).
Figure 4. 1 Schematic depicting the stages of cell differentiation from stem cells.
On the left, Lineage specification and commitment from stem cells and on the right, its
corresponding cell surface markers at each stage of development. Adapted from (Mandel
& Grosschedl, 2010)

61

The regulation of HSC self-renewal is not fully understood, but recent studies have
underscored the importance of cell cycle, apoptosis, and oxidative stress response in HSC
homeostasis. In the chapter 2 and 3, we show how MYSM1 is an essential and intrinsic
requirement for NK cell development. Concurrently, another group (Anastasia Nijnik et
al., 2012) reported the role of MYSM1 in BM hematopoiesis and function with a specific
defect at the early stage of stem cell differentiation. Although MYSM1 deficiency does
not affect commitment to NK cell lineage we were curious to identify the specific
developmental defect at the early stages of HSC differentiation. Hence, we sought to
identify more precisely the defective subset within the HSC compartment of MYSM1-/-
mice; since this was not detailed out by Nijnik et al. In the current chapter, we enumerate
the critical role of MYSM1 in HSC maintenance, self-renewal, and differentiation and
function elaborately (Wang Tao & Nandakumar et al., 2013).

4.2 Materials and Methods
4.2.1 Animals.
MYSM1-deficient (-/-) mice (MYSM1-KO first floxed mice) is generated through a “KO
first” strategy as described in 1.5. Briefly, they were generated by crossing MYSM1
mRNA truncation-first floxed mice (MYSM1
tm1a/tm1a
) with MMTV-cre mice in the
B6129F1 background or Tek-cre in the BL/6 background for complete deletion of the
floxed MYSM1 exon without any possible transcriptional leakage of the splice acceptor-
capture and RNA poly(A) termination strategy designed in the MYSM1-targeted vector
(Jiang XX, et al. 2011)(Wang Tao & Nandakumar et al., 2013). MMTV-Cre mice and
62

Tek-Cre have a widespread pattern of Cre expression in various cells including
hematopoietic cells, B and T cells and their progenitors (Kasper et al., 2006; Kim et al.,
2008).

4.2.2 Cell preparation
Single cell suspensions were obtained as described in 2.2.2

4.2.3 Flow cytometry and Cell sorting
Sample preparation, cytometric analysis, and sorting were performed as described in
2.2.3(Wang Tao & Nandakumar et al., 2013). The following antibodies from BD
Pharmingen (San Diego, CA), eBioscience (San Diego, CA) and BioLegend (San Diego,
CA) were used for flow cytometry: anti-mouse lineage cocktail (145-2C11, RB6-8C5,
M1/70, RA3-6B2, Ter-119), anti-Sca1 (anti-Ly6A; D7), anti-CD117 (anti-c-Kit; 2B8),
anti-CD127 (anti-IL-7Rα; A7R34), anti-CD150 (mShad150), anti-CD48 (HM48-1), anti-
CD135 (Flt3;A2F10.1), anti-CD34 (RAM34), anti-B220 (RA3- 6B2), anti-CD3e (145-
2C11), anti-CD4 (L3T4), anti-CD8a (53-6.7), anti-TER-119 (TER-119), anti-Gr1 (RB6-
8C5), anti-CD41(MWReg30), anti-CD11b (M1/70), anti-CD45.1 (A20), anti-CD45.2
(104), rat IgG2a k isotype (R35-95), rat IgG2b k isotype (A95-1), rat IgG1 k isotype (R3-
34), rat IgG1 λ isotype (A110-1), hamster IgG1 k isotype (A19-3), and mouse IgG2a k
isotype (G155-178). Data were collected on a FACSCanto II (BD Pharmingen, San
Diego, CA) and were analyzed with FlowJo software (TreeStar, Ashland, OR). For cell
progenitor population sorting, cells from BM were first depleted of mature hematopoietic
63

cells with a lineage cell depletion kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and
then isolated by FACSAria cell sorter (BD Pharmingen, San Diego, CA)(Wang Tao &
Nandakumar et al., 2013).

4.2.4 Cell-proliferation and cell cycle studies
In vivo incorporation of BrdU into LSK cells was assessed using the FITC BrdU Flow kit
(BD Pharmingen, San Diego, CA). Mice were intraperitoneally injected with 2 mg ml
-1
of
BrdU for 5 days and then sacrificed. BM cells were prepared and stained with antibodies
and analyzed by flow cytometry. In some experiments, LSK cells were first sorted and
infected with lentivirus or retrovirus constructs for indicated time. Cells were then
incubated with BrdU at a final concentration of 10 µM for 30 min before staining and
analysis. For cell cycle analysis, mice received a single intraperitoneal injection of BrdU
at the dose of 2 mg per recipient. 1 h later, mice were sacrificed and BM cells were
stained and analyzed by flow cytometry (Wang Tao & Nandakumar et al., 2013).

4.2.5 Colony assays
For mixed colony assays, 1x10
3
sorted LSK cells were plated in methylcellulose medium
(M3234; Stem Cell Technologies, Vancouver, BC, Canada) supplemented with 3 U/ml
rhEPO, 10 ng/ml IL6, 10 ng/ml rmIL3 and 50 ng/ml rmSCF (all from Peprotech, Rocky
Hill, NJ ). For the BFU-E assay, 1x10
3
sorted MEP cells were plated in methylcellulose
medium supplemented with 2 U/ml rhEPO and 50 ng/ml rmSCF. Colonies were scored
64

by light microscopy 12 d after plating according to the manufacturer’s instructions (Wang
Tao & Nandakumar et al., 2013).

4.2.6 Treatment with 5-FU
Mice were given a single intraperitoneal dose of 5-FU (75 mg per kg body weight) on
day 0. Blood was obtained from mice or mice were sacrificed on days 0, 6, and 18 after
injection, and BM and LSK compartments were counted and analyzed by flow cytometry.
The Hemavet 950 LV system (Drew Scientific, Dallas, TX) was used for differential
blood count analysis. In some experiments, 5-FU was administered intraperitoneally to
recipients (150 mg per kg body weight on a bi-weekly basis) and survival was
monitored(Wang Tao & Nandakumar et al., 2013).

4.2.7 Pyronin and Hoechst staining
The quiescence of the freshly isolated HSC was determined by staining with Hoechst
33342 (Molecular Probes, Eugene, OR) and pyronin Y (Sigma, St. Louis, MO). BM cells
were resuspended in PBS containing 2% (vol/vol) FCS and 10 µM Hoechst 33342. Cells
were then incubated for 30 min at 37° C, then washed and subsequently resuspended in
PBS supplemented with 20 mM HEPES, pH 7.4, glucose (1 mg/ml), 10% (vol/vol) FCS,
10 µM Hoechst 33342 and pyronin Y (1 µg/ml). Cells were incubated for an additional
15 min at 37° C, then were washed and stained for analysis by flow cytometry. Pyronin Y
fluorescence was detected at 575 nm in the linear range(Wang Tao & Nandakumar et al.,
2013).
65

4.2.8 Intracytoplasmic staining
For detection of γH2AX by flow cytometry, BM cells were first pre-stained with surface
markers then fixed and made permeable with a Phosflow kit (BD Pharmingen, San
Diego, CA). The cells were then stained with PE-conjugated antibodies against H2AX
phospho-Ser139 (20E3, Cell Signaling Technology, Danvers, MA) according to the
manufacturer’s instructions (BD Pharmingen, San Diego, CA)(Wang Tao & Nandakumar
et al., 2013).

4.3 Results
4.3.1 MYSM1 controls HSC differentiation during its transition to MPPs
Nijnik A et al.
32
have recently reported the role of MYSM1 in BM hematopoiesis and
function. In this study, we attempted to investigate the importance of MYSM1 in HSC
differentiation and function in a more detailed manner. To do this, we used MYSM1
knockout-first (MYSM1
−/−
) mice, as described previously (X. X. Jiang et al., 2011). As
reported before, MYSM1
−/−
mice showed a reduction in the absolute numbers of many
hematopoietic lineage cells by various degrees (X. X. Jiang et al., 2011; A. Nijnik et al.,
2012) (Fig C.1), hence, we interrogated if this reduction in cell numbers were due to
some defects in the HSC compartment of MYSM1
−/−
bone marrow (BM)(Wang Tao &
Nandakumar et al., 2013). We first checked MYSM1 expression level in BM and sorted
lineage-negative (Lin
−
: CD11b
−
Gr-1
−
B220
−
CD3ε
−
Ter119
−
), lineage-positive (Lin
+
:
CD11b
+
/Gr-1
+
/B220
+
/CD3ε
+
/Ter119
+
), Lin
−
Sca-1
+
c-Kit
+
(LSK), long-term HSC (LT-
HSC; CD150
+
CD48
−
LSK cells), short-term HSC (ST-HSC; CD150
+
CD48
+
LSK cells),
66

and multipotent progenitors (MPPs; CD150
−
CD48
+
LSK cells) from the BM of C57BL/6
wild-type mice. Real-time PCR analysis showed that MYSM1 mRNA expression was
much higher in the stem cell and progenitor compartments, especially in LT-HSC and
ST-HSC, than in BM and Lin
+
cells (Fig C.2, a), indicating the importance of MYSM1 in
HSC maintenance and function(Wang Tao & Nandakumar et al., 2013).

We then analyzed and found that MYSM1-deficient mice had a more than 4-fold
decrease in the total BM cell numbers and a 5-fold reduction in the Lin
−
BM cells (Fig
4.2a & Fig C.2, b). In MYSM1-deficient mice, a distinct reduction in the absolute
numbers of LSK cells, which include the HSC and their earliest progenitors, was
observed (Fig 4.2b)(Wang Tao & Nandakumar et al., 2013). Further analysis of the LSK
compartment indicated that MYSM1
−/−
mice had reduced absolute numbers of
CD34
−
Flt3
−
, CD34
+
Flt3
−
, and CD34
+
Flt3
+
LSK cells despite an increase in the proportion
of CD34
−
Flt3
−
and CD34
+
Flt3
−
LSK subsets. Strikingly, the more mature multipotent
progenitors, CD34
+
Flt3
+
LSK cells, were severely decreased both in proportion and
numbers and almost absent in the MYSM1
−/−
Lin
−
compartment (Figs 4.2c & 4.2d). We
observed similar trend by CD150- and CD48-based immunophenotyping of LT-HSC,
ST-HSC, and MPPs, except for an insignificant alteration in the cell numbers of LT-HSC
(Fig. 4.2e & 4.2f)(Wang Tao & Nandakumar et al., 2013).

LSK cells can be further subcategorized on the basis of their expression of CD150,
CD48, CD34, and Flt3 into the following five subsets: a most primitive HSC subset
67

(CD34
−
Flt3
−
CD150
+
CD48
−
LSK), and the increasingly differentiated MPP1
subset (CD34
+
Flt3
−
CD150
+
CD48
−
LSK), MPP2 subset (CD34
+
Flt3
−
CD150
+
CD48
+
LSK),
MPP3 subset (CD34
+
Flt3
−
CD150
−
CD48
+
LSK), and MPP4 subset
(CD34
+
Flt3
+
CD150
−
CD48
+
LSK)(Rathinam, Matesic, & Flavell, 2011; A. Wilson et al.,
2008). Our analysis showed that although there was not a significant change in the most
primitive HSC and MPP1 subsets, the absolute numbers of MPP2, MPP3, and MPP4
dramatically decreased (Figs 4.2g & 4.2h)(Wang Tao & Nandakumar et al., 2013).
Specifically, the reduction was severe in the most mature MPP4 compartment with 20
folds reduction in the percentages and 750 folds reduction in the cells numbers of
MYSM1
−/−
mice compared to its wild-type littermates (Figs 4.2g, 4.2h & C.2, e). We
further noticed that the reduction of MPP4 resulted from a distinct decrease of the
Flt3
+
LSK cells (Fig, C.2, f). Flt3’s expression enhances lymphoid repopulating potential
but not of that of the long-term repopulating potential (Adolfsson et al., 2001). Reduced
Flt3 expression in this compartment may signify an early lesion in lymphoid
development. Consistently, MYSM1
−/−
mice have been reported to show impaired
lymphopoiesis (X. X. Jiang et al., 2011; A. Nijnik et al., 2012). Together, these data adds
more to the finding of Nijnik et al by showing that MYSM1 is required for differentiation
of HSC, specifically during the transition to MPPs (Wang Tao & Nandakumar et al.,
2013).
Figure 4.2 MYSM1 deficiency results in a reduction of HSC and its progenitors
Absolute number of (a) BM cells and (b) LSK cells per femur (hind leg) of 8~12-week-
old wild-type and MYSM1
−/−
mice; n =9 mice per group. (c) Distribution of LSK cells
and various LSK subsets in the wild-type and MYSM1
−/−
BM: Lin negative gated cells
were assessed based on their expression of Sca-1 and c-Kit (top), LSK gated cells were
further defined on based on the expression of CD34 and Flt3 (bottom). Numbers adjacent
68

Figure 4.2 continued

to outlined areas indicate frequency. (d) Absolute number of LSK subsets per femur in
the wild-type and MYSM1
−/−
mice based on the gates in c; n = 8-10 mice per group. (e)
Distribution of cells in LSK subsets in the wild-type and MYSM1
−/−
mice, assessed based
on the expression of CD150 and CD48. (f) Absolute number of LSK subsets per femur in
the wild-type and MYSM1
−/−
mice based on the gates in e; n = 8-10 mice per group. (g)
Expression of Flt3 and CD34 in the wild-type and MYSM1
−/−
BM LSK subsets gated as
in e. (h) Absolute number of LSK subsets per femur in wild-type and MYSM1
−/−
mice (n
= 8-10 per group), stained as in g. *P < 0.05, **P < 0.01, ***P < 0.001 (Wang Tao &
Nandakumar et al., 2013)

4.3.2 Loss of MYSM1 drives HSCs from quiescence to rapid cycling
In steady-state, most cells in the HSC pool are quiescent, and maintenance of HSC’s
quiescence is critical for controlling the stem cell pool. When we measured total
RNA/DNA contents in the LSK cells and its subsets by Pyronin Y/Hoechst staining, we
69

found that MYSM1 deficiency significantly reduced, but not increased, the proportion of
LSK cells in the G0 phase while there was a significant increase in the proportion of LSK
cells in the G1 phase (Figs 4.2a, b & Fig C.3). To further verify this data, we measured
the rate of HSC proliferation by BrdU incorporation. Figs 4.2c, 4.2d & 4.2e show that, 5-
day post BrdU injection, more LSK cells in the MYSM1
−/−
mice incorporated BrdU, and
this was more evident in the most primitive MYSM1
−/−
CD150
+
CD48
−
LSK LT-HSC.
Consistent to these data, we also noticed an increase in the mRNA levels of cell cycle
genes, cyclin D1 and cyclin E2 and a decrease in the mRNA levels of p15 and p16 in the
MYSM1-/- LSK cells in comparison to that of wild-type cells (Figs 4.2f). Collectively,
our data indicates that MYSM1 maintains HSC in the quiescent stage, preventing them
from entering the cycling phase and that MYSM1’s deletion drives HSC from quiescence
to rapid cell cycling(Wang Tao & Nandakumar et al., 2013).
Figure 4.3 Loss of MYSM1 drives HSC from quiescence to rapid proliferation
(a-b) Quiescence of HSC was evaluated with Hoechst 33258/Pyronin Y staining in the
BM of wild-type and MYSM1-/- mice. Wild-type or MYSM1−/− BM cells were stained
for HSC surface antigens followed by Hoechst 33258/Pyronin Y staining. Representative
FACS plots (left) of cells depicting G0 (bottom left quadrant), G1 (top left quadrant), and
S/G2/M (top right quadrant) in LSK cells (a), and LSK subsets (b). Bar graph (right)
shows the percentage of cells in G0 phase for each individual subpopulation. (c). Wild-
type, MYSM1−/− mice received 2 mg BrdU intraperitoneally daily for 5 days.
Incorporation of BrdU was analysed by FACS in BM LSK and its subsets (d and e).
FACS plots of cell cycle kinetics of wild-type and MYSM1−/− LSK cells (d). Cell cycle
analysis in CD150+CD48−LSK and CD34−Flt3−LSK cells (e). Real-time PCR analysis
of cyclins, cyclin-dependent kinases (Cdks), and Cdk inhibitors in LSK cells (f). Data is
presented relative to the expression of Gapdh. Data are representative of three
independent experiments. *P < 0.05, **P < 0.01(Wang Tao & Nandakumar et al., 2013).

70

Figure 4.3 continued

4.3.3 Loss of MYSM1 results in failed recovery of HSC pool
To test whether MYSM1
−/−
HSC are likely more actively cycling and thus resulting in an
exhaustion of the quiescent stem cell reservoir, we used 5-fluorouracil (5-FU), a chemical
sensitive to cycling cells but not to the quiescent HSC. We administered a single dose of
5-FU (75 mg/kg, i.p) to 8-weeks-old wild-type and MYSM1
−/−
mice and performed serial
peripheral bleeds to monitor for leucopenia. Compared with wild-type controls, severe
leucopenia was observed in 5-FU-treated MYSM1
−/−
mice (Figure 4.4a). While the BM
71

cells and LSK cells from wild-type mice exhibited complete recovery 18 days after the
administration of 5-FU, cells of 5-FU-treated MYSM1
−/−
mice remained constantly
reduced (Figure 4.4b). Examination by FACS analysis revealed that in response to 5-FU,
LSK cells derived from wild-type mice exhibited hyper proliferation on day 6, reverted
back to normal and showed complete recovery on day 18, in contrast, cells derived from
MYSM1
−/−
mice were constantly proliferating and exhausting the stem cell reserves
leading to a steady reduction in the LSK and more mature Lin
−
Sca-1
−
c-kit
+
cell counts
(Figure 4.4c). We repeated the same experiment with 5-FU injections (150 mg/kg, i.p)
administered biweekly to 8-weeks-old wild-type and MYSM1
−/−
mice. Interestingly, 100
% of MYSM1
−/−
mice died before the second 5-FU treatment, in contrast all wild-type
mice survived after 6 cycles of 5-FU injections (Figure 4.4d). Similarly, when mice were
exposed to a lethal dose of 8 Gy irradiation, all of the MYSM1
−/−
mice died within 13
days, in contrast to the wild-type mice that survived much longer (Figure 4.4e). Taken
together, these results showed that in the absence of MYSM1, HSC proliferate rapidly
and exhaust the stem cell reserves leading to a defective stem cell renewal capacity, BM
exhaustion, and early death (Wang Tao & Nandakumar et al., 2013).
Figure 4.4 Exhaustion of Hematopoietic stem cell in the absence of MYSM1.
(a-c) 8 week-old wild-type and MYSM1
−/−
mice were injected with a single dose of 5-FU
(75 mg/kg, i.p.). At each indicated time, peripheral blood and BM were obtained to
monitor hematopoiesis recovery. The counts of WBC are shown as percentages of initial
baseline values for each group of mice; n = 5 mice group (a). Absolute number of BM
(left) and LSK cells (right) of wild-type and MYSM1
−/−
mice (b). Distribution of LSK in
wild-type and MYSM1
−/−
BM(c). (d) Survival outcome of wild-type and MYSM1
−/−
mice
after sequential 5-FU treatment (150 mg/kg, i.p.) (log-rank nonparametric test) and
presented as a Kaplan-Meier survival curve; n = 10 mice per group. (e) Survival outcome
of wild-type and MYSM1
−/−
mice after a lethal dose of 8 Gy irradiation; n = 5 mice per
group. Data are representative of two independent experiments. **P < 0.01, ***P
<0.001(Wang Tao & Nandakumar et al., 2013).
72

Figure 4.4 continued

4.3.4 MYSM1 deficiency causes increased apoptosis in HSCs
We then re-examined if reduction in the HSC pool size of MYSM1
−/−
mice is also
contributed by their increased apoptotic rate, as reported by Nijnik at al (A. Nijnik et al.,
2012). Indeed, we observed an average of 22.57% apoptosis in the MYSM1
−/−
LSK cells,
which was more than 6 folds higher than that of the wild-type mice (3.35%) (Figs 4.5a, b
& c). Strikingly, in the more mature CD34
+
Flt3
+
LSK progenitors, we observed more than
80% cell death, which was consistent with the sharp reduction in both the frequencies and
cell numbers of these cells in the MYSM1
−/−
mice (Fig 4.2c & Fig C.2, c). In addition
73

our data displayed clear signs of increased apoptosis, DNA damage and oxidative stress
in the MYSM1-/- LSK and Lin
-
cells such as: obvious reduction in the levels of anti-
apoptotic proteins, clear increase in the levels of pro-apoptotic proteins, increase in the
activity of caspase 3/7, reduction in the mitochondrial potential, increase in the rate of
apoptosis post-irradiation, up-regulation in the levels of ROS and increase in the levels
rH2AX marks (Figs 4.5d, e, f, g & h). Taken together, our data suggest that besides
controlling the quiescence and driving them into cycling, MYSM1 also balances the rate
of apoptosis of HSC and other progenitors for maintenance of the HSC pool size (Wang
Tao & Nandakumar et al., 2013).
Figure 4.5 MYSM1 deficiency leads to increased HSC apoptosis.
(a-c) Quantitative analysis of apoptotic cells among LSK (a) and LSK subsets (b and c)
cells from WT and MYSM1-/- mice. Apoptotic cells were detected by staining with
annexin V and 7AAD (a,b) or annexin V and DAPI (c). FACS plots (left) show
representative results of annexin V staining. Bar graphs (right) show the frequencies of
apoptosis of each individual subpopulation. (d) Apoptosis-related gene expression in WT
and MYSM1-/- Lin
-
cells. Actin serves as a loading control throughout. (e) Caspase-3 and
-7 activities. Freshly isolated LSK cells from WT and MYSM1-/- mice were plated in 96-
well plates and treated with Caspase-Glo
®
3/7 Reagent (Promega). Luminometer readings
were taken 1 hour later. (f) Mitochondrial membrane potential. BM cells were stained
with surface marker and DiIC1 (5) and mitochondrial membrane potential was monitored
by FACS. Red = cells from WT mice; black = cells from MYSM1-/- mice.(g) Percentages
of Annexin V positive LSK cells 4 hours after irradiation. Data is normalized to untreated
samples to take only the effects induced by irradiation into account. (h) Reactive oxygen
species (ROS) levels of LSK and LSK subsets cells were measured using DCFDA ROS-
sensitive fluorescent dye. (i) Levels of DNA damage γH2AX marker in WT and MYSM1
-/- LSK cells. MFI: median fluorescence intensity. Data are means ± SD. *P < 0.05, **P
< 0.01, ***P < 0.001(Wang Tao & Nandakumar et al., 2013).

74

Figure 4.5 continued

4.3.5 MYSM1-Deficient HSCs display intrinsic functional defects and impaired
Engraftment

To test whether besides a severe quantitative reduction in HSC, if these cells were also
affected in their functions, we sorted LSK cells and assessed the colony-forming units on
an in vitro differentiating medium. MYSM1
−/−
LSK cells overall generated very few
colonies (Fig 4.6a) in comparison to that of the wild-type cells and preferentially
75

generated colonies of granulocytes and macrophages and lost their ability to generate
erythroid colonies (Figs 4.6b, c)(Wang Tao & Nandakumar et al., 2013). Consistent with
this, we observed higher frequencies of granulocytes and macrophages in the MYSM1-
deficient mice (Fig. 4.6d). We next used competitive transplantation assays to compare
the in vivo functions of MYSM1
−/−
LSK cells. Each groups of lethally irradiated mice
(CD45.1) received 1×10
3
sorted LSK cells from wild-type or MYSM1
−/−
mice (CD45.2)
along with 2 ×10
5
competitor BM cells (CD45.1). The chimerism was followed in
peripheral blood at 4, 8, 12, and 16 weeks post-transplantation. Loss of MYSM1 did not
affect the homing of HSC after BM transplantation (Fig 4.6e), but markedly affected their
hematopoietic-reconstitution ability not only in peripheral blood and BM, but also in
thymus, spleen and other peripheral tissues (Figs 4.6 f, g, h & i and Fig C.3)(Wang Tao &
Nandakumar et al., 2013). These data demonstrate that normal HSC activity is critically
dependent on MYSM1, and MYSM1-deficient HSC display intrinsic functional defects
such as defective differentiation potential and impaired engraftment.
Figure 4.6 Cell-intrinsic defects of MYSM1-deficient HSC.
(a) Mixed colony assays with 1X10
3
WT and MYSM1-/- LSK cells. Colonies were scored
by light microscopy 12 d after plating (b) Frequencies of granulocyte, macrophage,
granulocyte and macrophage, burst-forming unit-erythroid (BFU-E) and granulocyte,
erythroid, macrophage in CFU assay as described in (a). (c) BFU-E assay with 1×10
4

sorted MEP cells from WT or MYSM1-/- mice. BFU-E colonies were scored by light
microscopy 12d after plating. (d) BM cells from WT and MYSM1-/- mice were stained
with different cell surface markers and analyzed with FACS. (e) BM homing assay.
2×10
7
BM cells from WT or MYSM1-/- mice (CD45.2) were transplanted into lethally
irradiated recipients (CD45.1). 16 hours later, mice were sacrificed and femurs and tibias
were flushed, Lineage-positive cells were depleted using MACS microbeads, Lineage-
negative cells were stained for LSK and analyzed by FACS (f-i) Donor chimerism in
competitive repopulation assay. 1×10
3
LSK cells sorted from wild-type or MYSM1-/-
mice (CD45.2) were transplanted into lethally irradiated recipients (CD45.1) together
with 2×10
5
competitor BM cells (CD45.1). (f) Percentages of donor-derived cells were

76

Figure 4.6 continued

followed in PB at 4, 8, 12, and 16 weeks after transplantation. Each dot indicates an
individual recipient mouse (n=5). (g) Percentages of donor-derived myeloid, B, and T
cells in PB 16 wks after transplantation. (h) Percentages of donor-derived LSK cells in
BM 16 wks after transplantation. (i) Percentages of donor-derived cells (CD45.2) in BM,
Spleen, thymus, and PB 4-week after transplantation. Data are representative of three
independent experiments. Shown are means ± SD. *P < 0.05, ***P < 0.001(Wang Tao &
Nandakumar et al., 2013).

4.4 Conclusions
The blood cells are constantly produced in the bone marrow through hematopoiesis and
its disruption results in severe life threatening disorders, including immunodeficiencies,
anemias, bone marrow failures, and malignancies (A. Nijnik et al., 2012). Even though
77

few proteins with H2A-DUB activity including ubiquitin-specific proteases (Usp3, 12,
16, 21, 22, and 46)(Joo et al., 2011; Vissers, Nicassio, van Lohuizen, Di Fiore, &
Citterio, 2008) and the Myb-like SWIRM and MPN domains containing protein 1 known
as MYSM1 (Zhu et al., 2007) have been identified the physiologic functions of histone
H2A deubiquitinating enzymes (H2A-DUBs) in vivo in a mammalian organism have not
been previously investigated(A. Nijnik et al., 2012). However H2A-DUBs role in the
transcription regulation of Hox gene expression in Xenopus, Drosophila development and
in responses to injury of the hepatocytes (Nakagawa et al., 2008) is known (A. Nijnik et
al., 2012).

We and others have identified MYSM1 as a novel regulator of bone marrow stem cell
function and lymphocyte differentiation (X. X. Jiang et al., 2011; A. Nijnik et al., 2012).
Nijnik A et al. have reported the role of MYSM1 in BM hematopoiesis and function;
however, the detailed role of MYSM1 exclusively in HSC’s biology has not been
investigated yet. In this study, we first identified the defective HSC differentiation into
MPPs in the MYSM1-/- mice. Although this is already shown by Nijnik et al., detailed
characterization with phenotypic markers and specific identification of the defective
MMP4 subsets in the MYSM1-/- mice was not detailed out in their analysis. Consistent
with their finding, HSC deficiency in MYSM1-/- mice was contributed by an increase in
apoptosis and MYSM1 regulated this HSC function in a cell intrinsic manner(Wang Tao
& Nandakumar et al., 2013). Additionally, we elaborate on this by showing that HSC
deficiency in MYSM1-/- mice apart from increase in apoptosis is also contributed by
78

inability of the MYSM1-deficient HSCs to remain quiescent. MYSM1-/- HSCs are
actively cycling causing an exhaustion of stem cell reserves in the MYSM1-/- mice. In
conclusion, our study elaborates on the initial reports of Nijnik A et al (A. Nijnik et al.,
2012) by enumerating the critical role of MYSM1 in HSC maintenance, survival and
homeostasis(Wang Tao & Nandakumar et al., 2013).

79

Chapter 5. The control of HSC homeostasis by MYSM1-mediated
Epigenetic regulation

5.1 Introduction
HSC are responsible for giving rise to all lineages of blood cells. To sustain blood cell
production throughout the lifetime of an individual, a steady state condition is established
during postnatal life, in which HSC self-renewal and differentiation are carefully
regulated to maintain the HSC pool(K. W. Orford & Scadden, 2008; S. H. Orkin & L. I.
Zon, 2008; Wang Tao & Nandakumar et al., 2013). Numerous genes and signaling
pathways, including HoxB4, Notch1, Gfi1, Bmi-1, and the Wnt signaling pathway, have
been implicated in this process(Antonchuk, Sauvageau, & Humphries, 2002; Brun et al.,
2004; Hock, Hamblen, et al., 2004; Iwama et al., 2004; J. Lessard & Sauvageau, 2003;
Park et al., 2003; van Es & Clevers, 2005; Wang Tao & Nandakumar et al., 2013; Zeng,
Yucel, Kosan, Klein-Hitpass, & Moroy, 2004). Recently, accumulating evidence indicate
that molecules involved in epigenetic and chromatin modifications are also critical for
HSC self-renewal and differentiation(Broske et al., 2009; Rebel et al., 2002; Trowbridge,
Snow, Kim, & Orkin, 2009; Wang Tao & Nandakumar et al., 2013; Yoshida et al., 2008).

Recent studies have shown that Polycomb group (PcG) proteins such as Bmi1, Mel18 and
Rae28 and their interaction are critical for HSC self-renewal and lineage restriction (J. A.
Lessard & Crabtree, 2010). Bmi1 plays an important role in regulating the self-renewal
and proliferative activity of stem and progenitor cells, specifically neural stem cells (Park
et al., 2003). Conditional inactivation of Tel (Translocation Ets leukemia; also known as
80

Etv6 [Ets variant gene 6]), an Ets (E-26 transforming-specific)-related transcriptional
repressor in HSCs rapidly leads to the depletion of Tel/Etv6-deficient bone marrow
(Hock, Meade, et al., 2004). Pbx1 is preferentially expressed in LT-HSCs compared to
more mature short-term HSCs and multipotent progenitor cells and conditional knockout
of Pbx1 results in impaired HSC quiescence (Ficara, Murphy, Lin, & Cleary, 2008).
HoxB4 is another essential factor for HSCs self-renewal both in vivo and in vitro
(Sauvageau et al., 1994). Two groups working independently determined that Gfi1, a
zinc-finger repressor, controls self-renewal of HSCs by restraining their proliferative
potential (Ficara et al., 2008; Hock, Hamblen, et al., 2004; Zeng et al., 2004). P53 have
been identified to have important roles in the proliferation, differentiation, apoptosis, and
aging of hematopoietic cells (Dumble et al., 2007). HSC are protected from oxidative
stress through upregulation of genes involved in their detoxification by the members of
FoxO subfamily of forkhead transcription factors (Tothova & Gilliland, 2007).

Despite increasing numbers of key players in regulating HSC fate decision have been
identified, their roles in HSC biology and their interplay remain poorly understood. We
and others have found that MYSM1 is required for BM hematopoiesis and lymphocyte
differentiation, especially in B cell development (X. X. Jiang et al., 2011; A. Nijnik et al.,
2012; Wang Tao & Nandakumar et al., 2013).
Figure 5. 1 Requirements of Transcription Factors in Hematopoiesis.
The stages at which hematopoietic development is blocked in the absence of a given
transcription factor, as determined through conventional gene knockouts, are indicated by
red bars. The factors depicted in black have been associated with oncogenesis. Those
factors in light font have not yet been found translocated or mutated in human/mouse
hematologic malignancies. Adapted from (Stuart H. Orkin & Leonard I. Zon, 2008)
81

Figure 5.1 continued

Nijnik A et al. have reported the role of MYSM1 in BM hematopoiesis and function.
However, the detailed role of MYSM1 exclusively in HSC’s biology and the mechanisms
associated with it has not been investigated yet. In our present study, we enumerate the
critical role of MYSM1 in HSC maintenance, self-renewal, differentiation and function in
a more detailed manner and provide a possible mechanism by which MYSM1carries out
this function (Wang Tao & Nandakumar et al., 2013).
82

5.2 Materials and Methods
5.2.1 Animals.
MYSM1-deficient (-/-) mice (MYSM1-KO first floxed mice) is generated through a “KO
first” strategy as described in 1.5.

5.2.2 Cell preparation
Single cell suspensions were obtained as described in 2.2.2

5.2.3 Flow cytometry and Cell sorting
These methods are described in 2.2.3

5.2.4 Immunoblot analysis and immunoprecipitation
Immunoblot analyses were performed as described in 3.2.5(Wang Tao & Nandakumar et
al., 2013). Primary antibodies were as follows: anti-Bcl-2 (610538), anti-Bcl-xl (556361),
anti-Bax (554106), and anti-Bak (556396, all from BD Pharmingen, San Diego, CA);
anti-Xiap (2042), anti-c-Myc (2278, all from Cell Signaling Technology, Danvers, MA);
and anti-flag (F1804, Sigma, St. Louis, MO). For coIP assay, proteins were
immunoprecipitated with antibody to flag tag with a commercially available kit according
to the manufacturer’s instructions (Sigma, St. Louis, MO).

83

5.2.5 RNA extraction and real-time PCR
Total RNA was isolated with an RNeasy Mini kit (Qiagen, Alameda, CA), then cDNA
was synthesized with iScript™ Advanced cDNA Synthesis Kit (Bio-Rad, Hercules, CA).
PCR was performed in duplicate with a CFX96 PCR system and iTaq™ Universal
SYBR® Green Supermix according to the manufacturer’s instructions (Bio-Rad,
Hercules, CA)(Wang Tao & Nandakumar et al., 2013).

5.2.6 Chromatin Immunoprecipitation
Chromatin was immunoprecipitated according to the manufacturer’s instructions (Cell
Signaling) (Sharabi et al., 2008)(Wang Tao & Nandakumar et al., 2013). In brief, cells
were crosslinked with 1% (vol/vol) formaldehyde. Chromatin was isolated, digested by
micrococcal nuclease (MNase), sheared by sonication, and immunoprecipitated with
antibodies. Immunoprecipitated DNA was washed and eluted according to the
manufacturer’s instructions. Eluted DNA and sheared input material was analyzed by
PCR or ligated to an adaptor and amplified by PCR according to the manufacturer’s
protocol (Illumina, San Diego, CA). The following antibodies were used: anti-MYSM1
(custom-made), anti-Gata2 (sc-9008x) and anti-Scl (sc-12984x) are both from Santa Cruz
Technology (Dallas, TX), anti-PU.1 (2258) is from Cell Signaling Technology (Danvers,
MA), anti-Runx1 (ab23980), anti-H3K4me (ab8895), anti-H3K4me3 (ab1012), anti-
H3K27me3 (ab6002), anti-H3K27Ac (ab4329) and anti-H3K9Ac (ab4441) are from
Abcam (Cambridge Science Park, Cambridge, UK).

84

5.2.7 Luciferase reporter assays.
The 1.4 kb Gfi1 promoter sequence (starting from -3.4 kb) and the 0.5 kb Gfi1 core
promoter sequence (starting form -2.3kb) were cloned into pGL3-basic plasmid(Wang
Tao & Nandakumar et al., 2013). The 0.5 kb Gfi1 enhancer sequence (starting from -
35kb) was cloned into pGL3-control plasmid. Luciferase reporter assays were performed
as described previously (Sharabi et al., 2008). 293T cells (2x106 cells/well) were seeded
in 12-well plates and cultured for 24 h in DMEM supplemented with 10% fetal bovine
serum (FBS) and 2 mM L-glutamine. Cells were washed with PBS before transfection.
For promoter activity assay, cells were cotransfected with pGL3b, pGL3b-(-3.4kb) or
pGL3b-(-3.4kb min pro) and MYSM1 expression vector or control vector with
lipofectamin2000 (Invitrogen). For enhancer activity assay, cells were cotransfected with
pGL3c or pGL3c-(-35kb), in/without the presence of siControl, siMYSM1 and/or Gata2
and Runx1 expression vector. Cells were cultured for 48 h and luciferase assays were
performed by using Dual-Luciferase Reporter Assay System (Promega, Madison,
Wisconsin) following the manufacturer’s instruction. Measurements of firefly and renilla
luciferase activities were used to determine the standard deviation of the firefly to renilla
luciferase ratio.

5.2.8 PCR array
Freshly isolated cells from bone marrow were stained with LSK markers and sorted by
FACSAria cell sorter (Wang Tao & Nandakumar et al., 2013). Then, total RNA was
isolated using an RNeasy Mini kit (Qiagen, Alameda, CA). cDNA was synthesized and
85

real-time PCR was performed by using Mouse Hematopoiesis RT² Profiler™ PCR Array
kit (PAMM-054Z; SABioscience, Frederick, MD) following the manufacturer’s
instruction. Data were analysed with PCRArray Data Analysis Template Excel
downloaded from webpage http://www.sabiosciences.com/pcrarraydataanalysis.php

5.3 Results
5.3.1 Gfi1 gene expression is regulated by MYSM1 in HSC
To gain an understanding of how MYSM1 functions in HSC, we performed a PCR array
analysis of 84 genes related to the development of blood-cell lineages from HSCs in
sorted wild-type and MYSM1-/- LSK cells. There were relatively limited differences in
the gene expression profile between wild-type and MYSM1-/- LSK cells (10/84, Fig 5.2)
(Wang Tao & Nandakumar et al., 2013). However, we noticed some differential gene
expression in some cytokines (Pf4, Csf2), transcription factors (Cbfb, Cebpg), matrix
metalloproteinase 9, Notch signaling molecule (Notch4), Wnt signaling molecule (Apc),
and surface antigen Cd14 in the MYSM1-/- LSK cells compared to that of wild-type cells
(Tables D.1 & D.2). Due to limited set of genes profiled on our PCR array, we checked
the expression of additional genes associated with HSC maintenance and differentiation
by qPCR. Our analysis on selected genes confirmed the increase in mRNA levels of
components of Notch-signaling pathway and identified more than 60% decrease in the
mRNA levels of Gfi1 in the MYSM1-/- LSK cells (Figure 5.2b)(Wang Tao &
Nandakumar et al., 2013). Notch is a crucial signaling pathway involved in stem-cell
maintenance and survival(Bigas, D’Altri, & Espinosa, 2012). Gfi1 is a zinc finger
86

transcriptional repressor originally recognized for its role in T cell differentiation
and lymphomas (Duan & Horwitz, 2003). Later studies have shown that Gfi1 is also an
intrinsic regulator of HSC function (Duan & Horwitz, 2005; Hock, Hamblen, et al.,
2004). Loss of Gfi1 causes defective ST-HSC and MPP development, an increase in
cycling cells within the HSC pool, and an increase in the apoptotic rate of HSC(Hock,
Hamblen, et al., 2004; Khandanpour et al., 2011; Zeng et al., 2004). Notably, Gfi1
−/−

HSC lose the ability to maintain long-term hematopoiesis due to an increase in
proliferation and eventual exhaustion of the stem cell pool (Hock, Hamblen, et al., 2004;
Zeng et al., 2004). Our analysis here using MYSM1-deficient HSC has identified many
phenotypes similar to that of Gfi1-deficient HSC. In addition, when MYSM1 was over
expressed in Lin
−
cells, an increase in the mRNA levels of Gfi1 was detected (Figure
5.2c). On the basis of these observations coupled with a marked decrease in the mRNA
levels of Gfi1 in the MYSM1-/- mice, we hypothesized that MYSM1 may possibly
regulate Gfi1 and that its deficiency may be one of many reasons for the defective HSC
homeostasis in the MYSM1-/- mice (Wang Tao & Nandakumar et al., 2013).
Figure 5.2 Differential Gene expression change between wild-type and MYSM1-/-
LSK cells.
(a) Mouse RT2 ProfilerTM PCR array was performed with sorted LSK cells from wild-
type and MYSM1-/- mice. The Bblack line indicates no change in gene-expressionfold
changes ((2 ^ (-DCt)) of 1. The Ppink lines indicate the fold-change (2 ^ (-DCt)) of
greater than or equal to 2 in gene expression threshold. (b) Real- time PCR analysis of
Notch signaling molecules and important HSC transcriptional factors in wild-type and
MYSM1-/- LSK cells. Data presented relative to the expression of Gapdh and are from
three independent expriments. (c) Sorted LSK cells from wild-type and MYSM1−/−
mice were infected with lentivirus encoding GFP (pCDH-GFP) or MYSM1 (pCDH-
MYSM1). Real- time PCR analysis of Gfi1 expression was assessed 72 hrs later by real-

87

Figure 5.2 continued

time PCR performed 72 h later. The Ct values are normalized to Gapdh and are
presented relative to wild-type (value set as 1). Data are mean+SD of triplicate
determination and are representative of three independent expriments (b-c), Data
presented relative to the expression of Gapdh and are from two independent expriments.
*P < 0.05(Wang Tao & Nandakumar et al., 2013).

5.3.2 Gfi1 is a direct transcriptional target of MYSM1and is regulated by MYSM1 and
its coordinated action with Gata2 and Runx1.

To investigate whether MYSM1 regulates Gfi1 expression by binding to the regulatory
elements of Gfi1 in HSC, we performed ChIP assays along the Gfi1 regulatory elements.
A panel of PCR primers to encompass the -3.4kb promoter and -35kb enhancer of the
Gfi1 locus was designed (Fig 5.3a)(Wang Tao & Nandakumar et al., 2013).
Immunoprecipitation with the MYSM1-specific antibody, but not with negative-control
88

IgG, enriched the sequences located at the Gfi1 promoter and enhancer regions in wild-
type Lin
−
progenitors (Fig 5.3b)(Wang Tao & Nandakumar et al., 2013), demonstrating
the direct association of MYSM1 with the Gfi1 regulatory elements.

Previous studies have characterized the enhancer 35 kb upstream of Gfi1, for its activity
in the early hematopoietic cells (N. K. Wilson et al., 2010). This enhancer is bound and
controlled by key HSC regulators, including Scl/Tal1, PU.1/Sfpi1, Runx1, and Gata2 (N.
K. Wilson et al., 2010). We tested whether MYSM1 interacts with these transcriptional
factors to regulate Gfi1 enhancer and its expression. We performed coIP assays in 293T
cells and we were surprised to detect direct interactions between MYSM1 and all of these
transcriptional factors in 293T cells (Figure 5.3c)(Wang Tao & Nandakumar et al., 2013).
However, when we further examined the association of these transcriptional factors along
the Gfi1 regulatory elements in wild-type and MYSM1
−/−
Lin
−
cells by ChIP assays,
significant reductions only in the binding of Gata2 and Runx1 and not of that of Scl and
PU.1 to -35kb enhancer of Gfi1 locus was observed in MYSM1
−/−
cells. No differences
were detected in transcriptional factor-binding in the +23kb control region of Runx1
locus (Fig 5.3d). Because no obvious changes in the mRNA expression of Gata2 and
Runx1 were found in MYSM1
−/−
cells (Fig 5.2c), this reduction of transcription factor
binding indicates reduced recruitment of these transcriptional factors to the Gfi1 enhancer
in the MYSM1
−/−
Lin
−
cells (Wang Tao & Nandakumar et al., 2013).

89

To further confirm the direct binding of MYSM1 to Gfi1 regulatory elements and its
control of Gfi1 expression, we performed reporter assay in 293T cells. MYSM1 alone did
not enhance Gfi1 expression through its promoter region (Fig 5.3e). However, Gata2 and
Runx1 transactivated Gfi1 expression via its -35kb enhancer and MYSM1 knockdown
significantly reduced this transactivation (Fig 5.3f). This data coupled with the interaction
of Gata2 and Runx1 with MYSM1 may indicate that MYSM1 regulates Gfi1 expression
via interaction and coordination with Gata2 and Runx1. However, the fact that Gata2 and
Runx1 association with Gfi1 regulatory elements were reduced in the absence of
MYSM1 indicates the existence of a concerted molecular-mechanism between MYSM1
and these transcriptional factors (Wang Tao & Nandakumar et al., 2013). Although
MYSM1 requires the interaction with Gata2 and Runx1 for transactivation of Gfi1, Gata-
2 and Runx-1 in turn requires MYSM1 for its stable association with the Gfi1 regulatory
elements, likely due to localized nucleosomic alterations created through its histone-
deubiquitinase actity on the Gfi1 locus (Wang Tao & Nandakumar et al., 2013)
Figure 5.3 Gfi1 is a target of MYSM1
(a) Schematic diagram of Gfi1 locus encompassing the promoter and enhancer region ;
arrows indicate positions of the primers used for ChIP assays. (b) ChIP assays of wild-
type Lin
-
cells using anti-MYSM1 and anti-IgG. The precipitated DNA was analyzed by
real-time PCR using primers along the Gfi1 promoter and enhancer sequence..The
relative amount of immunoprecipitated DNA is presented as a percentage of input DNA
(c) In vitro Co-IP experiment in 293T cells. A flag-tagged MYSM1-encoding plasmid
was co-transfected with Myc tagged Gata2, Runx1, PU.1 or Scl in 293T cells. Cell
lysates were immunoprecipitated using anti-Flag antibody. The immunoprecipitates were
examined by Western blotting using anti-Myc and anti-Flag antibody. 10% of cell lysates
was used as input. Data are representative of two independent experiments. (d) ChIP
assays in wild-type and MYSM1-/- Lin
-
cells using antibodies against Gata2, Runx1,
PU.1, Scl, and IgG antibodies. The precipitated DNA was analyzed by real-time PCR
using primers along the -35kb Gfi1 enhancer region. Primers along the +23kb Runx1

90

Figure 5.3 continued
regulatory sequence was used as a positive control. The relative amount of
immunoprecipitated DNA is presented as a percentage of input DNA .Data are
representative of three independent experiments. (e) The -3.4kb Gfi1 promoter and its
core region (-3.4kb min pro) were cloned into pGL3 basic luciferase vector. Promoter
activity was examined in the presence of pcDNA or pcDNA-MYSM1 48 hrs after
transfection in 293T cells. (f) pGL3-enhancer control (pGL3c) luciferase vector or
pGL3c-(-35kb) luciferase vector which contains the -35kb Gfi1 enhancer was co-
transfected with siRNA control (siControl), siMYSM1 or vectors encoding Gata2 and
Runx1 into 293T cells. Luciferase activitity was recorded 48 h after transfection. Data is
normalized to Luciferase and are representative of two independent experiments. *P <
0.05(Wang Tao & Nandakumar et al., 2013).

Fig. 5.3.3 Gfi1 partially restores the function of MYSM1-/- HSC
If this hypothesis was true, forced expression of Gfi1 should restore the impaired function
of HSC. To test this, we transplanted LSKFlt3
-
(CD45.2) cells from WT and MYSM1-/-
91

mice and LSKFlt3
-
cells (CD45.2) transduced with Gfi1 (pMIG–Gfi1) from MYSM1-/-

mice along with WT CD45.1 BM cells into lethally irradiated CD45.1 recipient mice.
Donor chimerism was followed 6 weeks post-transplantation. MYSM1-/- donor cells
were not detectable after transplantation, whereas the donor MYSM1-/- cells expressing
Gfi1 was present indicating a rescue of engraftment and self-renewal of MYSM1-/-

HSC
on forced expression of Gfi1 (Figs 5.4a & b)(Wang Tao & Nandakumar et al., 2013). Not
only that, these cells developed and expressed Flt3 in the recipient mice, indicating a
rescue of Flt3 levels on forced expression of Gfi1 in contrast to MYSM1-/-

cells with
defective Gfi1 expression (Fig 5.4c & d). Furthermore, while MYSM1-/-

LSK cells
could not reconstitute into different lineages in the recipient mice after transplantation,
MYSM1-/- cells expressing Gfi1 were able to do so, indicating that forced Gfi1
expression in the MYSM1-/- cells at least partially restores the function of MYSM1-/-
HSC (Fig 5.4e & f), very likely by protecting against apoptosis and stem cell exhaustion
caused by excessive proliferation. We tested this in vitro and consistent to our
speculation, the proportion of cells in the G0 phase increased and the apoptotic rate
decreased when MYSM1
−/−
LSK cells were transduced with pMIG-Gfi1-GFP, but not
with control retroviruses (Fig 5.4g & h). A lower proliferation rate and a reduction in the
proportion of cells in the S phase were also detected in the Gfi1-transduced MYSM1
−/−

LSK cells compared with GFP-transduced control cells (Fig 5.4i & j). Collectively, our
data demonstrate that MYSM1-mediated Gfi1 transcription is a cell-intrinsic requirement
for HSC survival, proliferation and differentiation (Wang Tao & Nandakumar et al.,
2013).
92

Figure 5.4. Forced expression of Gfi1 partially restores the function of MYSM1-
deficient HSC.
(a-f) 1×10
3
LSKFlt3
-
cells sorted from wild-type or MYSM1
−/−
mice (CD45.2) and
MYSM1
−/−
LSKFlt3
-
cells transduced with pMIG-Gfi1 were transplanted into lethally
irradiated recipients (CD45.1) together with 2×10
5
competitor

BM cells (CD45.1). (a)
Flow cytometric analyses of WT, MYSM1-/- CD45.2 cells and donor-derived chimerism
(CD45.2) 6 weeks post-transplantation in the recipient mice transplanted with WT,
MYSM1-/-cells and MYSM1-/-cells expressed with Gfi1 (pMIG –Gfi1 MYSM1-/-). (b)
Percentages of donor-derived cells (CD45.2) in BM. Each dot indicates an individual
recipient mouse. (c-d) Flow cytometric analyses and percentages of donor-derived
LSKFlt3
+
cells in the recipient mice 6 weeks after transplantation. Each dot indicates an
individual recipient mouse. (e-f) Flow cytometric analyses and percentages of donor-
derived CD45.2 or CD45.1 cells gated on B220
+
B-cells, CD3
+
T-cells and CD11b
+
Gr1
+

myeloid cells in the recipient mice 6 weeks after transplantation. Each dot indicates an
individual recipient mouse. (a-f) Data are representative of two independent experiments
with n=5 mice per group in the 1
st
experiment and n=3 mice per group in the 2
nd

experiment. Shown are means ± SD of 1
st
experiment with n=5 mice per group, ***P <
0.001. (g-j) Sorted MYSM1
−/−
LSK cells were infected with retroviral vectors encoding
either Gfi1 and GFP (pCDH-Gfi1-GFP) or GFP alone (pCDH-GFP) in vitro. Hoechst
33258/Pyronin Y staining (g), annexin V staining (h), BrdU incorporation assay (i) or cell
cycle analysis (j) were performed 48 h after infection to monitor G0 phase, apoptosis,
proliferation, and cell cycle profile respectively. (g-j) Data are representative of two
independent experiments, *P < 0.05(Wang Tao & Nandakumar et al., 2013).

93

Figure 5.4 continued

94

5.3.4 MYSM1 Orchestrates Histone Modifications at the Gfi1 Locus
To investigate how MYSM1 activates Gfi1 transcription, we first examined the levels of
ubH2A at the Gfi1 enhancer. Our data showed that ubH2A levels, as well as the
recruitment of histone-ubiquitinases Ring1b and Bmi1 was increased in the Gfi1 -3.4kb
region of MYSM1
−/−
Lin
−
progenitors, compared to that of the wild-type cells (Figs 5.5a-
d)(Wang Tao & Nandakumar et al., 2013). We then analyzed additional histone
modifications at the Gfi1 locus that may be impacted by loss of MYSM1 in the
Lin
−
progenitors. ChIP results showed that while the permissive H3K4me3 levels were
decreased, the repressive H3K27me3 levels were significantly elevated at the Gfi1
enhancer region of MYSM1
−/−
Lin
−
progenitors (Figs 5.5 e&f). Notably, the histone
modifications were enriched in the same region where ubH2A, Ring1b and Bmi1 were
enriched along the Gfi1 promoter locus. Interestingly, the recruitment of paused RNA
polymerase (pol) II was also increased in MYSM1
−/−
Lin
−
progenitors (Fig 5.5g). This
indicates that Gfi1 gene is poised for future activation in the absence of MYSM1-
mediated deubiquitinatinase activity at its enhancer region. Collectively, these
data indicate that MYSM1 orchestrates histone modifications and facilitates the
recruitment of critical transcription factors to the Gfi1 locus. MYSM1 deficiency results
in repressive histone modifications and impaired recruitment of key transcription factors
causing an inhibition of Gfi1transcription activation (Wang Tao & Nandakumar et al.,
2013).

95

Figure 5.5 MYSM1 Orchestrates Histone Modifications at the Gfi1 Locus.
ChIP assays were performed in wild-type and MYSM1
−/−
Lin
−
cells using anti- IgG (a),
anti-ubH2A (b), anti-Ring1b (c), anti-Bmi1 (d) anti-H3K4me3 (e), anti-H3K27me3 (f)
and anti- RNA pol II (g) antibodies. The precipitated DNA was analyzed by real-time
PCR using primers along the the Gfi1 promoter region (-2.2kb). Primer amplifying the
coding region (4
th
exon) of Gfi1 is used as a negative control. The relative amount of
immunoprecipitated DNA is presented as a percentage of input DNA. Data are
representative of three independent experiments,*P < 0.05(Wang Tao & Nandakumar et
al., 2013).

5.4 Conclusion

We have previously developed mice deficient in the MYSM1 gene, which encodes
histone H2A deubiquitinase, and examined a role for MYSM1 in B cell development by
using this mice (X. X. Jiang et al., 2011). In this study, we focused on a role of MYSM1
specifically in hematopoietic stem cells. We performed detailed characterization on the
activity of hematopoietic stem cells in the MYSM1-deficient mice and showed that the
hematopoietic stem and progenitor subpopulations tended to be actively cycling and also
96

exhibits an increased apoptotic rate, giving rise to hematopoietic stem cell
deficiency(Wang Tao & Nandakumar et al., 2013). However, a generic hematological
characterization in MYSM1-deficient mice has already been published by Nijnik et al.
(A. Nijnik et al., 2012). Nevertheless, we performed a series of cellular and molecular
analyses focusing on a role in the hematopoietic stem cell function in more elaborate
fashion, the results of which is presented in the previous chapter. Additionally, in this
chapter, we provide a new finding that Gfi1 is a candidate gene responsible for the
hematopoietic stem cell defect in MYSM1-deficient mice. We performed ChIP analysis
and showed molecular evidence indicating that MYSM1 is directly involved in regulation
of Gfi1 transcription through its epigenetic regulatory activity. We also showed that
active cycling and increase in apoptosis, found in hematopoietic stem cell and progenitor
subpopulations from MYSM1-deficient mice, was genetically suppressed by retroviral
transduction of Gfi1. Not only has that Gfi1 partially restores the function of MYSM1-/-
HSCs through genetic complementation with Gfi-1 gene(Wang Tao & Nandakumar et al., 2013).

We further showed that Gata2 and Runx1 transactivated Gfi1 expression via its -35kb
enhancer and MYSM1 knockdown significantly reduced this transactivation. This data
coupled with the interaction of Gata2 and Runx1 with MYSM1 may indicate that
MYSM1 regulates Gfi1 expression via interaction and coordination with Gata2 and
Runx1. Our further analysis on how MYSM1 regulated Gfi-1 expression showed that
MYSM1 controls Gfi-1 transcription by modulating histone modifications and directing key
transcriptional factor recruitment to the Gfi1 locus in HSCs. Thus our study elaborates on the
97

initial reports of Nijnik A et al by enumerating the critical role of MYSM1 in HSC homeostasis
and illustrates a possible mechanism associated with it(Wang Tao & Nandakumar et al., 2013).

98

Chapter 6. Overall Summary and Future Perspectives
Although use of knockout mice had enabled the identification of transcription factors for
Natural killer cell development in the past years, unveiling the regulation of events that
control hematopoietic cell development and specific stages of cell differentiation still
remains a challenge. Specifically, studies on epigenetic regulation of the transcription
factors required for cell development is an area of extensive investigation. Our lab is
interested in identifying regulators in the differentiation and functions of immune cells.
We unexpectedly found that the histone H2A deubiquitinase MYSM1 plays an essential
role in control of gene transcription and differentiation of various cell lineages. By
generating and studying MYSM1 knockout (KO) mice, we found that MYSM1 plays an
essential role in the epigenetic control of B cell development, which is manifested by a
marked decrease in peripheral B cells and a block in the differentiation of pro-B cells in
KO mice(X. X. Jiang et al., 2011).

In chapter 2 and 3 of this thesis, we have identified the essential and intrinsic role of a
histone H2A deubiquitinase, MYSM1 for NK cell maturation (Nandakumar et al., 2013).
Our study outlines this role of MYSM1 in three parts: One, MYSM1 intrinsically controls
the maturation of NK cells, downstream IL-15 signaling but is dispensable for NK
lineage commitment. Two, our mechanistic studies revealed that MYSM1 interacts with
NFIL3 and this interaction is critical for the recruitment of these proteins to the Id2 locus
for the activation of its transcription during NK cell development and that the
transcription factor ID2’s expression is impaired in MYSM1-deficient NK cells. Three,
99

our mechanistic study also revealed that in the absence of MYSM1, Id2 locus is poised
in its repressed state for future activation and that MYSM1-mediated epigenetic
alterations may suspend its chromatin from a poised to that of an activated state for
promoting NK cell development (Nandakumar et al., 2013).

Our mechanistic study revealed that MYSM1 is essential for the development of mature
NK cells by promoting the transcription of ID2. Several lines of evidence support this
conclusion (Nandakumar et al., 2013): first, the expression of ID2 was significantly
reduced in MYSM1-/- mNK and NK precursor cells; second, the forced expression of
MYSM1 rescued the defective expression of ID2 in MYSM1-/- NK cells; third, the
association of MYSM1 with the ID2 locus was detected by chIP assays; fourth, altered
histone modifications and paused RNA polymerase II occupancy demonstrates the
existence of a poised Id2 locus in the MYSM1-/- NK cells; and, fifth, the forced
expression of ID2 rescued the defective development of mature NKs from MYSM1-/- NK
precursors. Besides the evidence presented in this study, our conclusion is further
supported by the similarity of the NK cell phenotypes of MYSM1-/- and Id2 -/- mice.
Both these KO mice showed impaired maturation of NK cells, but the early stages of
development remained unaffected (M. D. Boos et al., 2007). The thymic NK cell
development was not compromised in both Id2 -/- and MYSM1-/- mice. We also observed
fewer and smaller lymph nodes (LNs) and Peyer’s patches (PPs) in MYSM1-/- mice (data
not shown), which resembled the defective development of LNs and PPs in Id2 -/- mice
(M. D. Boos et al., 2007). Thus, the block in NK cell maturation observed in MYSM1-/-
100

mice is likely due to the defective expression of ID2 in MYSM1-/- mNK and their
precursor cells.

Although our study did not identify much reduction in the transcript levels of many
known NK development transcription factors except that of ID2, T-BET and GATA3, we
cannot exclude the possibility that MYSM1 may directly or indirectly regulate additional
genes apart from Id2 during NK cell development (Nandakumar et al., 2013). For
example, we observed a loss of Flt3

expression in CLPs and this data of ours was
consistent to that of Nijnik et al, who demonstrated a loss of Flt3
+
KLS cells in MYSM1-/-
mice (Nandakumar et al., 2013; Anastasia Nijnik et al., 2012). Previously, Flt3 signaling
has been implicated in the development and/or maintenance of Flt3
+
MPPs, CLPs, B, T,
NK, and DC lineage cells (Cheng et al., 2009; McKenna et al., 2000). However, two
independent groups recently demonstrated the downregulation of Flt3 and upregulation of
CD122 as sequential steps in NK cell commitment from Flt3
+
CLPs (Carotta, Pang, Nutt,
& Belz, 2011; Fathman et al., 2011). Our data infact shows that reduction of Flt3
+
CLPS
in the MYSM1-/- mice did not compromise the generation of pre-NKPs and rNKPs.
Another group showed similar evidence in which the reduction of Flt3
+
CLPs in
hoxa9−/− mice did not compromise their ability to generate NKPs or even mature NKs
cells(Gwin, Dolence, Shapiro, & Medina, 2013). However, the precise role of MYSM1
through Flt3-dependent or independent regulatory circuits in regulation of NK cell
development may need more rigorous investigation (Nandakumar et al., 2013).

101

Our further investigation on how MYSM1 is selectively targeted to the Id2 locus
provides some clues, at least partially, that MYSM1 interacts and co-recruits with the
DNA-binding NFIL3 protein to Id2 locus. We also found that this interaction is critical
for the recruitment of NFIL3 to the Id2 locus, possibly due to the MYSM1-induced
chromatin alterations that could enhance NFIL3 binding in the Id2 locus. However, it is
interesting to notice that Nfil3-/- mice showed a defective NK cell development at both
the iNK and mNK stages of development (Gascoyne et al., 2009), while the MYSM1-/-
mice were defective only during the transition from iNK to mNK cells. It is unclear why
MYSM1 is not involved in NK cell development during transition from NKP to iNK
cells. It may be possible that organized epigenetic signals for the coordination of NFIL3
and MYSM1 are only composed at the later stages of NK cell development. What other
proteins help in targeting MYSM1 to the Id2 locus and if NFIL3 requires any epigenetic
factors other than MYSM1 to be recruited to the Id2 locus during the early stages of NK
cell development needs more investigation. Clearly, an interesting area of future studies
is to fully understand how MYSM1 interacts with different partners for selective
activation of its target genes in different lineages of cells or at different developmental
stages of cells from the same lineage(Nandakumar et al., 2013).

Specific chromatin marks keep master regulators of differentiation silent yet poised for
further induction (Oguro et al., 2010). Our data indicates that Id2gene is poised in its
repressed state for future activation and that MYSM1-mediated epigenetic alterations
may suspend its chromatin from a poised to that of an activated state. This data converge
102

well with a previous report that showed RNA polymerase pausing as one of the down-
stream consequences of histone H2A monoubiquitination (Stock et al., 2007). Although
ID2 is not essential for early NK cell development and concurrently MYSM1-/- mice
shows defect only in NK cell maturation and not it’s lineage commitment, ID2’s
expression is one of the first indications of NK cell lineage specification (M. D. Boos et
al., 2007). It will be interesting to identify the precise developmental point when
MYSM1 composes the epigenetic signals for the induction of Id2 gene for NK lineage
specification and development.

Nevertheless, this study demonstrates for the first time an important and intrinsic role of
MYSM1 in NK cell development through an epigenetic control of ID2 transcription,
critical for NK cell development(Nandakumar et al., 2013).

Nijnik A et al. recently reported a role of MYSM1 in BM haematopoiesis with a H2A-
DUB targeted mouse line MYSM1
tm1a/tm1a
(A. Nijnik et al., 2012). Although MYSM1
deficiency does not affect commitment to NK cell lineage our next goal was to identify
the specific development defect at the early stages of HSC differentiation. This formed
the basis for the second half of this thesis, where we sought to identify more precisely the
defective subset within the HSC compartment of MYSM1-/- mice; since this was not
detailed out by Nijnik et al. Hence in chapter 4 and 5 of this thesis, we investigated the
role of MYSM1, specifically in HSC homeostasis in an elaborate manner and found the
103

impairment in MYSM1-controlled Gfi1 regulation as one possible cause for the defective
HSC function in MYSM1-deficient mice(Wang Tao & Nandakumar et al., 2013).

Nijnik A et al. showed an increased proportion of LSK subsets in the total and Lin
−
BM
cells (A. Nijnik et al., 2012), which was also evident in our results. However, inconsistent
with Nijnik A’s observation that there was no obvious change in the absolute numbers
between wild-type and Mysm
tm1a/tm1a
LSK cells, we found a significant reduction in
MYSM1
−/−
LSK cells and this reduction was most conspicuous in the more mature
CD150
−
CD48
+
or CD34
+
Flt3
+
LSK cells in our mouse line. This may be due to variations
in the genetic background of mice or different ages of mice used in experiments. Both
Nijnik A et a.l and our group detected an increased apoptosis in the MYSM1
−/−
LSK cells
and its subsets; this was regarded as a consequence of elevated genetic instability and
oxidative stress. In addition to this finding, we found that MYSM1
−/−
LSK cells were
more sensitive to external stress such as irradiation and deregulation of Gfi1 and its
downstream targets such as bax (Nakazawa et al., 2007) may have contributed to this
abnormality in MYSM1-/- HSC. Not only that, our data provides more rigorous evidence
for the defects observed in HSC quiescence, self-renewal and maintenance of its pool size
in the MYSM1
−/−
mice. Based on our experimental evidences and phenotypic similarities
between MYSM1-/- and Gfi1-/- mice, we also present here a mechanism of Gfi1
repression as a possible cause for the defective HSC biology in the MYSM1
−/−

mice(Wang Tao & Nandakumar et al., 2013).

104

Quiescence is critical for the maintenance, survival and self-renewal of HSC. Published
studies of mice deficient in p21, p53, Gfi1, PTEN, Foxos, Pbx1, Mi2β, TSC1, PML, or
Fbw7 have shown that a loss of quiescence and unscheduled HSC proliferation results in
the loss of self-renewal ability or stem cell exhaustion(K. W. Orford & Scadden, 2008;
Trumpp, Essers, & Wilson, 2010; A. Wilson, Laurenti, & Trumpp, 2009). We found that
MYSM1-deficient HSC had hyper-proliferative properties due to their inability to remain
quiescent and unlike c-Cbl-deficient mice(Rathinam, Thien, Langdon, Gu, & Flavell,
2008), Erg1-deficient mice(Min et al., 2008) and Itch-deficient mice(Rathinam et al.,
2011), in which HSC continuous and excessive proliferation resulted in a larger HSC
pool but without ‘stem cell exhaustion’, MYSM1-deficient mice had a reduced pool of
HSC and failed to recover in response to stresses such as 5-FU and irradiation due to
premature proliferative exhaustion. Importantly, MYSM1-deficient HSC was
incompetent in repopulating the hematopoietic system in competitive BM transplantation
assays, indicating that the self-renewal of these HSC was impaired(Wang Tao &
Nandakumar et al., 2013).

HSC quiescence, self-renewal, and differentiation are well coordinated by transcription
factors (A. Wilson et al., 2008; A. Wilson et al., 2009), of which the zinc finger protein
growth factor independence 1 (Gfi1) is one example. Gfi1 was regarded as one of the
most critical regulators in maintaining functional HSC (Duan & Horwitz, 2005; Hock,
Hamblen, et al., 2004; Zeng et al., 2004). In searching for the molecular role of MYSM1
in HSC using gene expression analysis, we identified Gfi1 as a possible target of
105

MYSM1 (Wang Tao & Nandakumar et al., 2013). Importantly, Gfi1 rescued the
defective function of MYSM1-/- HSC, at least to a partial level. In addition to our
experimental evidences that prove the Gfi1 regulation by MYSM1, we noticed many
phenotypic similarities between MYSM1- and Gfi1-deficient HSC. In both Gfi1
−/−
and
MYSM1
−/−
mice, the absolute numbers of LSK cells and Flt3 positive progenitors were
significantly reduced, HSC were unable to maintain quiescence and increased apoptosis
was evident in HSC. Moreover, both deficient mice failed in long-term hematopoiesis
(Duan & Horwitz, 2005; Hock, Hamblen, et al., 2004; Zeng et al., 2004). Our further
mechanistic investigation revealed the concerted action between MYSM1, Gata2 and
Runx1 at the Gfi1 enhancer element to promote its transcription. MYSM1 association
with the Gfi1 locus correlated with active histone modifications(Wang Tao &
Nandakumar et al., 2013).

Collectively, these results clearly illustrated that MYSM1 regulates Gfi1 transcription by
orchestrating histone modifications and transcription factor recruitment in/to the Gfi1
locus. Although Gfi1 repression may be one possible reason for the defective HSC in
MYSM1-/- mice, one cannot exclude the MYSM1-dependent contribution of other genes
in this function, such as the signaling molecules and other cytokine genes identified
through our gene-expression analysis. Further studies are needed to elucidate the
complete molecular mechanism associated with the MYSM1 function in HSC.
Nevertheless, our study elaborates on the initial reports of Nijnik A et al (A. Nijnik et al.,
106

2012) by enumerating the critical role of MYSM1 in HSC homeostasis and illustrates a
possible mechanism associated with it(Wang Tao & Nandakumar et al., 2013).

With thorough understanding of the physiological and molecular role of MYSM1, it can
be used as targets to strategically manipulate and enhance the endogenous levels of NK
cells, their progenitor stem cells and their cell-mediated functions for the prevention and
treatment of various immunological disorders since aberrant regulation of NK and HSC
cell development can lead to immune deficiency, autoimmunity and immune cell
malignancies that are difficult to diagnose and treat.

107

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120

Appendices
Appendix A: Supplementary information for Chapter 2
Figure A.1 Confirmation of the reduction in MYSM1 mRNA levels in MYSM1-/-
NK cells and T cell frequencies and numbers in WT vs MYSM1-/- mice.
A. Real-time PCR analysis of the expression of MYSM1 in wild-type and MYSM1-/- Lin
-
CD3
-
CD122
+
NK1.1
+
NK cells from bone marrow (BM) and spleen. mRNA expressions
were normalized to GAPDH and values are presented as relative expression with that of
the mRNA levels of wild-type NK cells (values set to 1). Data are mean + SEM of
triplicate determinations from three experiments (one per bar; n = 3 mice per genotype)

B. Representative flow cytometric profiles of ratio of CD4
+
and CD8
+
T cells C. average
of the frequencies of CD4
+
and CD8
+
T cells E. average of the total cell numbers of CD4
+

and CD8
+
T cells in the indicated organs of WT and MYSM1-/- mice D. Representative
flow cytometric profiles of T cell development at DN1 (CD44
+
CD25
-
), DN2
(CD44
+
CD25
+
), DN3 (CD44
-
CD25
+
) and DN4 (CD44
-
CD25
-
) stages gated on CD4
-
CD8
-
thymocytes in the WT and MYSM1-/- mice. ‘DN’ stands for ‘double negative’
thymocytes. Data shown are mean of n>6 mice per group are representative of more than
3 independent experiments *, P < 0.05; **, P < 0.01; ***, P < 0.001.

121

Figure A.1 continued

Figure A.2 Thymic NK cells were not defective in MYSM1-/- mice.
Thymocytes from WT and MYSM1−/− mice were stained for CD122, DX5, and IL-7R.
Data shown are A. representative flow cytometric profiles of DX5
+
IL7R
+
thymic NK
cells gated on Lin
−
CD122
+
thymocytes from 3 independent experiments, n=2 mice per
group B. Quantification of the average percentages of thymic NK cells .

Figure A.3 Flt3
-
IL-7R
+
CLPs were increased in MYSM1-/- mice.
A. Representative flow cytometric analyses of bone marrow Lin
-
IL-7R
+
Sca1
+
c-Kit
int

common lymphoid progenitors (CLPs) and Flt3
-
and

Flt3
+
CLP subsets gated on Lin
-
IL-
7R
+
Sca1
+
c-Kit
int
CLPs of MYSM1-/- mice and WT littermates. Numbers adjacent indicate
percents of indicated populations. Quantification of the average frequencies of bone
marrow B. CLPs or C. Flt3
+
IL-7R
+
and

Flt3
-
IL-7R
+
CLPs from 3 independent
experiments, n=5 mice per group.
122

Figure A.3 continued

Figure A.4 Transition to immature Lin-CD122+NK1.1+NKp46- NK cells is
unaffected in MYSM1-/- mice.
A. Representative flow cytometric profiles of NK1.1
+
NKp46
-
and NK1.1
+
NKp46
+
NK
cells and further analysis of mature NK subsets: NKp46
+
DX5
+
, CD27
+
CD11B
+
and
CD27
-
CD11B
+
cells gated from NK1.1
+
NKp46
+
NK cells within the CD122
+
B220
-
CD3
-

BM lymphocytes. Data shown are representative of 3 independent experiments, with n=3
mice group in each experiment.

123

Figure A.5. Additional analysis for the cell-intrinsic role of MYSM1 in NK cell
maturation in vitro and in vivo
A. Mixed chimera model proves the intrinsic requirement of MYSM1 for NK cell
maturation. Sublethally-irradiated 10- to 12- week-old WT B6.SJL-PtprcPep3/BoyJ
recipient mice (CD45.1) were transplanted with 3000 Lin
-
CD122
+
NK1.1
-
DX5
-
NKPs
from WT CD45.1 bone marrow mixed with 1500 NKPs from CD45.2 WT or MYSM1-/-
mice. Donor-derived lineage reconstitution was evaluated in the spleen 21days after
transplantation by FACS. The specific gates are indicated in the text above the plot and
by arrows. Data shown are representative of 2 independent experiments, with n=5 mice
group in each experiment. B-C. NK generation from KLS cells in vitro. FACs-sorted
10,000 Lin
-
c-KIT
+
Sca-1
+
KLS from WT and MYSM1-/- mice were cultured on OP9
stroma in a NK-cell conditioned media for 14 days and then harvested for assessing the
NK cell out-growth by flow cytometric analysis, B. representative FACS plots of
NK1.1
+
DX5
+
cells gated on CD3
-
B220
-
cells generated in vitro; specific gates are
indicated above the plots C. Mean proportions of NK1.1
+
DX5
+
cells of the total CD3
-
B220
-
cells generated in vitro. Data shown are mean + SEM of two independent
experiments *, P < 0.05; **, P < 0.01; ***, P < 0.001. D-G. MYSM1 functions
downstream IL-15 during NK cell development FACs-sorted WT NKPs transduced with
LV GFP or over-expressed with LV MYSM1 were cultured on OP9 stroma with
cytokines: KL, IL-7 (first week only), FL, IL-2 but in the absence of IL-15 for 14 days
and then harvested for assessing the NK cell out-growth by D-E. Flow cytometric
analysis as explained in A-B. Data shown are mean + SEM of 4 independent experiments
F. MYSM1 mRNA levels of in vitro-derived and post-sorted NK1.1
+
NK cells from WT
NKPs over-expressed with LV MYSM1 cultured with NK cell cytokines on OP-9 stroma
in the presence and absence of IL-15 or G. MYSM1 mRNA levels of freshly-sorted and
exvivo cultured Lin
-
CD3
-
CD122
+
NK1.1
+
NK cells cultured in the presence and absence
of IL-15 (30ng/ml) for 6 hours. mRNA expressions were normalized to GAPDH and
values are presented as relative expression with that of the mRNA levels of NK cells
derived or cultured in the absence of IL-15 (values set to 1). Data are mean + SEM of
triplicate determinations from one of 4 independent experiments. *, P < 0.05; **, P <
0.01; ***, P < 0.001.

124

Figure A.5 continued

Figure A.6 Sorting gating scheme and validation of purity of the sorted cells used in
our experiments
Representative FACS profiles of gating schemes used for sorting the Lin
-
CD122
+
DX5
-
NK1.1
-
NKPs for in vitro NK cell generation and transplantation assays and Lin
-
CD122
+
NK1.1
+
NK cells for chIP assays. 7AAD was included to exclude dead cells.
Reanalysis after sorting is also shown; the purity was reproducibly more than 98%.

125

Figure A.6 continued

126

Appendix B: Supplementary information for Chapter 3
Figure B.1 qRT-PCR analyses of NK cell development transcription factors in
sorted WT and MYSM1-/- mature NK cells (CD122
+
Lin
-
NK1.1
+
DX5
+
).
Real-time PCR analyses for the indicated transcription factors of sorted mNKs from the
femurs and tibias of bone marrow pooled from 10-15 MYSM1-/- and WT mice as
explained in Fig. B.1. Data are mean + SEM of triplicate determinations from one of two
independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Figure B.2 qRT-PCR analyses of ID2 in sorted WT NK cell and NK cell progenitor
subsets from bone marrow.
Data shown are representative of one of two independent experiments, shown are mean +
SEM of triplicate determinations. Data are mean + SEM of triplicate determinations from
one of 5 independent experiments.

127

Figure B.3 Enrichment of known NK cell specific interactions in the chromatin
fractions prepared for chIP assays.
ChIP assays of sorted WT and MYSM1-/- NK cells (Lin
-
CD3
-
CD122
+
NK1.1
+
) using anti-
RUNX3 and anti-IgG to check for its binding to A. Cd122 promoter B. NKp46 promoter.
Shown are representative chips (percent input) of WT and MYSM1-/- NK cells along
Cd122 and NKp46 loci and these interactions were verified in every newly prepared
chromatin fractions.

128

Table B.1 Primers used for qRT-PCR of NK cell development-related factors

129

Table B.1 Primers used for chIP analysis of Id2 locus

130

Appendix C: Supplementary information for Chapter 4
Figure C.1 Reduction in the absolute numbers of hematopoietic lineage cells in
MYSM1-/- mice
(a) Absolute cell number of spleen (left) and thymus (right) in WT and MYSM1
-/-
mice.
(b) Percentages of indicated cell population from WT and MYSM1-/- mice. (c)
Representative flow cytometry profiles of bone marrow (BM), spleen (SP), thymus (Thy),
and peripheral blood (PB) from WT and MYSM1
-/-
mice. (d) Percentages of B220
+
B
cells, CD3
+
T, CD4
+
T cells, CD8
+
T cells and Gr1
+
CD11b
+
cells in indicated tissues of
WT and MYSM1
-/-
mice (n = 4 per group). (e) HE staining of WT and MYSM1
-/-
femurs.
(f) Blood counts of RBC, WBC and PLT in WT and MYSM1
-/-
mice (n=6 per group). **P
< 0.01, ***P < 0.001.

131

Figure C.2 Characterization of MYSM1 in HSC and its compartments
(a) MYSM1 mRNA levels in hematopoietic cell subpopulation from WT mice
determined by qRT-PCR analysis. (b) Absolute number of lineage-negative (lin-) cells
per femur in WT and MYSM1-/- mice (n =9). (c-e) Percentages of various LSK subsets
among total WT and MYSM1-/- BM cells. (f) Flt3 expression in WT and MYSM1-/- lin-
and LSK cells.

Figure C.3 Loss of quiescence in MYSM1-deficient HSCs
(a) Quiescence of lin
-
c-kit
+
sca-1
-
progenitors and (b,c) HSC subsets in WT or MYSM1
-/-

BM cells. BM cells were stained for HSC surface antigens followed by Hoechst
33258/Pyronin Y staining. Data are representative of three independent experiments. *P
< 0.05, **P
< 0.01.

132

Figure C.4 MYSM1-deficient HSCs display defective engraftment
(a) 1×10
6
BM cells from WT or MYSM1
-/-
mice (CD45.2) were transplanted into lethally
irradiated recipients (CD45.1) together with 2×10
5
competitor BM cells (CD45.1).
Percentages of donor-derived BM cells, B cells, T cells and myeloid cells 2wks post
transplantation were analyzed. (b) 1×10
3
LSK cells sorted from WT or MYSM1
-/-
mice
(CD45.2) were transplanted into lethally irradiated recipients (CD45.1) together with
2×10
5
competitor BM cells (CD45.1). Percentages of donor-derived B cells, T cells and
myeloid cells were followed in PB at 4, 8, 12, and 16 weeks after transplantation. (c,d)
1×10
6
Fetal liver cells from WT or MYSM1
-/-
mice (CD45.2) were transplanted into
lethally irradiated recipients (CD45.1) together with 2×10
5
competitor BM cells
(CD45.1). (c) Percentages of donor-derived T cells, B cells and myeloid cells in PB 16
weeks after transplantation. (d) Percentages of donor-derived LSK cells in BM 16 weeks
after transplantation. Data are representative of two (a,c,d) or three (b) independent
experiments. Shown are means ± SD. **P < 0.01, ***P < 0.001.

133

Figure C.4 continued

134

Appendix D: Supplementary information for Chapter 5
Table D.1 List of genes changed more than 2 fold in the Mouse RT
2
Profiler
TM
PCR
array

2^- Δ Ct Fold Change
Mysm1
-/-
WT Mysm1
-/-
/WT
Cbfb 6.95E-02 1.06E-02 6.57
Mmp9 5.38E-02 1.03E-02 5.24
Pf4 3.39E-01 6.93E-02 4.89
Notch4 2.04E-03 5.14E-04 3.97
Apc 6.41E-02 1.89E-02 3.38
Hprt 1.14E+01 3.54E+00 3.21
Hdac9 1.85E-02 8.53E-03 2.16
Cebpg 2.20E-02 4.89E-02 -2.22
Cd14 1.62E-02 6.05E-02 -3.73
Csf2 2.73E-03 1.12E-02 -4.11

Table D.2 Mouse hematopoiesis RT
2
Profiler
TM
PCR array data

135

Table D.2 continued

136

Table D.3 Primers used for qRT-PCR of HSC development-related factors

Abstract (if available)

Abstract Histone modifications play critical roles in regulating hematopoietic cell development and differentiation. The importance of several histone H2A ubiquitinases and deubiquitinases (ubiquitin-specific proteases) in transcription regulation have been demonstrated recently. In our recent study, we found that Myb-like, SWIRM, and MPN domains-containing protein 1 (MYSM1), a histone H2A deubiquitinase (2A-DUB), is required for early B cell development by de-repressing EBF1 transcription (Jiang et al., 2011). ❧ So far, very little is known about the epigenetic control of NK cell development. In the first half of the thesis, we present our finding that NK cell development is severely impaired in mice deficient (-/-) in the histone H2A deubiquitinase MYSM1 (Nandakumar et al., 2013). We demonstrated that MYSM1 is required for NK cell maturation but not for NK lineage specification and commitment. We also found that MYSM1 intrinsically controls this NK cell maturation. Mechanistic studies revealed that the expression of transcription factor Id2, a critical factor for NK cell development, is impaired in MYSM1-/- NK cells. MYSM1 was found to interact with NFIL3 (E4BP4), a critical factor for mouse NK cell development and the recruitment of NFIL3 to the Id2 locus is dependent on MYSM1. Further, we observed that MYSM1 is involved in maintaining an active chromatin at the ID2 locus to promote NK cell development. Hence this study, for the first time, uncovers the critical epigenetic regulation of NK cell development by histone H2A deubiquitinase, MYSM1 through the transcriptional control of transcription factors important for NK cell development.

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Asset Metadata

Creator Nandakumar, Vijayalakshmi (author)

Core Title The essential role of histone H2A deubiquitinase MYSM1 in natural killer cell maturation and HSC homeostasis

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Genetic, Molecular and Cellular Biology

Publication Date 10/30/2013

Defense Date 10/16/2013

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag epigenetics,histone debiquitination,MYSM1,natural killer cell,OAI-PMH Harvest

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Chen, Si-Yi (committee chair), Akbari, Omid (committee member), Machida, Keigo (committee member), Ouellette, Andre J. (committee member), Yuan, Weiming (committee member)

Creator Email viji_kct@yahoo.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-342553

Unique identifier UC11297095

Identifier etd-Nandakumar-2120.pdf (filename),usctheses-c3-342553 (legacy record id)

Legacy Identifier etd-Nandakumar-2120.pdf

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Document Type Dissertation

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Rights Nandakumar, Vijayalakshmi

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Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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