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Mysm1, a histone de-ubiquitinase, is essential for dendritic cell development and function
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Mysm1, a histone de-ubiquitinase, is essential for dendritic cell development and function
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Content
MYSM1, A HISTONE DE-UBIQUITINASE, IS ESSENTIAL FOR
DENDRITIC CELL DEVELOPMENT AND FUNCTION
By
Hae Jung Won
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements of the Degree
DOCTOR OF PHILOSOPHY
(Molecular Microbiology and Immunology)
December 2013
Copyright 2013 Hae Jung Won
i
ACKNOWLEDGEMENTS
First and foremost I would like to acknowledge my mentor: Dr.Si-Yi Chen, thank you for
giving me opportunities to challenge new field of the study and creating working
environment where I could train myself to become an independent scientist. Thank you
for all of the advice and guidance you provided while I navigated my graduate career.
I would like to thank my dissertation committee members, Dr. James Ou, Dr. Andre
Oullette and Dr. Alan Epstein, along with program director Dr. Stanley Tahara for your
guidance advice throughout my graduate education.
Finally, I would like to thank my lab mates for being the best supports for me as
colleagues and friends.
ii
TABLE OF CONTENTS
Acknowledgements………………………………………………………………………...i
Abstract……………………………………………………………………………………v
Chapter 1: Introduction………………………………………………………………. 1
1.1 Epigenetic regulation of hematopoiesis………………………………………….. 1
1.2 Role of Mysm1 in hematopoiesis……………………………………………………..2
Chapter 2. Developmental defect of dendritic cell in Mysm1-/- mouse………………4
2.1 Introduction………………………………………………………………………..4
2.2 Materials Methods………………………………………………………………...6
2.3 Result…………………………………………………………………………….10
2.3.1 Generation of Mysm1-/- mice………………………………………….10
2.3.2 Mysm1 is required for cDC and pDC development…………………...11
2.3.3. Intrinsic role of Mysm1 in DC development………………………….12
2.3.4 Decrease of DC progenitors in Mysm1-/-……………………………...16
2.3.5 Mysm1 is selectively required for Flt3L-induced DC
development from early hematopoietic precursors…………………………..18
2-4 Discussion………………………………………………………………………..19
Chapter 3. Mysm1 induces DC differentiation by up-regulating Flt3……………….21
3.1 Introduction ……………………………………………………………………...21
3.2 Materials and Methods …………………………………………………………..23
3.3 Results …………………………………………………………………………...26
3.3.1 Mysm1 up-regulates expression of Flt3 through direct
binding at the promoter locus ……………………………………………….26
3.3.2 Mysm1 regulates modifications of histone, RNA
polymerase at the Flt3 promoter region ……………………………………..29
3.3.3 Mysm1 is required for the recruitment of PU.1 to
the Flt3 locus ………………………………………………………………...30
3.4 Discussion ……………………………………………………………………….33
Chapter 4. Mysm1 attributes to DC fate decision in CMP ………………………….36
4.1 Introduction ……………………………………………………………………...36
4.2 Materials and Methods …………………………………………………………..37
4.3 Results …………………………………………………………………………...39
4.3.1 Development of monocyte, macrophage, and granulocyte
is not altered in Mysm1-/- …………………………………………………..39
4.3.2 Mysm1 induces DC lineage specification from CMP by
activating Flt3 expression …………………………………………………...40
4.3.3 Mysm1 does not associate with Pu.1 at the GM-CSF
receptor and M-CSF receptor loci …………………………………………...43
4.4 Discussion ……………………………………………………………………….46
iii
Chapter 5: Mysm1 controls DC function ……………………………………………49
5.1 Introduction ……………………………………………………………………...49
5.2 Materials Methods ………………………………………………………………50
5.3 Results …………………………………………………………………………...54
5.3.1 Mysm1-/- DCs are less mature than WT DCs ………………………...54
5.3.2 Maturation defect in Mysm1-/- DC …………………………………...58
5.3.3 Mysm1-/- DC suppresses allogeneic T cell reaction
by inducing Treg …………………………………………………………….59
5.3.4 Inducing Ag-specific T cell proliferation is impaired
in Mysm1-/- splenic DCs ……………………………………………………62
5.3.5 Mysm1-/- DC elicits reduced systemic
Immunostimulatory potency ……………………………………………….63
5.3.6 Deletion of Mysm1 results in global changes of gene
expression in BMDC ………………………………………………………...65
5.4 Discussion ……………………………………………………………………….67
Chapter 6: Concluding remarks ……………………………………………………..72
References …………………………………………………………………………...75
iv
LIST OF FIGURES
Figure 1-1. Epigenetic regulation of transcription ……………………………………1
Figure 2-1. Schematic diagram of Dendritic Cell (DC) development ………………..4
Figure 2-2. Mysm1 knockout mouse ………………………………………………..10
Figure 2-3. Mysm1 is required for DC development ………………………………..12
Figure 2-4. Intrinsic role of Mysm1 in DC development …………………………...13
Figure 2-5. Decrease in DC progenitors …………………………………………….15
Figure 2-6. Mysm1
-/-
DC progenitors failed to differentiate into DC upon Flt3L
stimulation in vitro …………………………………………………………………………..17
Figure 3-1. Mysm1 regulates Flt3 expression ……………………………………….27
Figure 3-2. Altered histone modification and RNA polymerase recruitment at Flt3 locus
in Mysm1 KO ………………………………………………………………………...…28
Figure 3-3. Mysm1 mediates localization of Pu.1 at the Flt3 locus, but not at the GM-
CSF αR and M-CSFR loci ……………………………………………………..………...32
Figure 4-1. Mysm1 is not required for Development of monocyte, macrophage, and
granulocyte …………………………………………………………………………..39
Figure 4-2. Mysm1 induces DC lineage specification by selectively upregulating Flt3
expression …………………………………………………………………………...42
Figure 4-3. Mysm1 mediates localization of Pu.1 at the Flt3 locus, but not at the GM-
CSF αR and M-CSFR loci …………………………………………………………...44
Figure 4-4. Mysm1-dependent/-independent Pu.1 transcriptional complexes ……...47
Figure 5-1. Decreased expression of MHCII and co-stimulatory molecules in Mysm1-/-
DCs ………………………………………………………………………………..55
Figure 5-2. Immature phenotype of Mysm1 DC …………………………………...56
Figure 5-3. Maturation defect in Mysm1-/- DC ……………………………………..57
Figure 5-4. Mysm1 KO DC attenuates allogeneic CD4 T cell proliferation in MLR.
………………………………………………………………………………………..60
Figure 5-5. Mysm1-/- DC represses Allogeneic T cell activation and induces
Treg…………………………………………………………………………………61
Figure 5-6. Mysm1-/- DCs were deficient in T cell priming. ……………………….63
v
Figure 5-7. Systemic Immunostimulatory potency is reduced in Mysm1 KO DC…..64
Figure 5-8. Deletion of Mysm1 result in changes in global gene expression profile in
BMDC ……………………………………………………………………………….65
Figure 6-1 Transcriptional regulation of Flt3 by Mysm1……………………………72
Figure 6-2. CMP lineage differentiation; Mysm1-dependent/-independent
pathway……………………………………………………………………………... 73
vi
Abstract
The mechanisms controlling the development of dendritic cells (DCs) remain
incompletely understood. Using a genetic knockout mouse model, we identified the
histone H2A deubiquitinase Mysm1 as a novel critical regulator in DC differentiation.
Mysm1
−/ −
mice showed a global reduction of DCs in lymphoid organs whereas
development of granulocytes and macrophages were not severely affected.
Hematopoietic progenitors and DC precursors were significantly decreased in Mysm1
−/ −
mice, and common myeloid progenitors (CMP) were defective in Flt3L-induced, but not
in GM-CSF-induced DC differentiation in vitro. Molecular studies demonstrated that the
developmental defect of DCs in Mysm1
-/-
CMPs is a result of decreased Flt3 expression
and that Mysm1 de-represses transcription of the Flt3 gene by directing histone
modifications at the Flt3 promoter region. Two molecular mechanisms were found to be
responsible for the selective role of Mysm1 in DC differentiation: the selective expression
of Mysm1 in a subset of CMPs, and the different requirement of Mysm1 for Pu.1
recruitment to the Flt3 locus and both GM-CSF and M-CSF receptor loci. In conclusion,
this study reveals an essential role of the histone H2A deubiquitinase Mysm1 in Flt3
transcription and DC development and provides novel mechanism of epigenetic control
in DC development via histone modifications.
Dendritic cell undergo maturation upon encountering pathogens which is critical for
initiating adoptive immune response by upregulating co-stimulatory markers and
cytokine secretion. However, epigenetic control of DC maturation has been poorly
studied so far. We show herein that histone deubiquitinase, Mysm1, positively regulates
DC maturation. In Mysm1
-/-
mice, steady state DCs and GM-CSF derived BMDC display
relatively immature phenotypes including low level of MHCII and co-stimulatory
molecules and increased phagocytosis and migration. In addition, Mysm1
-/-
BMDCs were
vii
impaired in inducing expression of MHCII and co-stimulatory molecules upon LPS
stimulation. Mysm1
-/-
splenic DCs suppress allogeneic T cell reaction by inducing Treg
differentiation. Moreover, we found that antigen specific immune stimulatory potency
was reduced in Mysm1
-/-
DCs. MicroArray data indicates that deletion of Mysm1 results
in global changes of gene expression in BMDC many of which were related to
cytokine/chemokine regulatory pathways. Collectively, this study suggests a novel
mechanism for regulation of DC maturation by epigenetic control.
1
Chapter 1: Introduction
1.1 Epigenetic regulation of hematopoiesis
Figure 1-1. Epigenetic regulation of transcription. (Wong et al., 2007)
Epigenetic regulation plays pivotal roles in controlling lineage-specific gene expression
in hematopoiesis. DNA strands wrap around histone molecules forming a condensed
structure called nucleosomes. Nucleosome is dynamic that it opens or closes the
structure by modifying histones and/or DNA to allow or denies the access of
transcriptional machinery (Wong et al.). Transcription factors bind to regulatory regions
of their target genes in a DNA sequence-specific manner and recruit cofactors, which
are, in most cases, multi-protein complexes capable of enhancing or inhibiting gene
transcription by modifying chromatin structure and accessibility of transcription
machineries to gene regulatory loci (Figure 1-1). Such chromatin modifications include
2
DNA methylation and histone modifications, including acetylation, methylation,
phosphorylation, SUMOylation, and ubiquitination (Rice et al., 2007). Histone
ubiquitination is a reversible, post-translational modification and so far, ubiquitinations of
H2A K119 and H2B K120 have been studied most extensively. About 5-15% of histone
2A (H2A) is monoubiquitinated in mammalian cells, making it the most abundant
ubiquitinated protein in the nucleus. Unlike other protein ubiquitinations, histone
monoubiquitination is not linked to protein degradation, but instead plays important roles
in transcriptional control (Atanassov et al., 2011). Also, it has been implicated in cell
cycle regulation, X-chromosome inactivation, and DNA repair (Komander et al., 2009).
1.2 Role of Mysm1 in hematopoiesis
Mysm1 belongs to the Mov34/MPN/PAD-1 family, which includes histone deubiquitinase,
with possible Zn
2+
-dependent deubiquitinase activity. The gene is identified by Zhu et al.
(Zhu et al., 2007) and its histone deubiquitinase activity has been shown by siRNA study.
Co-immunoprecipitation and peptide mass fingerprinting identified the histone acetylase
p300/CBP-associated factor (PCAF) as a binding partner, implicating a correlation
between histone acetylation and ubiquitination. Deubiquitination may promote H1
dissociation from nucleosomes that is often linked with gene activation; however, the
cellular and physiological functions of Mysm1 are still largely unknown. Recently, we
reported the role of Mysm1 in B cell development by demonstrating that Mysm1 de-
represses transcription of EBF1, a master transcription factor for B cell lineage
commitment and early development, and that a deletion of Mysm1 interferes with early B
cell commitment in mice (Jiang et al., 2011). Furthermore, Nijnik et al. demonstrated the
role of Mysm1 in maintaining bone marrow hematopoietic stem cell function and
development of lymphoid and erythroid lineages (Nijnik et al., 2012). Thus, these
3
previous studies suggest that Mysm1 is required for the epigenetic regulation of
hematopoiesis and prompted us to further investigate the physiological function of
Mysm1 in the development of other hematopoietic lineages and in this study, we
demonstrated essential role of Mysm1 in dendritic cell development and function.
4
Chapter 2. Developmental defect of dendritic cell in Mysm1-/- mouse
2.1 Introduction
Diverse subsets of dendritic cells (DCs) exist in various tissues throughout the body and
perform different functions in steady state and immune responses. DCs have a short
half-life and constantly need to be replenished from bone marrow-derived hematopoietic
stem cells and/or progenitor cells in both lymphoid and non-lymphoid tissues (Liu and
Nussenzweig, 2010; Merad and Manz, 2009). DCs can be derived from both common
lymphoid progenitor (CLP) and common myeloid progenitor (CMP) (Figure 2-1). While
CMP-derived pathways have been extensively studied, the mechanisms regulating the
final differentiation of myeloids, DCs, monocytes, and granulocytes, are still not very
clear. Macrophage-dendritic cell progenitors (MDP) are the first precursors of DCs
downstream CMPs, and as the name implicates, differentiate into both macrophages
and dcs. Common dendritic cell progenitors (CDP) are DC-restricted precursors which
originate from MDPs without monocyte potential. CDPs are an immediate precursor of
pre-cDCs and pre-pDCs, which are the terminal differentiation of DCs in secondary
lymphoid organs and bone marrow, respectively, but are not yet mature (Watowich and
Liu, 2010).
5
Figure 2-1. Schematic diagram of Dendritic Cell (DC) development
6
2.2 Materials Methods
Generation of Mysm1 KO-first floxed mice and Mysm1 KO mice. Mysm1 targeted
vector is composed of an FRT flanked splice acceptor (En2 SA), lacZ, and neomycin
and poly(A) sequence followed by a loxP site. An additional loxP site is inserted
downstream of the targeted MYSM1 exon (E3). The Mysm1 mRNA truncation-first
strategy was based on inserting a cassette into an intron of an intact target gene that
produces a truncated mRNA 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, preventing the gene from being transcribed into mRNA
downstream of the cassette site (Figure 2-2A) (Testa et al.). Subsequent Cre expression
results in the deletion of the floxed MYSM1 exon 3 by crossing with cre transgenic mice.
The Mysm1 mRNA truncation-first floxed sperm in the C57BL/6J background
(Mysm1_A04; Mysm1
tm1a( ∆MP)Wtsi
MGI#: 2444584) were provided by the KOMP
Repository at UC Davis. In vitro fertilization, microinjection, chimera production, and the
generation ofMysm1 mRNA truncation-first floxed mouse founders were carried out at
the USC Transgenic Mouse Core Facility. In all experiments, WT littermates (
+/+
) were
used for controls. Mice were maintained in a pathogen-free barrier facility, and all
experiments were performed in accordance with the University of Southern California
Institutional Animal Care and Use Committee.
Flow cytometry and cell sorting. Single-cell suspensions of bone marrow (BM) and
spleens were prepared and were first stained for 20 min at 4° C with CD16/CD32 Fc-
blocking antibody (2.4G2), unless indicated otherwise, in flow cytometry buffer, followed
by incubation with a ‘cocktail’ of antibodies conjugated to fluorescein isothiocyanate
(FITC), phycoerythrin (PE), peridinine chlorophyll protein complex–cyanine 5.5 (PerCP-
7
Cy5.5), phycoerythrin-indotricarbocyanine (PE-Cy7), allophycocyanin (APC), or
allophycocyanin-indotricarbocyanine (APC-Cy7). For each staining, at least 100,000
events were collected for analysis. The following antibodies from BD Biosciences,
eBioscience, or BioLegend 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-7R α; A7R34), anti-PDCA1(129c), anti-CD8a (53-
6.7), anti-Gr-1 (RB6-8C5), anti-CD11b (M1/70), anti-CD11c (N418), anti-F4/80 (BM8),
anti-CD115 (12-3A3-1B10), anti-CD45RA (HI100), anti-SIRPa (P84), anti-IA/IE (anti-
MHCII, M5/114.15.2), anti-CD103 (2E7), anti-Flt3 (2AF10), 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) and were analyzed with FlowJo software (TreeStar).
For cell progenitor population sorting (Lin
-
cells), cells from BM were first depleted of
mature hematopoietic cells using a lineage cell depletion kit (Miltenyi Biotec), and were
then sorted by flow cytometry using the anti-mouse lineage cocktail. Cell populations
were isolated by FACS Aria III as follows: from bone marrow, LSK cells (Lin
−
Sca1
+
c-Kit
+
),
CLP cells CLP (Lin
-
IL7R
+
Sca1
int
cKit
int
), CMP cells (Lin
-
SCA-1
-
c-kit
CD34
+
CD16/32
mid
Flt3
+/-
), and Lin
-
CD115
+
(Waskow et al., 2008).
BM-derived DC culture. Murine BM-derived DCs were prepared as described
previously (Sharabi et al., 2008). Briefly, murine BM was flushed from the hind limbs,
passed through a nylon mesh, and depleted of red blood cells with RBC lysis buffer (BD
Bioscience, 555899). After extensive washing with RPMI-1640, cells were cultured with
RPMI-1640 supplemented with 10% FBS, and recombinant mouse GM-CSF/ml (20
ng/ml; PeproTech) and recombinant mouse IL-4 (20 ng/ml; PeproTech) or recombinant
mouse Flt3L (100 ng/ml; eBioscience). For GM-CSF culture, the supernatant was
8
removed and replaced with fresh media containing mGM-CSF and mIL-4 on days 2 and
4 of culture. Non-adherent granulocytes were removed after 48 hours of culture, and
fresh media was added. After 7 days of culture, more than 80% of the cells expressed
characteristic DC-specific markers as determined by FACS. For Flt3L culture, the cells
were cultured for 8 days without disruption. All cultures were incubated at 37° C in 5%
humidified CO
2
.
Bone marrow transplantation. Unfractionated BM cells (2x10
6
) from WT and KO
CD45.2 mice were transplanted into lethally irradiated (9.5 Gy) wild type C57BL/6
(CD45.1) mice through retro-orbital injection. PE-conjugated CD45.2 and FITC-
conjugated CD45.1 (BD Bioscience) were used to determine the donor- or recipient-
derived population by flow cytometry analysis (Sharabi et al., 2008). Peripheral blood,
spleen, and bone marrow were harvested and analyzed by flow cytometry.
Lentivirus and retrovirus production and transduction. Recombinant lentiviral
vectors were produced as described in our previous publications (Shen et al., 2004).
Lentivirus supernatants were prepared by transient cotransfection of 293T cells with
package plasmids VSVg, Rev, Gag/Pol, and lentiviral constructs encoding Mysm1-eGFP
(LV- Mysm1) or eGFP alone (LV-GFP). Viral supernatants were collected after 60 to 72
hours. Retroviruses were produced as described previously (Jiang et al., 2011). Briefly,
Phoenix cells were transfected with plasmids that contain a specific gene expression
vector (pMIG) encoding Flt3 followed by an IRES-GFP cassette (Carotta et al., 2010).
The empty pMIG vector expressing GFP alone acted as a control. pMIG-Flt3 was a gift
from Dr. Stephen L. Nutt (The Walter and Eliza Hall Institute of Medical Research,
Parkville, Victoria, Australia). Viral supernatants were harvested after 48-72 hours. For
transduction, bone marrow cells, Lin
-
cells, or CMP cells were cultured overnight in
9
RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100
μg/ml streptomycin, 2 mM L-glutamine, 50 μM β-mercaptoethanol. Lentiviral or retroviral
supernatants were applied to culture dishes pretreated with RetroNectin (TaKaRa) and
centrifuged at 3,000 rpm for 90 minutes, then incubated at 37° C in the presence of
polybrene (4 μg/ml) for an additional 24 hours. Cells were then washed and
resuspended in fresh media.
10
2.3 Result
2.3.1 Generation of Mysm1-/- mice
Figure 2-2. Mysm1 knockout mouse (Jiang et al.)
(A) Diagram of the MYSM1 targeted. pA: polyA; FRT: Flippase recognition target site;
SA: splice acceptor. (B) Genotyping of wild-type (WT), heterozygous (+/-) and
homozygous (-/-) MYSM1 KO-mice. (C) MYSM1 protein expression in WT and Mysm1-/-
mice bred from MYSM1 KO mice by Western blot of splenocytes and thymocytes with a
custom-made rabbit polyclonal anti-MYSM1 antibody against a pool of peptides derived
from MYSM1. (D) Quantitative RT-PCR analysis of the expression of MYSM1 in sorted
HSC (Lin-c-kit+Sca1+), B220+ B cells and CD3+ T cells from homozygous Mysm1-/-
mice and WT littermates from one of three independent experiments. Relative mRNA
levels were normalized to Hprt expression and are presented relative to WT mice (set as
1). **P < 0.01.
To investigate the physiological role of MYSM1, we generated Mysm1 mRNA truncation-
first- floxed mice from the Mysm1-targeted sperms in the C57BL/6J background
provided by the Knockout Mouse Project (KOMP) Repository (Figure 2-2 A). The
absence of Mysm1 mRNA was confirmed by genomic polymerase chain reaction (PCR)
and Southern blot analyses. Homozygous Mysm1
−/ −
mice were fertile and viable,
11
although they had truncated tails and growth retardation (Figure 2-2 B). Heterozygous
mice did not differ in morphology, growth, and viability from their wild-type (WT)
littermates. MYSM1 protein expression was drastically downregulated in the spleen and
thymus as well as other tissues of the Mysm1
−/ −
mice, as determined by immunoblot
analysis (Figure 2-2 C). MYSM1 mRNA in sorted B cells, T cells, and hematopoietic
stem cells (HSCs) was reduced by more than 80% in the Mysm1
−/ −
mice, as detected by
qRT-PCR (Figure 2-2 D).
2.3.2 Mysm1 is required for cDC and pDC development.
To investigate dendritic cell development from Mysm1-/- mice, various immune organs
were harvested from 4-6 week old Mysm1
-/-
or WT littermates, and the development of
myeloid populations including dendritic cells (DC) was analyzed by flow cytometry (Fig.
2-3A-D). Both conventional DCs (cDC; CD11c
hi
MHCII
+
) and plasmacytoid DCs (pDC;
CD11c
mid
PDCA1
+
) (Fig. 2-3A) were significantly decreased in Mysm1
-/-
mice both in
frequency as well as absolute number in spleen (Fig. 2-2B). Splenic cDCs were then
further analyzed for subsets and we found that the proportions of both CD8
+
and CD8
-
cDCs were not significantly changed (Fig. 2-3A and B). Since it has been reported that
plasmacytoid DCs fully develop in bone marrow and migrate to lymphoid organs and
periphery tissues, we analyzed the development of pDCs in bone marrow (Fig 2-3C and
D). The flow cytometry results showed a significant reduction of pDCs in Mysm1
-/-
bone
marrow.
12
Figure 2-3. Mysm1 is required for DC development
(A-H) Spleens and bone marrow of 4-6 week old wild type (WT) littermates or Mysm1
-/-
(KO) mice were analyzed by flow cytometry for indicated cell populations. (n=4) (A)
Representative flow cytometry of splenic cDCs (CD11c
high
MHCII
+
) and its subset; CD8
+
cDCs (CD11c
high
MHCII
+
CD8
+
CD11b
-
), CD8
-
cDCs (CD11c
high
MHCII
+
CD8
-
CD11b
+
), and
splenic pDCs (CD11c
int
PDCA1
+
). (B) Frequency and cell numbers of splenic cDCs and
pDCs from WT and Mysm1 KO mice. (C-D) Bone marrow pDCs (CD11c
+
PDCA1
+
) were
analyzed by Flow cytometry (C) and bar graph presented is the mean (± SEM) of
frequency and cell numbers of cDCs from WT and Mysm1
-/-
bone marrow.
2.3.3. Intrinsic role of Mysm1 in DC development
To test whether the defect in DC development in Mysm1
-/-
mice is a cell intrinsic effect,
bone marrow cells from CD45.2
+
Mysm1
-/-
or WT littermates were transplanted into
lethally irradiated CD45.1
+
recipient mice at 1:1 ratio with CD45.1
+
WT competitor cells.
At 3 weeks post transplantation, cells from spleen, bone marrow, and peripheral blood
13
Figure 2-4. Intrinsic role of Mysm1 in DC development
(A) CD45.2
+
(2x10
6
) WT or KO BM cells were transplanted into lethally irradiated
CD45.1
+
recipient mice at 1:1 ratios with CD45.1
+
WT competitor cells (2x10
6
). CD45.2
+
cell populations in recipients were determined by flow cytometry at 3 weeks post-
transplantation in bone marrow (BM), peripheral blood (PBL), and spleen (SP).
Representative flow cytometry data from 2 mice are shown. (B-D) Bone marrow cells
were harvested from femurs and tibiae of Mysm1
-/-
and WT littermates and 2x10
6
cells/ml were then plated in a 6-well plate. For Flt3L-induced DC culture, 100 ng/ml Flt3L
was added in DC culture media and cultured for 8 days without disruption (B and C). (D)
For GM-SCF-induced DC culture, 20ng/ml GM-CFS and 20 ng/ml IL-4 were added in DC
culture media and cultured for 5 days. Cells were harvested and stained with indicated
surface markers. Shown are representative flow cytometric analyses of pDCs
(CD11c
+
B220
+
), cDCs (CD11c
+
B220
-
or CD11c
+
CD11b
+
), monocytes (FSC
mid-high
SSC
low
),
and granulocytes (FSC
high
SSC
high
) from each culture condition. (E) Bone marrow from
KO mice were transduced with either lentivirus overexpressing mouse Mysm1 (LV-
Mysm1) or control lentivirus (LV-GFP) for 8 hrs. Cells were washed and cultured in
Flt3L- (100 ng/ml) supplemented media for 8 days and CD11c
+
DC differentiation was
analyzed by flow cytometry.
were harvested and analyzed by flow cytometry for reconstitution of DCs (CD11c
+
) (Fig.
2-4A). The results showed Mysm1
-/-
bone marrow (CD45.2
+
) failed to develop DCs. This
14
result implies the intrinsic role of Mysm1 in differentiation of BM progenitors into dendritic
cells. Flt3L is an essential and sufficient cytokine for DC differentiation and its absence
leads to a dramatic reduction of DCs in mice (McKenna, 2001). Flt3L induces both cDC
and pDC differentiation in vitro with >85% CD11c
+
in WT bone marrow, whereas GM-
CSF induces only cDCs which resemble the monocyte-derived inflammatory DC
phenotype (Gilliet et al., 2002; Serbina et al., 2003). To further examine the intrinsic role
of Mysm1, bone marrow cells from Mysm1
-/-
and WT littermates were cultured in Flt3L-
or GM-CSF/IL-4-supplemented media and DC differentiation was analyzed by flow
cytometry. Strikingly, Mysm1
-/-
bone marrow cells completely failed to differentiate into
cDC and pDC upon Flt3L stimulation (Fig. 2-4B). Conversely, the Flt3L cultured bone
marrow cells had granulocyte phenotypes as shown in SSC/FSC
high
and Gr1
+
populations (Fig. 2-4 C). In contrast, GM-CSF/IL4-induced DC differentiation was intact
in Mysm1
-/-
bone marrow (Fig. 2-4 D). To confirm that the failure of Mysm1
-/-
bone
marrow cells in Flt3L-induced DC culture was caused by an absence of Mysm1, we
reconstituted Mysm1 expression in Mysm1
-/-
bone marrow cells using a recombinant
lentiviral vector (LV) that co-expresses a full-length Mysm1 and GFP marker and
cultured in Flt3L supplemented media for 8 days and subjected to flow cytometry for DC
differentiation (Figure 2-4E). Mysm1-/- bone marrow transduced with LV-Mysm1 were, in
fact, able to differentiate into CD11c
+
DCs in Flt3L culture, indicating that the defect in
DC differentiation of Mysm1
-/-
bone marrow can be rescued by forced expression of
Mysm1.
15
Figure 2-5. Decrease in DC progenitors
Bone marrow cells were obtained from femurs and tibiae and stained for flow cytometric
analysis of indicated DC progenitor populations. Representative flow cytometry analysis
is shown. (A) Lineage markers, Sca1, cKit, CD34, and CD16/32 were used to analyze
HSC, CLP, MEP, GMP, and CMP populations. Hematopoietic precursor subpopulations
are shown as percentage of whole bone marrow cells and as absolute cell number (B).
n=4. BMs from WT or Mysm1
-/-
were analyzed for Lin
-
CD115
+
and CDP (Lin
-
CD115
+
c-
Kit
int
Flt3
+
) (C), and spleen was analyzed for Pre-cDC (CD11c
+
MHCII
-
SIRPa
+
Flt3
+
) by
flow cytometry (D). (E) Percentage of Lin
-
CD115
+
, CDPs, and pre-cDCs in BM are
shown (left panel, n=4), and absolute numbers of each population were calculated from
percentages indicated in the left panel (right panel). Data presented are mean values
(±SEM). n=4
16
2.3.4 Decrease of DC progenitors in Mysm1
-/-
Reduction of DC populations can be caused by a decrease of DC progenitor/precursor
cell populations and/or a loss of or impaired differentiation capacity of DC
progenitor/precursors. To determine whether the defect in DC development in Mysm1-
deficient mice is due to a decrease of hematopoietic progenitors or DC precursors, bone
marrow cells from Mysm1
-/-
and WT littermates were analyzed by flow cytometry (Fig. 2-
4). All of the hematopoietic progenitors tested, including HSCs (Lin
-
Sca1
+
cKit
+
), CLPs
(Lin
-
IL7R
+
Sca1
int
cKit
int
), CMPs (Lin
-
IL7R
-
Sca1
-
cKit
+
CD34
+
CD16/32
int
), and GMPs (Lin
-
IL7R
-
Sca1
-
cKit
+
CD34
+
CD16/32
hi
), were markedly decreased both in frequency and
absolute number in Mysm1
-/-
mice (Fig 2-5 A and B). Similarly, DC-committed precursor
populations, Lin
-
CD115
+
(MDP-enriched population (Waskow et al.) and common DC
progenitor (CDP; Lin
-
Flt3
+
CD115
+
cKit
int
), were significantly decreased in Mysm1
-/-
bone
marrow. Also, the migratory DC precursors pre-cDC (Lin
-
CD11c
+
MHCII
+
Flt3
+
SIRP α
neg-low
)
showed a marked reduction in Mysm1
-/-
spleen (Liu and Nussenzweig, 2010; Liu et al.,
2009) (Fig 2-5 C, D and F). These results indicate that Mysm1 controls the development
of both hematopoietic progenitors and DC precursors.
17
Figure 2-6. Mysm1
-/-
DC progenitors failed to differentiate into DC upon Flt3L
stimulation in vitro
(A-B) DC progenitor cells Lin
-
, LSKs (Lin
-
Sca1
+
cKit
+
), CLPs (Lin
-
IL7R
+
Sca1
int
c-Kit
int
), Lin
-
CD115
+
, CMPs (Lin
-
IL7R
-
Sca1
-
c-Kit
+
CD34
+
CD16/32
int
) were isolated from WT or Mysm1
-
/-
bone marrow by FACS and 10,000 progenitor cells were cultured with 1.5x10
5
CD45.1
BM feeder cells in the presence of Flt3L (100 ng/ml) (A) or GMCSF (20 ng/ml) and IL-4
(20 ng/ml) (B) in a 96-well plate for 8 days. Cells were harvested and analyzed by flow
cytometry for CD11c and CD45.1 surface markers. DC populations differentiated from
progenitors are gated by CD45.1
-
CD11c
+
. The number of DCs generated from each
culture was calculated and is presented as average number of DCs from 2-3 wells per
1,000 progenitors (C).
18
2.3.5 Mysm1 is selectively required for Flt3L-induced DC development from early
hematopoietic precursors
Although DC precursors are significantly reduced in Mysm1
-/-
mice, they are still present
at a low frequency. Therefore, we were prompted to test whether there is a
differentiation defect in Mysm1
-/-
DC precursors. Lin
-
, LSK, CMP, and Lin
-
CD115
+
populations from WT or Mysm1
-/-
bone marrow were sorted by FACS and cultured with
feeder cells from CD45.1
+
syngeneic mice in Flt3L- (Fig 2-6 A) or GM-CSF/IL-4-
supplemented media (Fig 2-6 B). Deletion of Mysm1 abolished generation of DCs from
all of the precursors tested in Flt3L culture, whereas the deletion had a less significant
effect on GM-CSF/IL4-induced DC differentiation in vitro, as shown by Mysm1
-/-
DC
precursors which still produced DCs in GM-CSF/IL4-supplemented media, albeit in
reduced numbers (Fig 2-6 C). These data indicate that Mysm1 plays pivotal roles in
Flt3L-induced DC development but may only have supportive roles in GM-CSF-induced
DC development.
19
2-4 Discussion
After screening the development of myeloid lineages, we observed significant decreases
in both pDC and cDC populations in spleen and pDC populations in bone marrow. We
ascertained the cell intrinsic effect of Mysm1 in DC development in vivo and in vitro
using a competitive bone marrow transplantation assay and in vitro DC culture.
Surprisingly, upon Flt3L stimulation, in vitro DC differentiation was completely abrogated
in Mysm1
-/-
bone marrow and considerable amount of cells from the culture showed
granulocyte-like phenotypes instead (high SSC/FSC and Gr1
+
), which implicates that the
myelopoiesis of Mysm1
-/-
bone marrow may skew toward granulocytes. Flt3L is essential
for steady state DC development, whereas GM-CSF induces only cDCs in vitro which
resemble a monocyte-derived inflammatory phenotype; an unresponsiveness of Mysm1
-
/-
bone marrow to Flt3L in vitro, but not to GM-CSF, is consistent with the decrease of
steady state DC in Mysm1
-/-
mice. To test whether the observed defect in DC
development is due to a defect in either development or function of DC progenitors, we
analyzed frequencies of DC progenitors and the capacity of these progenitors to
differentiate into DCs. We have found significant reduction in all DC progenitors, from
early progenitors including HSCs, LSKs, CLPs, and CMPs, to DC-committed precursors
including MDPs, CDPs, and pre-cDCs. Furthermore, we examined the capacity of a few
DC progenitors (Lin
-
, LSKs, CMPs, and Lin
-
CD115
+
) to differentiate into DCs, and we
found that all of the progenitors tested from Mysm1
-/-
bone marrow failed to generate
DCs upon Flt3L stimulation. Therefore, a defect in steady state DC development in
Mysm1
-/-
is cell intrinsic and caused both by decreased numbers and impaired DC
differentiation from common myeloid progenitors. Despite the drastic reduction in DC
progenitors, there is still a small number of DCs present in Mysm1
-/-
mice and in vitro
GM-CSF-induced DC differentiation remains intact. However, these Mysm1
-/-
DCs were
20
found to be defective in maturation after stimulation with various TLR agonists.
Moreover, the forced expression of Flt3 did not rescue the maturation defect in Mysm1
-/-
DCs (data not shown). Future studies are needed to investigate whether, in addition to
its role in DC differentiation, Mysm1 may also regulate DC maturation and function by
targeting other genes.
21
Chapter 3. Mysm1 induces DC differentiation by up-regulating Flt3
3.1 Introduction
DC differentiation is tightly regulated by the coordination of signal transduction pathways
and transcriptional networks. Transcription factors control DC commitment, lineage
specification, and survival, and their actions are induced or inhibited by extracellular
signals (Belz and Nutt, 2012; Satpathy et al., 2011). Various extracellular stimuli induce
progression of DC differentiation through progenitors by activating or de-activating
cytokine receptors. Major cytokine receptors known to play important roles in DC
development are macrophage colony-stimulating factor receptor (M-CSFR), granulocyte
macrophage colony-stimulating factor alpha receptor (GM-CSF R), and Fms-like
tyrosine kinase-3 (Flt3), which are highly expressed on DC progenitors (Schmid et al.,
2010). GM-CSF can derive DCs from murine bone marrow or human monocytes in vitro
(Ebner et al., 2001; Inaba et al., 1992; Sallusto and Lanzavecchia, 1994). However,
deletion of the GM-CSFR gene in mice does not result in a severe reduction (Vremec et
al., 1997) of DCs, which implicates a dispensable and redundant role of GM-CSF in DC
development. Recently, it has been reported that GM-CSF plays crucial roles in inducing
generation of TNF- α- and iNOS-producing DCs in infection (Xu et al., 2007). In contrast,
Flt3L is an indispensable cytokine in steady state DC development; Flt3L induces both
cDC and pDC development from murine bone marrow, whereas GM-CSF generates
only cDCs in vitro (Brasel et al., 2000; Gilliet et al., 2002; Xu et al., 2007). Flt3 and Flt3L
knock-out studies showed a dramatic reduction in the number of DCs in mice (Kingston
et al., 2009; McKenna, 2001; Waskow et al., 2008). Furthermore, bone marrow
progenitor cells gain DC-differentiating potential, along with the expression of Flt3, as all
DC progenitors and precursors express Flt3 (D'Amico and Wu, 2003; Karsunky et al.,
22
2003). Therefore, Flt3L-Flt3 is a dominant cytokine stimuli and signaling network for
steady state DC development. The mechanism by which expression of Flt3 is regulated,
however, is poorly understood.
23
3.2 Materials and Methods
Flow cytometry and cell sorting. Single-cell suspensions of bone marrow (BM) was
first stained for 20 min at 4° C with CD16/CD32 Fc-blocking antibody (2.4G2), unless
indicated otherwise, in flow cytometry buffer, followed by incubation with a ‘cocktail’ of
antibodies conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE),
peridinine chlorophyll protein complex–cyanine 5.5 (PerCP-Cy5.5), phycoerythrin-
indotricarbocyanine (PE-Cy7), allophycocyanin (APC), or allophycocyanin-
indotricarbocyanine (APC-Cy7). For each staining, at least 100,000 events were
collected for analysis. The following antibodies from BD Biosciences, eBioscience, or
BioLegend 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-7R α; A7R34), anti-CD11c (N418), anti-F4/80 (BM8), anti-CD115 (12-
3A3-1B10), anti-Flt3 (2AF10), 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)
and were analyzed with FlowJo software (TreeStar). For cell progenitor population
sorting (Lin
-
cells), cells from BM were first depleted of mature hematopoietic cells using
a lineage cell depletion kit (Miltenyi Biotec), and were then sorted by flow cytometry
using the anti-mouse lineage cocktail. Cell populations were isolated by FACS Aria III as
follows: from bone marrow, CMP cells (Lin
-
SCA-1
-
c-kit CD34
+
CD16/32
mid
Flt3
+/-
), and Lin
-
CD115
+
(Waskow et al., 2008).
Quantitative RT-PCR. Quantitative RT-PCR was performed as described previously
(Sharabi et al., 2008). Total RNA from isolated cells was purified with an RNeasy Minikit
(Qiagen) according to the manufacturer’s instructions. The iScript cDNA Synthesis kit
24
(BioRad) was used for reverse transcription. A SYBR Green PCR kit (BioRad) was used
for quantitative real-time PCR and results were quantified with a CFX96 Real Time PCR
Detection system (BioRad).
Protein immunoprecipitation and immunoblotting. Cells were lysed in lysis buffer (50
mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP-40) and incubated with anti-
Flag (F1804, Sigma), or control IgG at 4° C overnight, followed by adding protein A/G
PLUS-Agarose beads for additional 2h before washing three times with washing buffer.
Immunoprecipitates were boiled in SDS sample buffer and resolved by 4-12% Bis-Tris
NuPAGE gel (Invitrogen), transferred to PVDF membrane and probed with an anti-PU.1
antibody (T-21, sc-352).
Chromatin Immunoprecipitation. Chromatin was immunoprecipitated according to the
manufacturer’s instructions (Cell Signaling, 9002). Briefly, sorted cells were crosslinked
with 1% (vol/vol) formaldehyde at room temperature for 10 min, and incubated with
glycine for 5 min at room temperature. Cells were then sequentially washed in ice-cold
buffer A and buffer B, followed by MNase digestion. The nuclear pellet was suspended
in ChIP buffer and sheared by sonication, with an average size of sheared fragments of
about 300 base pairs (bp) to 800 bp. After centrifugation at 10,000 rpm for 10 min,
sheared chromatin was diluted in ChIP buffer and precleared by adding protein A/G plus
agarose beads (sc-2003) for 1 h at 4° C. The beads were discarded and the supernatant
was then incubated with one of the following antibodies: uH2A (05-678), H3K27me3 (ab-
6002), PU.1 (T-21, sc-352), Mysm1, or control anti-IgG (Cell Signaling) at 4° C overnight.
The next day, protein A/G plus agarose beads were added and incubated for 2 h at 4° C.
For anti-uH2A, anti-mouse IgM μ (12-488, Millipore) and protein A/G plus agarose beads
were added. Beads were harvested by centrifugation and were washed three times in
25
low salt washes and once in a high salt wash. Beads were then eluted with ChIP elution
buffer. The elutes and input were added to proteinase K and RNase A and heated at 65°
C for 2 h to reverse the formaldehyde cross-link. DNA fragments were purified with
columns and amplified by site-specific primers by quantitative real time PCR. For
sequential two-step ChIP experiments (Zhou et al., 2008), crosslinked chromatin was
immunoprecipitated with an anti-Mysm1 antibody or a control IgG (Cell Signaling, #9002).
Precipitated chromatin was then eluted in a solution of 30 mM DTT, 500 mM NaCl, and
0.1% SDS. Eluted chromatin was diluted 10-fold with ChIP buffer (Cell signaling, #9002)
and then re-immunoprecipitated with anti-Pu.1 or control IgG. The relative binding was
defined by determining the immunoprecipitation level (ratio of the amount of
immunoprecipitated DNA to that of the input sample) and then comparing to
corresponding 1
st
ChIP or 2
nd
ChIP control IgG immunoprecipitation level, which was set
as 1.0.
26
3.3 Results
3.3.1 Mysm1 up-regulates expression of Flt3 through direct binding at the
promoter locus
Flt3, the receptor for Flt3L, is variably expressed on early hematopoietic progenitors and
highly expressed on committed DC progenitors (Satpathy et al., 2011). Therefore, Flt3
has been used as a surface marker to identify the DC precursors CDP, MDP, and pre-
cDC (Liu and Nussenzweig, 2010). As shown previously (Fig. 3-1), a drastic decrease of
some DC precursors in KO mice is due to the lack of Flt3
+
populations. As expression of
Flt3 is essential for DC development in steady state, coupled with our observation that
Mysm1
-/-
bone marrow failed to differentiate into DCs upon Flt3L stimulation in vitro, we
were prompted to examine Flt3 expression in Mysm1
-/-
hematopoietic progenitors and
DC precursors. We measured expressions of Flt3 in Lin
-
and CMP at the protein and
mRNA levels. The flow cytometry results showed that expression of Flt3 on the cell
surface in KOs was almost completely abrogated (Fig. 3-1A) in all of the DC progenitors
examined. To test if the reduction occurred at the transcription level, the amount of Flt3
mRNA was measured by quantitative real-time PCR and the results showed a significant
down-regulation of Flt3 transcription in Mysm1
-/-
DC precursors (Fig 3-1B). Collectively,
the results suggest that Mysm1 is required for Flt3 expression.
27
Figure 3-1. Mysm1 regulates Flt3 expression
Expression of Flt3 was analyzed by (A) flow cytometry and (B) QPCR from Lin
-
and CMP
from WT and Mysm1
-/-
mice. mRNA expression was normalized to Glyceraldehyde 3-
phosphate dehydrogenase (GAPDH) mRNA amount (B). (C-D) Targeting of Mysm1 on
Flt3 gene locus is measured by Chromatin Immunoprecipitation (ChIP) assay. Lin
-
cells
were isolated from wild type bone marrow and chromatin was immunoprecipitated with
either anti-Mysm1 antibody or IgG. The pulled-down DNA was eluted and subjected to
real-time quantitative PCR analysis. (C) Sequence alignment of dog, human, monkey,
and mouse Flt3 genes. Transcription start site (ATG) and promoter site (P) are shown.
Primers for ChIP real-time PCR are designed from conserved region (boxed) among
species. (D) Binding of primers is analyzed by real time PCR.
Despite the critical role of Flt3 and its signaling pathway in hematopoiesis, the regulating
mechanism of Flt3 expression is poorly understood. To examine whether Mysm1 directly
regulates expression of Flt3, we surveyed the Flt3 gene for an Mysm1 binding site by
Chromatin Immunoprecipitation (ChIP) assay (Fig 3-1C and D) in Lin
-
populations
28
isolated from WT bone marrow. The genomic sequences of Flt3 from dog, human,
monkey, and mouse were aligned and primers were designed from highly conserved
regions including the promoter (promoter 1 and promoter 2) and the first exon (Exon)
(Fig. 3-1C). Mysm1-chromatin complex was immunoprecipitated by anti-Mysm1
polyclonal antibody and quantitative real-time PCR with site-specific primers were
performed to analyze the enrichment of the identified site. We found that the signal was
highly enriched at the Flt3 promoter site (Fig. 3-1D), indicating a direct Mysm1 binding at
the Flt3 promoter.
Figure 3-2. Altered histone modification and RNA polymerase recruitment at Flt3
locus in Mysm1 KO
ChIP analyses of WT or Mysm1
−/ −
BM Lin
−
cells via uH2A (A), H3K27me3 (B),
Phosphoserine-2 RNA Polymerase (C), or Phosphoserine-5 RNA Polymerase II (D). The
precipitated DNA was analyzed by quantitative PCR with primers amplifying Flt3
promoter regions (promoter 1 and 2), promoter deprived region (control primer), the Flt3
exon 1 region, and normalized to the input DNA. N/D: not detected.
29
3.3.2 Mysm1 regulates modifications of histone, RNA polymerase at the Flt3
promoter region
Since Mysm1 is known to have histone deubiquitinase activity, we hypothesized that the
histone ubiquitination status may be altered at the Flt3 promoter site in Mysm1
-/-
. To test
this hypothesis, we immunoprecipitated the ubiquitinated H2A (uH2A)-chromatin
complex from WT or Mysm1
-/-
bone marrow with an anti-ubiquitinated H2A (uH2A)
antibody, and enrichments in promoter sites, the first exon, and promoter desert region
(PDR) were compared (Fig 3-2A). The results showed that there was a significant
increase of uH2A at all of the sites tested. Since histone de-/ubiquitination have been
shown to coordinate histone modifications and interact with other histone modifiers
(Campos and Reinberg, 2009; Gelato and Fischle, 2008), we tested the status of other
histone modifications at the Flt3 regulatory locus by ChIP assay. Figure 3-2B shows that
there was a significant increase of repressive tri-methylated histone H3 at lysine 27
(H3K27me3) at the Flt3 promoter sites whereas no significant differences were found at
the promoter-deprived region ((-) control) or the first exon.
It has been previously reported that histone ubiquitination is involved in regulating
transcription by acting as a restraint for poised RNA polymerase II on gene promoters
and a block for transcriptional elongation (Atanassov et al., 2011; Zhou et al., 2008).
RNA polymerase II assembled at the promoter is phosphorylated at serine 5 of the
carboxy-terminal domain (CTD) repeat through transcription factor IIH (TFIIH). The CTD
is partially dephosphorylated at this position after it escapes into the elongation phase.
As elongation proceeds, the level of phosphorylation of the CTD at phosphoserine-2 (S2)
increases and peaks near the 3' end of a gene (Banchereau et al., 2000). To examine
the modifications on RNA polymerase II at the promoter and transcription stage of Flt3,
we pulled down chromatin by anti-RNA polymerase II antibodies that recognize either
30
the S2 or phosphoserine-5 (S5) version of RNA polymerase II and measured enrichment
of Flt3 promoter DNA by quantitative real-time PCR. The level of S2 RNA polymerase II
remained similar in WT and Mysm1
-/-
(Fig. 3-2C), but we observed an accumulation of
S5 RNA polymerase II at the Mysm1
-/-
Flt3 promoter site (Fig. 3-2D). This result
suggests that an increase of uH2A causes RNA polymerases to halt at the promoter
which, in turn, stops it from proceeding to elongation. Collectively, these data suggest
that Mysm1 not only deubiqutinates ubiquitinated histones at the Flt3 promoter region,
but also modulates the status of repressive histone modification, H3K27me3.
Furthermore, Mysm1 exerts de-repressive effects on Flt3 expression by promoting
transcription elongation and also recruits the transcription factor Pu.1 at the promoter.
3.3.3 Mysm1 is required for the recruitment of PU.1 to the Flt3 locus
Next, we tested whether this altered histone modification status can influence
recruitment of transcription factors at the Flt3 promoter site. Despite the crucial roles of
Flt3 signaling in DC development and pathogenesis of hematopoietic malignancies, the
regulating mechanisms of Flt3 expression and transcription factors that directly bind to
Flt3 regulatory regions remain largely unknown. Pu.1 has been proposed as a crucial
regulator of DC development since it is expressed in cDCs and pDCs (Back et al., 2005;
Nutt et al., 2005), and a deletion of Pu.1 causes defects in DC development (Anderson
et al., 2000; Guerriero et al., 2000). In addition, a recent study reported that Pu.1
controls expression of Flt3 by directly associating with the Flt3 promoter region (Carotta
et al., 2010). Therefore, we tested whether PU.1 recruitment to the Flt3 promoter is
altered in Mysm1
-/-
bone marrow by comparing PU.1 occupancy at the Flt3 locus in WT
and Mysm1
-/-
bone marrow via ChIP assay. Figure 3-3A shows that recruitment of PU.1
31
was significantly decreased at the Flt3 promoter site in Mysm1
-/-
. To further test direct
interaction of Mysm1 with Pu.1, we performed a co-immunoprecipitation assay. pCMV-
Mysm1-Flag and pCMV-Pu.1 plasmids were co-transfected into 293T cells and
immunoprecipitated using an anti-Flag antibody. The results revealed that Mysm1 and
Pu.1 are co-precipitated (Fig. 3-3B). Since both Mysm1 and Pu.1 directly bind to the Flt3
promoter region, we examined if there is an association of Mysm1 with Pu.1 at the Flt3
locus with a sequential 2-step ChIP assay. Accordingly, the results showed both Mysm1
and Pu.1 associate at the Flt3 promoter region (Fig. 3-3C).
Figure 3-3. Mysm1 mediates localization of Pu.1 at the Flt3 locus, but not at the
GM-CSF αR and M-CSFR loci
(A) ChIP analyses of the occupancy Pu.1 in WT or Mysm1
−/ −
BM Lin
−
cells. The
precipitated DNA was analyzed by quantitative RT PCR via 2 sets of primers amplifying
the Flt3 promoter region (promoter 1 and promoter 2) or the first exon (exon1) and
normalized with IgG control before comparison. (B) Sequential two-step ChIP assays of
32
WT BM Lin
−
progenitors were performed, showing the recruitment and Mysm1 and Pu.1
to the Flt3 promoter. Relative binding was defined by determining the
immunoprecipitation level (ratio of the amount of immunoprecipitated DNA to that of the
input sample) and then comparing to corresponding first ChIP or second ChIP control
IgG immunoprecipitation level, which was set as 1.0. (C) Co-immunoprecipitation assay
of Mysm1 and Pu1. pCMV-Pu.1 plasmid was co-transfected with wither pCMV-Flag-
Mysm1 or pCMV-FLAG expression plasmid into HEK293T cells and the cell lysates were
incubated with an anti-Flag antibody and immunoprecipitated proteins were analyzed by
anti-Pu.1 antibody.
33
3.4 Discussion
Flt3L is a major, non-redundant cytokine for DC development as shown in knock-out
studies in which mice lacking Flt3L or its receptor Flt3 have severely reduced cDCs,
pDCs, and interstitial dermal DCs (Kingston et al., 2009; McKenna, 2001; Waskow et al.,
2008). Expression of Flt3 pre-defines DC lineage potential of hematopoietic progenitors
as it has been concluded that DC development proceeds along a successive line of Flt3
+
progenitors (D'Amico and Wu, 2003; Karsunky et al., 2003; Mende et al., 2006).
Therefore, Flt3 is used as one of the surface markers to identify DC progenitors (CDP
and Pre-cDC) for flow cytometry analysis. Since we observed the global reduction of DC
populations, a complete abrogation of response to Flt3L stimulation in Mysm1
-/-
bone
morrow, and the down-regulation of DC progenitors in flow cytometry analysis was due
to a decrease or absence of Flt3 expression, we suspected that expression of Flt3 may
be altered in Mysm1
-/-
mice. We confirmed that expression of Flt3 was significantly
reduced in early progenitors: Lin
-
, LSK, and CMP and DC-committed progenitors, and
MDP in both protein and mRNA level. Despite its pivotal roles in hematopoiesis,
especially in DC development, the regulation of Flt3 expression at the molecular level is
poorly understood. Our ChIP data showed direct binding of Mysm1 to the Flt3 promoter
region. As mentioned earlier, histone modifications occur in a concerted manner in that a
change of one modification can facilitate the changes in chromatin architecture at the
promoter locus (Cairns, 2009). We observed an increase of monoubquitinated H2A
(uH2A) in Mysm1 at Flt3 promoter region accompanied by increased tri-methylation of
H3K27 (H2K27me3) in Mysm1
-/-
bone marrow, both of which favor transcription
repression. Since Mysm1 has histone deubiquitinase activity, it was expected to see an
increased level of uH2A, but the increase in H3K27me3 was caused by a change in the
histone ubiquitination status. We did not observe changes in levels of H3K4me3 or Acyl-
34
H3K9 (data not shown). Since it has been reported that H2A ubiquitination needs to be
removed by a deubiquitinase for H3K4 tri-methylation to occur in mammalian cells
(Nakagawa et al., 2008), we speculated that increase of uH2A may have interfered with
try-methylation of H3K4 at the Flt3 promoter locus. A previous report showed that
Mysm1 coordinates histone acetylation and deubiquitination (Zhu et al., 2007), but we
did not observe a decrease of histone acetylation at Flt3 promoter region, which
indicates that there may be redundant histone acetylases present. We also noticed there
is a significant increase in phosphoserine-5 RNA polymerase II at the Flt3 promoter
which is consistent with the previous report that ubiquitinated H2A halts the transcription
initiation machinery at the promoter and blocks elongation (Zhou et al., 2008). Therefore,
Mysm1 regulates expression of Flt3 by orchestrating histone modifications at the
promoter.
Dynamic structural changes in chromatin regulate the accessibility of transcription
factors to gene regulatory regions. Recently, Carrota et al. reported that the transcription
factor PU.1 regulates expression of Flt3 by direct association with the promoter (Carotta
et al., 2010). Here, we showed that recruitment of transcription factor Pu.1 was denied at
the Flt3 promoter in Mysm1
-/-
bone marrow. We further confirmed association between
Pu.1 and Mysm1 at Flt3 promoter by sequential 2-step ChIP assay. Pu.1 is a critical
transcription factor in hematopoietic lineage determination and has been reported to
modulate expression of nearly 3000 genes expressed in hematopoietic cells including
cell-surface proteins (CD11b), cytokines and their receptors (G-CSF, GM-CSF, M-CSF,
and Flt3) (Dakic et al., 2005; Hohaus et al., 1995; Kingston et al., 2009). In addition,
Pu.1 is expressed in mature monocytes and granulocytes, and essential for GM-CSF-
induced DC development from hematopoiesis progenitors. Since development of
35
monocytes and granulocytes and GM-CSF-induced DC development in vitro was not
severely affected by an absence of Mysm1, recruitment of Pu.1 to the gene regulatory
regions required for the processes above is likely to be intact. This also suggests the
gene-specific activity of Mysm1, but the mechanisms by which the specific activity of
Mysm1 is regulated is yet to be investigated.
36
Chapter 4. Mysm1 attributes to DC fate decision in CMP
4.1 Introduction
Our finding that Mysm1 is required for DC lineage prompted us to investigate of Mysm1
also controls development of other myeloid cell populations; macrophages, monocyte
and granulocyte, which share the same precursor, common myeloid progenitor (CMP)
with dendritic cells. Interestingly, we found that development of granulocyte and
macrophage lineage was not severely affected by deletion of Mysm1 in knockout mice
which begs a critical question: how is Mysm1 able to selectively drive the DC lineage
program from common myeloid progenitors? In this chapter, we examined molecular
mechanisms for the selective role of Mysm1 in DC differentiation.
CMPs are a heterogeneous population that can give rise to multiple myeloid lineages
(Lawrence and Natoli, 2011), however, the regulation of lineage specification is not well
understood. Approximately 40% of CMPs express Flt3, which can differentiate into DCs
upon Flt3L stimulation, and the remaining 60%, Flt3
-
CMPs, do not have DC potential
and give rise to GMPs and MEP. As shown previously, Mysm1 was found to be required
for Flt3 transcription and DC differentiation. Therefore, we hypothesized selective defect
of DC differentiation from CMP may be attributed by lack of the Flt3+ subset in CMP.
37
4.2 Materials and Methods
Flow cytometry and cell sorting Single-cell suspensions of bone marrow (BM) and
spleens were prepared and were first stained for 20 min at 4° C with CD16/CD32 Fc-
blocking antibody (2.4G2), unless indicated otherwise, in flow cytometry buffer, followed
by incubation with a ‘cocktail’ of antibodies conjugated to fluorescein isothiocyanate
(FITC), phycoerythrin (PE), peridinine chlorophyll protein complex–cyanine 5.5 (PerCP-
Cy5.5), phycoerythrin-indotricarbocyanine (PE-Cy7), allophycocyanin (APC), or
allophycocyanin-indotricarbocyanine (APC-Cy7). For each staining, at least 100,000
events were collected for analysis. The following antibodies from BD Biosciences,
eBioscience, or BioLegend were used for flow cytometry: anti-Gr-1 (RB6-8C5), anti-
CD11b (M1/70), anti-CD11c (N418), anti-F4/80 (BM8), anti-CD115 (12-3A3-1B10), 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) and were analyzed with
FlowJo software (TreeStar). For cell progenitor population sorting (Lin
-
cells), cells from
BM were first depleted of mature hematopoietic cells using a lineage cell depletion kit
(Miltenyi Biotec), and were then sorted by flow cytometry using the anti-mouse lineage
cocktail. Cell populations were isolated by FACS Aria III: Flt3+ and Flt3- CMP cells (Lin
-
SCA-1
-
c-kit CD34
+
CD16/32
mid
Flt3
+/-
).
Quantitative RT-PCR. Quantitative RT-PCR was performed as described previously
(Sharabi et al., 2008). Total RNA from isolated cells was purified with an RNeasy Minikit
(Qiagen) according to the manufacturer’s instructions. The iScript cDNA Synthesis kit
(BioRad) was used for reverse transcription. A SYBR Green PCR kit (BioRad) was used
38
for quantitative real-time PCR and results were quantified with a CFX96 Real Time PCR
Detection system (BioRad).
Lentivirus and retrovirus production and transduction. Recombinant lentiviral
vectors were produced as described in our previous publications (Shen et al., 2004).
Lentivirus supernatants were prepared by transient cotransfection of 293T cells with
package plasmids VSVg, Rev, Gag/Pol, and lentiviral constructs encoding Mysm1-eGFP
(LV- Mysm1) or eGFP alone (LV-GFP). Viral supernatants were collected after 60 to 72
hours. Retroviruses were produced as described previously (Jiang et al., 2011). Briefly,
Phoenix cells were transfected with plasmids that contain a specific gene expression
vector (pMIG) encoding Flt3 followed by an IRES-GFP cassette (Carotta et al., 2010).
The empty pMIG vector expressing GFP alone acted as a control. pMIG-Flt3 was a gift
from Dr. Stephen L. Nutt (The Walter and Eliza Hall Institute of Medical Research,
Parkville, Victoria, Australia). Viral supernatants were harvested after 48-72 hours. For
transduction, bone marrow cells, Lin
-
cells, or CMP cells were cultured overnight in
RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100
μg/ml streptomycin, 2 mM L-glutamine, 50 μM β-mercaptoethanol. Lentiviral or retroviral
supernatants were applied to culture dishes pretreated with RetroNectin (TaKaRa) and
centrifuged at 3,000 rpm for 90 minutes, then incubated at 37° C in the presence of
polybrene (4 μg/ml) for an additional 24 hours. Cells were then washed and
resuspended in fresh media.
39
4.3 Results
4.3.1 Development of monocyte, macrophage, and granulocyte is not altered in
Mysm1-/-
Since DCs are differentiated from common myeloid progenitors (CMP), which are
capable of generating multiple myeloid cell types (Iwasaki and Akashi, 2007), we
analyzed the development of other myeloid lineage cell populations. Interestingly, in
contrast to DC development, the development of granulocytes, monocytes, and
macrophages was rather intact or enhanced by the Mysm1 deletion (Fig 4-1 A-D). The
frequency of granulocytes (CD11b
+
Gr1
+
) was increased by approximately 3 fold whereas
monocytes (CD11b
+
Gr1
-
F4/80
-
) and macrophages (CD11b
+
Gr1
-
F4/80
+
) were not
significantly altered in Mysm1
-/-
spleen (Fig 4-1 A). However, absolute numbers of all the
populations examined were reduced due to the overall
Figure 4-1. Mysm1 is not required for Development of monocyte, macrophage, and
granulocyte (A-D) Granulocytes (CD11b
+
Gr1
+
), monocytes (CD11b
+
Gr1
-
F4/80
-
), and
macrophages (CD11b
+
Gr1
-
F4/80
+
) were analyzed by flow cytometry. (A) Representative
flow cytometric analysis of splenic granulocytes, monocytes, and macrophages. Bar
graph presented is the mean (± SEM) of cell numbers of each cell type (B). (C)
Representative flow cytometric analysis of bone marrow granulocytes, monocytes, and
macrophages. Bar graph presented is the mean (± SEM) of cell numbers of each cell
type (D).
40
decrease in number of splenocytes (Fig 4-1 B). In bone marrow, the frequencies of both
granulocytes and monocytes were found at similar levels to wild type (Fig. 4-1 C and D).
Collectively, these observations indicate that Mysm1 is required for cDC and pDC
development, but dispensable for the development of monocytes, macrophages, and
granulocytes.
4.3.2 Mysm1 induces DC lineage specification from CMP by activating Flt3
expression
To ascertain direct correlation between reduced level of Flt3 and DC development defect
in Mysm1
-/-
, we restored Flt3 in Mysm1
-/-
Lin
-
cells by transducing with a retrovirus
expressing either Flt3 or GFP control and culturing in Flt3L media for 8 days (Figure 4-
2A). Flow cytometry analysis for CD11c showed that forced expression of Flt3 restored
DC development in Mysm1
-/-
Lin
-
cells, which indicates that reduction of Flt3 mainly
attributed to the DC developmental defect in Mysm1
-/-
mice.
We have shown that the development of macrophages and granulocytes was not
significantly affected by deleting Mysm1 as compared to the dramatic reduction of DCs
(Fig 4-1) although all of these lineages are branched out from CMPs. CMPs can be
separated into two populations on an Flt3 expression basis: Flt3
-
CMPs and Flt3
+
CMPs.
It has been previously reported that the Flt3
+
portion of CMPs gives rise to DC
progenitors including MDPs and CDPs, since Flt3 is essential for DC development. Flt3
-
CMPs possess GMP potential that differentiates into macrophages and granulocytes
41
(D'Amico and Wu). As shown above, Mysm1 regulates Flt3 expression via a direct
association with its promoter region. Therefore, we hypothesized that Mysm1 contributes
DC lineage specification from CMPs by up- regulating Flt3. To test this hypothesis, we
first compared DC differentiation potential from Flt3
-
and Flt3
+
CMPs. We identified the
CMP population from WT bone marrow as described in Figure 3A, and sorted Flt3
-
and
Flt3
+
portions of CMPs by FACS. The ratio of Flt3
-
and Flt3
+
CMP was approximately
60:40, respectively, which is consistent with previous reports (D'Amico and Wu). The
sorted cells were then cultured in Flt3L-supplemented media for 8 days, then stained
with CD11c and Gr1 antibodies for flow cytometry analysis. As expected, differentiation
of DCs was significantly decreased from Flt3
-
CMPs as compared to Flt3
+
CMPs, in
which the majority of the cells gave rise to DCs (88%) (Fig 4-2B). Since we observed a
close correlation between Mysm1 and Flt3 expression, we suspected that Flt3
+
CMPs
may express a higher level of Mysm1. As such, we measured the mRNA level of Mysm1
in Flt3
-
and Flt3
+
CMPs by quantitative RT-PCR and found that Mysm1 expression was
indeed higher in the Flt3
+
CMP population (Fig 4-2C). To further investigate the role of
Mysm1 in DC differentiation from CMPs and Flt3 expression, we transduced WT Flt3
-
CMPs with a lentiviral vector expressing with GFP (LV-GFP) or Mysm1 (LV-Mysm1) and
cultured the cells in Flt3L supplemented media for 8 days. Forced expression of Mysm1
not only restored Flt3 expression in Flt3
-
CMPs but also induced DC differentiation (Fig
4-2D). Taken together, the results demonstrate that Mysm1 plays an important role in
DC lineage specification from CMPs by up-regulating Flt3 expression.
42
Figure 4-2. Mysm1 induces DC lineage specification by selectively upregulating
Flt3 expression
(A) Lin
-
cells were sorted from Mysm1
-/-
bone marrow and transduced with retrovirus
expressing GFP (RV-GFP) or Flt3 (RV-Flt3) for 12 hrs. The cells were then extensively
washed, resuspended in Flt3L (100 ng/ml) media (1x10
5
cells
/100ul) and cultured for 8
days. Surface expression of CD11c was analyzed by flow cytometry. (B-D) Mysm1
induces DC lineage specification from CMPs. CMPs from WT bone marrow was sorted
and divided into two populations by Flt3 expression: Flt3
-
CMPs and Flt3
+
CMPs. The
sorted cells were cultured in Flt3L-supplemented media for 8 days and surface
expression of CD11c and Gr1 was analyzed by flow cytometry (B) and Mysm1 level was
measured by quantitative RT-PCR (C). (D) Flt3
-
CMPs were infected with lentivirus
expressing either GFP or Mysm1 and cultured in Flt3L-supplemented media for 8 days.
Surface expression of CD11c and Flt3 was analyzed by flow cytometry.
43
4.3.3 Mysm1 does not associate with Pu.1 at the GM-CSF receptor and M-CSF
receptor loci
Pu.1 is a central regulator of many hematopoietic cell lineages and essential for final
differentiation of myeloid cells (Kastner and Chan). Both the GM-CSF receptor and M-
CSF receptor are required for macrophage and granulocyte development from myeloid
progenitors, and their expressions are also activated by Pu.1 which binds to their
promoter locus (Hohaus et al.) (Zhang et al.). Since we found that Mysm1 associates
with Pu.1 at the Flt3 promoter, we speculated that Mysm1 may interact with Pu.1 at GM-
CSF αR and M-CSFR promoter loci as well. To test the hypothesis, we first compared the
targeting of Pu.1 and Mysm1 at the promoter region of GM-CSF αR and M-CSFR by
ChIP analysis in WT Lin
-
bone marrow cells (Fig 4-3A). Pu.1 binding sites at GM-CSF αR
and M-CSFR promoter regions have been previously reported; therefore, we designed
site-specific primers encompassing Pu.1 binging sites to amplify promoter regions and
control primers from exon regions since it is highly unlikely that transcription factors
would bind to encoding sequences. The results showed high enrichment of both GM-
CSF αR and M-CSFR promoters from the anti-Pu.1 pull-down samplesSurprisingly, there
was no signal from anti-Mysm1 pull-down samples (Figure 4-3B), suggesting a different
role of Mysm1 in Pu.1 recruitment to the Flt3 compared to the GM-CSF αR and M-CSFR
loci. To further confirm that Mysm1 does not affect recruitment of Pu.1 to the GM-
CSF αR and M-CSFR promoters, chromatin from Mysm1
-/-
was pulled down by an anti-
Pu.1 antibody or IgG control and precipitation of GM-CSF αR and M-CSFR promoter was
surveyed by site-specific primers. The result showed high associations of Pu.1 at GM-
CSF αR
44
Figure 4-3. Mysm1 mediates localization of Pu.1 at the Flt3 locus, but not at the
GM-CSF αR and M-CSFR loci
(A) Targeting of Mysm1 or Pu.1 on GM-CSF α receptor and M-CSFR promoter locus is
measured by ChIP assay. Lin
-
cells were isolated from WT bone marrow and chromatin
was immunoprecipitated with anti-Mysm1, anti-Pu.1 antibody or IgG. The pulled down
DNA was eluted and amplified by real-time quantitative RT-PCR via primers spanning
the promoter or exon region. The signal was normalized to IgG control. (B) Targeting of
Pu.1 on GM-CSF αR or M-CSFR gene locus in Mysm1
-/-
Lin
-
cells is measured ChIP
assay. The signal was normalized to IgG control. (C) mRNA expressions of GM-CSF αR
and M-CSFR were measured in WT and Mysm1
-/-
Lin
-
cells.
45
and M-CSFR promoters in the absence of Mysm1 (Figure 4-3C), in contrast to the
reduced association of Pu.1 at the Flt3 locus in Mysm1
-/-
myeloid progenitors (Figure 3-
3A). Furthermore, we measured the mRNA expression levels of GM-CSF αR and M-
CSFR in WT and Mysm1
-/-
Lin
-
bone marrow cells by quantitative RT-PCR and found no
significant differences (Figure 4-3C). Collectively, the data indicates that Pu.1 binding to
the Flt3 locus is Mysm1-dependent, while Pu.1 binding to the GM-CSF αR and M-CSFR
loci is Mysm1-independent, which may form a molecular basis for the different
requirements of Mysm1 in in vitro Flt3- vs. GM-CSF-induced DC differentiation from their
progenitors, as well as in in vivo DC vs granulocyte and macrophage development.
46
4.4 Discussion
CMPs are a heterogenous population that approximately only 40% of CMPs express Flt3,
which can differentiate into DCs upon Flt3L stimulation. Interestingly, we found that
Mysm1 is highly expressed in Flt3
+
CMPs compared to that in Flt3
-
CMPs, and that
forced expression of Mysm1 in WT Flt3
-
CMPs upregulated Flt3 expression and
promoted DC differentiation. Taken together, our data suggest that the differential
expression of Mysm1 in a subset of CMPs leads to Flt3 transcription and subsequent DC
development. Although Flt3 is a key gene targeted by Mysm1 in DC progenitors, it is
likely that Mysm1 regulates the transcription of other target genes as well. Our
preliminary data showed a significant decrease in mRNA levels of Flt3 (-5.3), Irf8 (-4.8),
and E2-2 (-2.0) in Mysm1
-/-
HSC (LSK) cells by PCR array analysis of 22 transcription
factors implicated in DC development (Belz and Nutt). A pilot global analysis of gene
expression by microarray analysis also showed altered levels of many genes in Mysm1
-/-
HSCs (data not shown). A comprehensive identification and characterization of all
genes targeted by Mysm1 in DC progenitors is warranted in a future study.
Another molecular mechanism for the selective role of Mysm1 in DC differentiation is the
different requirements of Pu.1 for its recruitment to different gene loci. We found an
Mysm1-dependent recruitment of Pu.1 to the Flt3 locus for its transcription, but an
Mysm1-independent recruitment of Pu.1 to the GM-CSF R and M-CSFR loci (Fig 4.4.1).
We observed that 1) development of macrophages and granulocytes was not
significantly affected by an Mysm1 deletion, and 2) the reduction of DC-committed
precursors (CDPs, pre-cDCs) in Mysm1
-/-
was greater than the reduction of GMPs,
although both are downstream of CMPs. Thus, the differentiation of Mysm1
-/-
CMPs
47
Figure 4.4 Mysm1-dependent/-independent Pu.1 transcriptional complexes
skews toward macrophage/granulocyte development through an increased
differentiation of GMPs expressing little Flt3 (D'Amico and Wu), which is consistent with
the normal development of macrophages and granulocytes in Flt3 knockout mice
(Kingston et al., 2009), (McKenna), (Waskow et al.). Pu.1 is a critical transcription factor
in hematopoietic lineage determination and modulates expression of nearly 3,000 genes
expressed in hematopoietic cells including cell-surface proteins (CD11b) and cytokines
and their receptors (G-CSF, GM-CSF, M-CSF, and Flt3) (Dakic et al., 2005; Hohaus et
al., 1995; Kingston et al., 2009). Recently, Carrota et al. reported that the transcription
factor Pu.1 regulates expression of Flt3 by direct association with the promoter (Carotta
et al., 2010). In this study, we found that the recruitment of transcription factor Pu.1 to
the Flt3 promoter region and Flt3 transcription in CMPs is dependent upon Mysm1. Both
the GM-CSF receptor and M-CSF receptor, which are crucial receptors for
macrophage/granulocyte development and GM-CSF-induced DC differentiation, are also
activated by Pu.1. In contrast to its role in Pu.1-mediated Flt3 transcription, the
48
recruitment of Pu.1 to the GM-CSF αR and M-CSFR loci and the expression of GM-
CSF αR and M-CSFR were not compromised in Mysm1
-/-
CMPs. Thus, this study
indicates that, in addition to its differential expression in different subsets of CMPs,
Mysm1 selectively drives DC differentiation to CMPs due to the Mysm1-dependent or
independent recruitment of the transcription factor Pu.1 to different loci. This
unexpected finding certainly raises an additional question on how a transcription factor in
a progenitor cell is able to form varying transcriptional complexes for the targeting and
transcription regulation of different genes.
49
Chapter 5: Mysm1 controls DC function
5.1 Introduction
Dendritic cells (DCs) are professional antigen presenting cells that modulate innate and
adoptive immune response. DCs present in lymphoid organs and periphery tissues.
Immature DCs have abilities to uptake protein complexes, soluble antigens using
macropinocytosis, endocytosis, or an entire cell by phagocytosis. Upon antigen uptake
and activation of pattern recognition receptors (PRRs), DCs undergo maturation that
enhances their abilities to activate immune cells (Banchereau et al., 2000).
Phenotypically, mature DCs upregulate cell surface expression of maturation markers
including immunostimulatory molecules such as MHC class I and II molecules as well as
T cell co-stimulatory molecules such as CD80, CD83 and CD86 (Cresswell, 1994),
(Turley et al.), (Trombetta et al.), (Greenwald et al.). In addition, during DC maturation,
endocytosis, migration into T cell zones of lymphoid organs, and cytokine expression are
altered (Garrett et al.), (Kamath et al.). In secondary lymphoid organs such as the
spleens, mature DCs loaded with peptide antigen engage with naïve CD4+ T cells
through MHC class I or II and T cell co-stimulatory molecules. Upon the interaction with
DCs, naïve T cells differentiate into effector T cells which elicit different effector functions
including Th1, Th2, and Th17 depending on cytokines secreted from DCs and other
environmental stimuli. Regulation of DC maturation by orchestration of cytokines, growth
factors and transcription factors which turn on or off downstream signaling pathways
transduced by de-/phosphorylation of signaling molecules has been extensively studied
(Hammer and Ma). However, epigenetic control of DC maturation and function is largely
unknown. In this study, we demonstrated role of histone de-ubiquitinase, Mysm1, in DC
maturation and immunostimulatory function.
50
5.2 Materials Methods
Flow cytometric analysis. For intracellular staining, CD4+ or CD8+ T cells were
harvested from mesenteric LNs of immunized mice and cultured with gp120-loaded BM-
DCs for 8 hours at 37°C. For the final 6 hours of culture, GolgiPlug (BD Biosciences —
Pharmingen) was added to the supernatant. After surface staining with anti-CD8 or -CD4,
cells were permeabilized and stained for intracellular cytokines, as previously described
(Song et al.). For cell surface staining, cells were incubated with antibodies conjugated
with FITC, PE, or allophycocyanin (APC) in PBS containing 0.1% NaN
3
and 2% FCS.
mAbs specific for mouse CD8 (clone 53-6.7), CD11c (HL3), CD40 (3/23), CD80 (16-
10A1), CD86 (GL1), CD83, CCR7, MHC-II, and matched isotype controls were
purchased from BD Biosciences — Pharmingen or eBioscience. Stained cells were
analyzed on a FACSAria II (BD) flow cytometer and FlowJo software (TreeStar Inc.).
Quantitative RT-PCR. Quantitative RT-PCR was performed as described previously
(Sharabi et al., 2008). Total RNA from isolated cells was purified with an RNeasy Minikit
(Qiagen) according to the manufacturer’s instructions. The iScript cDNA Synthesis kit
(BioRad) was used for reverse transcription. A SYBR Green PCR kit (BioRad) was used
for quantitative real-time PCR and results were quantified with a CFX96 Real Time PCR
Detection system (BioRad).
Bone marrow-derived DC culture. Murine BM-derived DCs were prepared as
described previously (Sharabi et al., 2008). Briefly, murine BM was flushed from the hind
limbs, passed through a nylon mesh, and depleted of red blood cells with RBC lysis
buffer (BD Bioscience, 555899). After extensive washing with RPMI-1640, cells were
51
cultured with RPMI-1640 supplemented with 10% FBS, and recombinant mouse GM-
CSF/ml (20 ng/ml; PeproTech) and recombinant mouse IL-4 (20 ng/ml; PeproTech). The
supernatant was removed and replaced with fresh media containing mGM-CSF and mIL-
4 on days 2 and 4 of culture. Non-adherent granulocytes were removed after 48 hours of
culture, and fresh media was added. After 7 days of culture, more than 80% of the cells
expressed characteristic DC-specific markers as determined by FACS. Cultures were
incubated at 37° C in 5% humidified CO
2
.
Transfection and immunization of BM-DCs. Mouse BM-DCs were generated by
culturing with GM-CSF and IL-4 as described above and were pulsed with OVA protein
for 6 hours, followed by ex vivo maturation with LPS (100 ng/ml, Sigma-Aldrich). The
DCs were then injected into C57BL/6 mice (The Jackson Laboratory) via footpad. Some
of the immunized mice were treated with LPS (30 μg/mouse) or poly(I:C) (50 μg/mouse)
intraperitoneally 3 times on days 1, 2, and 3 after DC immunization.
ELISPOT ELISPOT assays of isolated splenocytes were performed as described in our
previous studies (Song et al.). DCs pulsed with ovalbumin (Sigma-Aldrich) were used for
T cell stimulation.
ELISA assays To detect cytokine production in BM-DCs and T cells, the culture
supernatants were harvested and detection performed with commercial cytokine kits
(BD).
In vitro T cell proliferation assays CD4+ T cells were purified from OT-II transgenic
mice (The Jackson Laboratory) using CD4
+
T cell isolation kit (Miltenyi Biotec). For
antigenic stimulation of TCR transgenic OT-II cells, 5 × 10
4
purified T cells and 5 ×
10
3
BM-DCs pulsed with OT-II (ISQAVHAAHAEINEAGR) peptides (Shen et al.) were
52
placed in each well of a round-bottom 96-well microtiter plate in triplicate in 200 μl RPMI
1640 medium supplemented with 10% FCS, 4 mM L-glutamine, 1 mM sodium pyruvate,
100 U/ml penicillin and streptomycin, 10 mM HEPES, and 5 μM 2-ME. After 3–6 days of
coculture, the cells in the cocultures were subjected to flow cytometric assays. To
assess T cell proliferation and cytokine production, CD11c
+
BM-DCs were depleted by
using MACS columns, activated T cells were seeded into plates in the presence of anti-
CD3 (2.5 μg/ml) for 16 hours, and culture media were then collected for ELISA analysis
of the indicated cytokines (BD Biosciences — Pharmingen). T cell proliferation was
assessed by adding 1 μCi [
3
H]TdR per well for the last 8 hours of culture and measured
using a MicroBeta scintillation counter (TopCount NXT, Packard). Triplicate
determinations were performed and are representative of repeated experiments.
In vitro migration assay In vitro migration assay was performed in Transwell plates
(Costar) of 6.5-mm diameter with 5- μm pore filters separating the upper and lower
compartments of the Transwells. A total of 500,000 cells in 100 μl assay medium (0.5%
BSA/RPMI) was added to the upper compartment, and 600 μl assay medium with or
without chemokines or culture medium of BM-DCs was added to the lower compartment
in triplicate. After 3 hours incubation, the cells that migrated to the lower compartments
were measured.
OVA-FITC uptake assay 10
5
BMDCs were incubated in 96-well plate with 25 or 50ug/ml
FITC conjugated OVA peptide in 25’c or 37’c for 1hr. Cells were washed with PBS+
0.01% NaAzide to stop further uptake and FITC-OVA uptake was analyzed by Flow
cytometry.
53
FITC painting 2 month old WT or Mysm1-/ were painted with 200ul 1% FITC in acetone
and dibutyl phthalate (1:1) on the shaved abdomen. 72hrs later, Lymph nodes were
harvested, stained with CD11c. FITC uptake by DC, migration to lymph node, and
expression of CCR7 were analyzed by flow cytometric analysis.
Statistics Groups of three to eight mice were used for statistical analysis. P values were
calculated with Student's t-test
54
5.3 Results
5.3.1 Mysm1
-/-
DCs are less mature than WT DCs
We have previously reported that Mysm1 is required for DC development (unpublished
data). Although the number of DC was significantly decreased in Mysm1
-/-
mice, we were
wondering if individual DC is functioning normally compare to WT DCs. First, we
analyzed maturation markers; MHCII, CD40, and CD86 on DC by flow cytometry (Figure
5-1A). Surprisingly, Mysm1 splenic DCs were not only reduced by number, but also,
expression of activation markers was significantly decreased. In previous study, we
showed that Mysm1 bone marrow can differentiate into DCs in vitro upon GM-CSF
stimulation which resembles inflammatory DC (iDC) whereas Mysm1
-/-
bone marrow
failed to give rise to DCs upon Flt3L stimulation. Therefore, we tested expressions of
activation markers on GM-CSF induced DC. Bone marrow cells from WT or Mysm1
-/-
mice were cultured in GM-CSF and IL-4 supplemented media for 6 days and expression
of MHCII, CD80, CD40, CD86, and CD83 was analyzed by flow cytometry (Figure 5-1B).
The results showed that expressions of all the activation markers were almost
completely abrogated in Mysm1
-/-
DCs, which is consistent with phenotypes of DCs from
in vivo.
Since we observed that Mysm1
-/-
DCs express less maturation markers which is
immature phenotype of DC, we were prompted to test function of immature DC.
Dendritic cells present as immature form in periphery until it encounters pathogens. DCs
take up pathogens by phagocytosis and migrate to secondary lymphoid organs, spleen
or lymph nodes, where DCs engage with T cell thought antigen present molecule, such
as MHCII (Lee and Iwasaki). During migration, DCs go through maturation which is
characterized by increased expression of maturation markers and pro-inflammatory
55
cytokine secretion. Therefore, the functions of immature DCs are specialized to initiating
immune response including phagocytosis and migration. To test phagocytosis efficiency
of soluble antigen, WT or Mysm1
-/-
BMDC were cultured with OVA protein conjugated
with FITC and FITC level was analyzed by flow cytometry (Figure 5-2A). The result
Figure 5-1. Decreased expression of MHCII and co-stimulatory molecules in
Mysm1-/- DCs
(A) Flow cytometric analysis of WT and Mysm1 KO splenocytes. Splenocytes were
obtained from WT and Mysm1 KO mice. Expression level of CD11c, MHCII, CD40, and
CD86 were analyzed by flow cytometry. (B) BMDCs were cultured in DC culture media
supplemented with GM-CSF and IL4 for 5 days and expression of MHCII, CD80, CD40,
CD86 and CD83 was analyzed by flow cytometry analysis.
56
Figure 5-2. Immature phenotype of Mysm1 DC
(A)10
5
BMDCs were incubated in 96-well plate with 25 or 50ug/ml FITC conjugated OVA
peptide in indicated temperature for 1hr. Cells were washed with PBS+ 0.01% NaAzide
to stop further uptake and FITC-OVA uptake was analyzed by Flow cytometry. (B)
Migration of BMDC of WT or KO in response to RANTES. Migration rate is the ratio of
the number of cells in the bottom chamber in the test case to the number of cells in the
absence of the chemokine. (C) FITC painting. 2 month old WT or Mysm1-/- (MMTV
background) were painted with 200ul 1% FITC in acetone and dibutyl phthalate (1:1) on
the abdomen. 72hrs later, Lymph nodes were harvested, stained with CD11c, and FITC
uptake and migration (C), and expression of CCR7 (D) were analyzed by FACS.
57
Figure 5-3. Maturation defect in Mysm1-/- DC
(A)BMDCs were cultured in culture media supplemented with GM-CSF and IL4 for 5
days. 0.5 million BMDCs were plated in 48-well plate and stimulated with 100ng/ml LPS.
(A) Cells were harvested at indicated time points and mRNA expression of Mysm1 was
measured by quantitative real-time PCR. mRNA expression was normalized to
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA amount (B) 24hr post
LPS stimulation, cells were harvested, stained with various maturation markers, and
analyzed by flow cytometry. Numbers presented are Mean Fluorescence Index (MFI). (C)
Fold change in MFI of indicated surface markers after LPS stimulation for 24 hours from
WT and Mysm1-/- DCs. (D) the levels of IL6, IL12p40, IL12p70, and TNF in culture
supernatant were measured by ELISA. Representative of 4 different experiments with
similar results.
58
showed that phagocytosis was increased in Mysm1
-/-
BMDCs by approximately 10% and
20% when the cells were cultured with 25ug/ml and 50ug/ml, respectively. We also
tested chemotaxis of BMDCs using RANTES to test migration capacity (Figure 5-2B).
The migration rate was increase in Mysm1
-/-
BMDC by approximately 10%. Lastly, we
examined phagocytosis and migration in vivo by FITC painting (Figure 5-2C). FITC paint
was applied on abdomen of WT or Mysm1
-/-
mice and FITC level was analyzed in lymph
node DC. FITC level was significantly increased in Mysm1
-/-
, which indicates increased
phagocytosis and migration. To support enhanced migration, a cytokine receptor CCR7
level was measured on WT and Mysm1
-/-
DC from lymph node by flow cytometry (Figure
5-2D). Consistent with previous result, the level of CCR7 was significantly increased in
Mysm1
-/-
DC. Collectively, the results suggest that DCs are less mature in the absence of
Mysm1.
5.3.2 Maturation defect in Mysm1-/- DC
Given that deletion of Mysm1 causes immature phenotypes of DC in vivo and in vitro, we
speculated Mysm1 might be required for maturation of DC. We first measure expression
level of Mysm1 in WT BMDC upon LPS stimulation at different time points (Figure 5-3A).
LPS induced expression of Mysm1 by 3 fold 12hr post stimulation which implicates that
Mysm1 may play important roles in DC maturation induced by TLR agonist, LPS. To
ascertain requirement of Mysm1 in DC maturation, we compared expression of
activation markers between WT and Mysm1
-/-
BMDC after LPS stimulation (Figure 5-3B).
Surprisingly, expression levels of activation markers tested were significantly lower in
Mysm1
-/-
BMDC than WT BMDC. In addition, we normalized increase of activation
marker expression after LPS stimulation to expression before stimulation (Figure 5-3C)
59
and we found that the fold increase of Mysm1
-/-
BMDC was significantly low in MHCII,
CD80, and CD86. Since another important change in mature DC is increased pro-
inflammatory cytokine, we examined cytokine secretion levels after LPS stimulation
(Figure 5-3D). Interestingly, there was no significant reduction of cytokine secretion from
Mysm1
-/-
BMDCs. Taken together, the results indicates that Mysm1 regulates DC
maturation upon TLR agonist stimulation, LPS, by inducing expression of activation
markers, but not cytokine production.
5.3.3 Mysm1-/- DC suppresses allogeneic T cell reaction by inducing Treg
To investigate the ability of Mysm1
-/-
DC to stimulate T cell proliferation, we performed
mixed lymphocyte reaction (MLR) cultures (Figure 5-4). CD4+ T cells were isolated from
spleens of BALB/c mice. Splenic DCs were isolated from WT or Mysm1
-/-
(C57BL/6
background) spleens using microbeads.CD4+ T cells were co-cultured with serially
diluted WT or Mysm1
-/-
splenic DC for 3 days. CD4+ T cell proliferation was increased
as DC: T cell ratio increases in both WT and Mysm1
-/-
DC cultures (Figure 5-4A). We
found that T cell proliferation was reduced by approximately half when it was cultured
with Mysm1
-/-
splenic DC in 1:1 ratio. To confirm reduced proliferation of CD4+ T cells in
Mysm1
-/-
co-culture, we analyzed cell cycle status of CD4 T cells by BrDU staining
(Figure 5-4B and C). Flow cytometry analysis showed that T cells in S phase were
significantly reduced in Mysm1-/- DC co-culture whereas frequency of apoptotic T cell
was increased. Taken together, Mysm1
-/-
DC is impaired in stimulating allo-CD4+ T cell
proliferation in MLR.
60
Figure 5-4. Mysm1 KO DC attenuates allogeneic CD4 T cell proliferation in MLR.
(A) 1x10
5
CD4+ T cells from Balb/C were cultured with serially diluted WT or Mysm1-/-
splenic DC (CD11c+) for 3 days in a 96-well plate. Proliferation of T cells was
determined by uptake of [3H] thymidine for last 16hr of the culture. The figure represent
triplicate measurements. (B) Cells were harvested and stained with BrdU and 7AAD for
cell cycle analysis. S; S phase, A; apoptotic, G; G0/G1 phase, M: M myelosis. The bar
graph represent percentage of CD4+ T cells in S phase of Apoptosis from flow
cytometric analysis.
61
Figure 5-5. Mysm1-/- DC represses Allogeneic T cell activation and induces Treg
1x10
5
CD4+ T cells from Balb/C were cultured with serially diluted WT or Mysm1-/-
splenic DC (CD11c+) for 3 days in a 96-well plate. Cells were harvested and stained for
analysis of T cell activation. Expression of CD25 (A) and CD69 (B) in CD11c- CD4+ T
cell population was analyzed by flow cytometry. (C) Expression of FoxP3 was analyzed
within CD4+CD25+ gated population.
We further investigated activation status of Allogeneic CD4+ T cells after co-culture with
WT or Mysm1
-/-
splenic DCs to test the ability of Mysm1-/- DCs in stimulating cell
activation by staining cells with CD25 or CD69 antibody (Figure 5-5). Flow cytometry
analysis results showed that allogeneic CD4+ T cells cultured with Mysm1
-/-
DCs
expressed less CD25 and CD 69 surface markers and the reduction fold decreased as
DC ratio decreases (Figure 5-5A and B). We also examined if Mysm1
-/-
induces Treg
differentiation in allogeneic T cell co-culture by CD25 and Foxp3 staining (Figure 5-5C).
62
Among CD4+ CD25+ populations, FoxP3 expression was significantly increased in
allogeneic T cells cultured with Mysm1
-/-
splenic DCs. Collectively, the results suggests
that Mysm1
-/-
splenic DCs repress allogeneic T cell activation and induce Treg
differentiation in MLR.
5.3.4 Inducing Ag-specific T cell proliferation is impaired in Mysm1-/- splenic DCs
Since one of the primary functions of mature DCs is to induce Ag-specific T cell
proliferation and activation, we next compared the ability of WT and Mysm1
-/-
to trigger
Ag-specific T cell proliferation (Figure 5-6). Splenic CD4 T cells from OTII transgenic
mice were co-cultured with WT or Mysm1
-/-
splenic DCs loaded with OVA323-339
peptide and activated with LPS. After 72 hrs, cells were harvested and proliferation was
measure by 3H incorporation (Figure 5-6A). The result showed that proliferation was
markedly reduced in response to LPS-stimulated- and non- stimulated Mysm1
-/-
splenic
DCs.
Since DC plays critical roles in determining Th1/Th2 T cell response, we next examined
whether impaired ability of Mysm1
-/-
to stimulate Ag-specific T cell response skews to
either Th1 or Th2 response by measuring T cell cytokine levels (Figure 5-6B).
Supernatant from the co-cultures were collected and assayed for IFN-r and IL4 levels by
ELISA. As shown in Fig, the level of IFNr was markedly decreased in Mysm1
-/-
co-culture
whereas secretion of IL4 did not show significant reduction, which implicates Mysm1
-/-
DC is impaired in stimulating Th1 response.
63
Figure 5-6. Mysm1 KO DCs were deficient in T cell priming.
(A) Ag specific T cell proliferation. WT or Mysm1 Mysm1-/- splenic DCs were pulsed with
OVA peptide 323-339, stimulated by LPS for 24 hrs and co-cultured with CD4+ T cells
from OTII spleen with indicated ratio for 3 days. Proliferation of T cells was determined
by uptake of [3H] thymidine for last 16hr of the culture. (B) Supernatant were harvested
from the co-culture and level of IFN and IL4 was measured by ELISA.
5.3.5 Mysm1-/- DC elicits reduced systemic Immunostimulatory potency
For evaluation of the immunostimulatory potency of Mysm1
-/-
DCs, BMDCs from WT or
Mysm1
-/-
were loaded with recombinant OVA protein and matured with LPS ex vivo
(Figure 5-7). Groups of mice were then immunized with WT, Mysm1
-/-
DCs or
64
Figure 5-7. Systemic Immunostimulatory potency is reduced in Mysm1 KO DC.
C57/BL6 mice were immunized with OVA-pulsed WT or Mysm1 KO BMDC
(1x10
6
/mouse) twice with 2 week interval. 10days after the second immunization, mice
were sacrificed and various organs were harvested for analysis. (A) Splenocytes were
pooled from each group of mice and level of IFNsecreting cells were measured by
ELISPOT assays. The bar graph represent mean of triplicate +- SD. (B and C) Draining
Lymph nodes from immunized mice were harvested and restimulated with OVA protein
for 6 hrs. Expression levels of cytokines, IFNr, IL2, and TNFa on (B) CD4 T cell and (C)
CD8 T cells were analyzed by Intracellular staining.
PBS via footpad twice with a week interval. ELISPOT assay showed that immature
Mysm1
-/-
DCs failed to elicit OVA-specific CTL response and LPS stimulated Mysm1
-/-
DC showed approximately 30% decrease in OVA-specific CTL response than did the
control WT BMDCs (Figure 5-7A). Intracellular cytokine staining (ICS) demonstrated
lower percentages of IFN- γ+, IL-2+, or TNF- α+ in both CD8+ T cells in the draining LNs
of Mysm1-/- BMDC mice compared with those in WT BMDC–immunized mice (Figure 5-
65
7B and C). Taken together, the results demonstrated that Immunization of Mysm1
-/-
BMDCs induces weaker Ag-specific immunity.
5.3.6 Deletion of Mysm1 results in global changes of gene expression in BMDC
Figure 5-8. Deletion of Mysm1 result in changes in global gene expression profile
in BMDC
RNA was isolated from LPS stimulated (24hrs) WT and Mysm1-/- BMDCs using
standard RNA extraction protocol and 10ng of RNA were amplified for labeling with Cy3
or Cy5. The labeled cRNAs were hybridized to Agilent Whole Mouse Genome Oligo
microarrays. (A)Genes that are increased or decreased by 2 fold or greater were subject
to KEGG pathway analysis for biological function. (B) Genes that had fold reduction
greater than 10 are listed.
66
Since main function of Mysm1 is histone de-ubiquitination, it is likely that absence of
Mysm1 would affect expression of many genes. Indeed, we previously showed that
Mysm1 targets and regulates transcription of different genes in various cell lineages. To
explore the mechanism by which Mysm1 regulates maturation of DC, we compared
gene expressions between WT and Mysm1
-/-
BMDC after 24hr LPS stimulation by
Microarray analysis (Figure 5-8). The results showed that over 1000 genes were
decreased by 2 fold or greater and over 140 genes were decreased by 5 fold or greater
in Mysm1
-/-
BMDC. Furthermore, we found that over 1200 genes were up-regulated by 2
fold or greater in Mysm1
-/-
BMDC. Using KEGG pathway analysis, we categorized the
genes showing change of 2 folds or greater by biological functions. The results showed
that genes regulating cytokine, chemokine signaling pathways, MAPK, JAK-STAT
pathways are down-regulated and genes regulating metabolic pathways, focal adhesion,
and complement cascade are up-regulated (Figure 5-8A). Therefore, this data suggests
that deletion of Mysm1 results in global changes in gene expression in DC by direct or
indirect mechanisms which leads to maturation defect by downregulation of genes
involved in critical maturation process, such as cytokine, chemokine, MAPK and JAK-
STAT signaling pathways.
67
5.4 Discussion
In 2011, Nobel Prize in physiology or Medicine awarded Dr. Ralph Steinman for his
discovery of dendritic cells in 1973 and described DCs as “gatekeepers of immune
system” (Steinman and Cohn). As the description implies, dendritic cells are specialized
family of antigen presenting cells (APCs) responsible for coordinating innate and innate
immunity. Central to the ‘gatekeeper’ function of dendritic cells is to surveillance stimuli
from microenvironment, such as antigens, chemokine, and cytokines and triggering a
cellular differentiation program termed ‘maturation’ which activates adoptive immune
response (Hammer and Ma). In this study, we showed, for the first time, epigenetic
control of DC maturation and adoptive immune response by a histone deubiquitinase,
Mysm1. Zhu et al. identified Mysm1 in 2007 reporting its biochemical function and tumor
suppressive role in prostate cancer cell line (Zhu et al.). In addition, critical roles of
Mysm1 in stem cell maintenance and hematopoietic lineage differentiation including B
cell, NK cell, and DCs have been recently reported by our group and others (Nijnik et al.),
(Jiang et al.). We expanded our interest of study to role of Mysm1 in dendritic cell
function.
While screening phenotypes of dendritic cells from Mysm1
-/-
mice, we observed that
expression of MHCII and maturation markers; CD40 and CD86 is significantly reduced in
steady state splenic DCs in Mysm1
-/-
. Expression of maturation markers in steady state
may be imprinted during DC development by transcription factors or cytokines,
intracellular ubiquitinligases, and other molecules (Shortman and Liu), (Hammer and
Ma). As reported previously, deletion of Mysm1 results in defect in DC development,
thus, it will be interesting to investigate the correlation between developmental defect
and expression of maturation. Also, we found that expression of MHCII was markedly
decreased not only in DC populations but also in the whole splenocytes. Therefore, we
68
surveyed mRNA expression of transcription factors regulating MHCII and found that
expression of CIITA, a master regulator of MHCII, was significantly reduced in Mysm1
-/-
splenocyte and bone marrow cells (data not shown). Therefore, we also examine
association of Mysm1 at CIITA regulatory locus by ChIP assay; however, we did not find
binding of Mysm1 at the CIITA locus tested which implicates that Mysm1 affects
expression of CIITA and MHCII by indirect mechanisms. As shown previously, Mysm1
-/-
bone marrow cells failed to differentiate into DC upon Flt3L stimulation in vitro due to
down-regulation on Flt3, however, they maintain the ability to differentiate into DC upon
GM-CSF stimulation which resembles inflammatory dendritic cells (Shortman and Naik).
Although they develop DC in normal ratio, the phenotype was significantly altered as
shown that expression of MHCII and co-stimulatory molecules; CD80, CD86, and CD40
was significantly decreased in Mysm1
-/-
GM-CSF induced DCs. Collectively, the results
indicate that Mysm1 regulates expression of MHCII and co-stimulatory molecules both in
steady-state and inflammatory DCs.
Upon recognition of pathogens thorough pattern recognition receptors (PRRs), DCs
undergo maturation. First, DC phagocytose and process antigens to peptide to present
on MHCI or MHCII molecule. Simultaneously, DCs upregulate expression of CCR7
receptors which induce migration of DC from tissues to lymph nodes. While migrating to
lymphatic organs, DCs translocate MHCII molecules from interacellular vesicles to cell
surface to more effectively present antigen peptides to naïve CD4+ T cells (Trombetta
and Mellman) and upregulate expression of co-stimulatory molecules and cytokines. To
test function of Mysm1
-/-
DC, we assessed step-wise transformation of DC upon
exposure to antigens or TLR ligands. The results indicate that the ability of Mysm1-/- DC
in antigen uptake and migration was either intact or enhanced. However, when Mysm1-/-
GM-CSF induced BMDCs were stimulated with LPS, it failed to upregulate to expression
69
of MHCII and co-stimulatory molecules. Conversely, secretion of pro-inflammatory
cytokines; IL-6, IL-12, and TNF, remained intact compare to WT BMDC. It is interesting
that deletion of Mysm1 causes defect only in up-regulation of MHCII and co-stimulatory
markers whereas other maturation processes, antigen uptake, migration, and cytokine
production, are now significantly affected by deletion of Mysm1. This data suggests that
DC maturation is a step-wise process that each step perhaps is regulated independently
via different mechanism. Therefore, selective mechanisms by which Mysm1 regulates
expression of maturation need to be investigated in the future.
Recent studies described that immature DCs elicit tolerogenic or regulatory function
(Mahnke et al.), (Kubach et al.). Since we observed immature phenotypes from Mysm1
-/-
DC, we speculate tolerogenic effect and performed MLR to examine Mysm1
-/-
DC in
stimulating allogeneic T cell proliferation. As expected, Mysm1
-/-
splenic DCs does not
stimulate T cell proliferation as effectively as WT DCs and T cells co-cultured with
Mysm1
-/-
DCs underwent apoptosis more frequently. Moreover, we found that Msysm1
-/-
DCs induced differentiation of Treg as shown by higher expression of Foxp3 in Mysm1
-/-
DC co-cultured T cells. The data implicates low level of maturation makers may attribute
to tolerogenic function of DCs and therapeutic use of Mysm1
-/-
DC for allogeneic
disorders such as graft versus host disease (GVHD).
Once DCs reach lymph node, DCs migrate into T cell area where DCs engage with
antigen specific naïve T cells. DCs provide 3 signals to induce effector T cell
differentiation. First signal is peptide antigen loaded on MHCII molecule which T cells
bind to thought TCRs. Second signal is provided by co-stimulatory molecules on DC
such as CD80 and CD 86 that stimulate CD28 on T cells. The third signal is cytokine
secreted from DCs which direct T cell differentiation into Th1, Th2, or Th17 (Constant
and Bottomly), however, the necessity of third signal in effector T cell differentiation is
70
still controversial. Since density of the peptide antigen presented and co-stimulatory
molecule is critical in stimulating T cells, we speculated that Mysm1
-/-
DC may be
defective in stimulating antigen specific T cells. As expected, Mysm1
-/-
DC did not
stimulate T cell proliferation and IFN production as effective as WT DCs. We noticed
that production of IL4 from T cells co-cultured with Mysm1
-/-
DC was intact which suggest
Mysm1
-/-
DC selectively affect Th1 T cell differentiation. In addition, immunostimulatory
potency of Mysm1
-/-
DC was reduced in vivo from immunized mice shown by deceased
of IFNr secreting T cells and reduced cytokine level from CD8+ T cells. Since Mysm1
-/-
showed decreased level of MHCII and co-stimulatory molecules but cytokine production
was intact upon TLR ligand stimulation, reduced potential to stimulate T cell
differentiation perhaps mainly accounts for decreased maturation markers. Further
studies need to be done to investigate detail mechanism how Mysm1
-/-
DC regulates
antigen specific T cell differentiation.
Histone 2A ubiquitination is very abundant histone modification that approximately 15%
of genes in mammals contain ubiquitinated histone 2A. Therefore, deletion of histone de-
ubiquitinase, Mysm1 should results in changes in expression of many genes by direct- or
indirect mechanisms. Our Microarray analysis from GM-CSF induced BMDC after 12 hr
showed that over 2000 genes are either up- or down-regulated in LPS-stimulated, GM-
CSF induced BMDC from Mysm1
-/-
. Since we found significant maturation defect from
GM-CSF induced BMDC from Mysm1
-/-
, we tested direct association of Mysm1 at loci of
a few candidate genes including CIITA and IRF8, both of which were significantly down-
regulated in Mysm1
-/-
BMDC, however, we failed to find direct association of Mysm1 with
these genes. Thus, detail molecular mechanisms by which Mysm1 regulates DC
maturation and function should be investigated. Interestingly, we found down-regulation
of many genes that are involves in DC function and maturation processes, such as
71
cytokine, chemokine signaling pathway, MAPK signaling, JAK-STAT signaling and
endocytosis from Microarray data which is consistent with the maturation defect
phenotype we observed from Mysm1
-/-
BMDC. Furthermore, among genes that are
down-regulated, many are related to infectious diseases, such as tuberculosis and
measles, and autoimmune arthritis which suggest new perspective for Mysm1
-/-
DCs for
clinical benefit.
72
Chapter 6: Concluding remarks
The physiological roles of histone de-ubiquitinase, Mysm1 in hematopoiesis have been
recently reported by a few groups, including ourselves. Our group demonstrated that
Mysm1 plays an essential role in early B cell development by de-repressing EBF1
transcription in B cell progenitors (Jiang et al., 2011). In addition, Nijnik et al. reported a
role for Mysm1 in the maintenance of BM stem cell function, in the control of oxidative
stress and genetic stability in hematopoietic progenitors, and in the development of
lymphoid and erythroid lineages (Nijnik et al., 2012). Both studies strongly implicate the
crucial functions of Mysm1 in hematopoiesis. Therefore, we expanded our Mysm1
knock-out mouse study to other hematopoietic lineages. This study demonstrated, for
the first time, epigenetic regulation of dendritic cell development and function by the
histone H2A deubiquitinase, Mysm1.
Figure 6-1 Transcriptional regulation of Flt3 by Mysm1
73
First, we demonstrated the essential role of histone H2A de-ubiquitinase Mysm1 in
steady state-DC development. Furthermore, we showed that Mysm1 regulates the
expression of cytokine receptor Flt3 by altering histone modifications at its encoding
locus and recruiting the transcription factor PU.1 to the promoter region (Fig 6-1).
Mysm1 modifies histone structure at Flt3 promoter locus that allows access of
transcriptional machinery composed of Pu.1 and other yet-unknown co-factors. We
found increased level of repressive histone modifications, H2A ubiquitination and H3K27
tri-methylations, and accumulation of Serine 2,5 phosphorylation on the RNA at the Flt3
promoter which is an indication of halt in transcription elongation. Overall, we concluded
that Mysm1 de-represses transcription of Flt3, therefore, promotes DC differentiation.
Figure 6-2. CMP lineage differentiation; Mysm1-dependent/-independent pathway.
74
Second, this study proposes a mechanism for lineage differentiation from CMP (Fig 6-2).
High level of Mysm1 was found in Flt3
+
CMP which leads to differentiation of DC from
CMP. In contrast, differentiation of monocyte, macrophage, and granulocyte, which do
not require Flt3, was Mysm1 independent. Therefore, we suggest that level of Mysm1 is
one of the lineage determining factors in CMP. Besides its distinct roles in hematopoietic
cell differentiation, Mysm1 was found to be important for HSC renewal and maintenance
(Wang et al., 2013, in press), (Nijnik et al., 2012). Further studies are clearly needed to
investigate how Mysm1 can play such diverse roles in various cell lineages and at
different stages of hematopoiesis.
Lastly, we suggested a role of Mysm1 in DC maturation. We observed Mysm1
-/-
splenic
DCs and bone marrow derived DCs have maturation defect upon TLR ligand stimulation
and their immunostimulatory potency was decreased in vitro and in vivo. Detail
molecular mechanisms by which Mysm1 regulates DC maturation and possible
therapeutic applications need to be investigated in the future.
75
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Abstract (if available)
Abstract
The mechanisms controlling the development of dendritic cells (DCs) remain incompletely understood. Using a genetic knockout mouse model, we identified the histone H2A deubiquitinase Mysm1 as a novel critical regulator in DC differentiation. Mysm1−/− mice showed a global reduction of DCs in lymphoid organs whereas development of granulocytes and macrophages were not severely affected. Hematopoietic progenitors and DC precursors were significantly decreased in Mysm1−/− mice, and common myeloid progenitors (CMP) were defective in Flt3L-induced, but not in GM-CSF-induced DC differentiation in vitro. Molecular studies demonstrated that the developmental defect of DCs in Mysm1-/- CMPs is a result of decreased Flt3 expression and that Mysm1 de-represses transcription of the Flt3 gene by directing histone modifications at the Flt3 promoter region. Two molecular mechanisms were found to be responsible for the selective role of Mysm1 in DC differentiation: the selective expression of Mysm1 in a subset of CMPs, and the different requirement of Mysm1 for Pu.1 recruitment to the Flt3 locus and both GM-CSF and M-CSF receptor loci. In conclusion, this study reveals an essential role of the histone H2A deubiquitinase Mysm1 in Flt3 transcription and DC development and provides novel mechanism of epigenetic control in DC development via histone modifications. ❧ Dendritic cell undergo maturation upon encountering pathogens which is critical for initiating adoptive immune response by upregulating co-stimulatory markers and cytokine secretion. However, epigenetic control of DC maturation has been poorly studied so far. We show herein that histone deubiquitinase, Mysm1, positively regulates DC maturation. In Mysm1-/- mice, steady state DCs and GM-CSF derived BMDC display relatively immature phenotypes including low level of MHCII and co-stimulatory molecules and increased phagocytosis and migration. In addition, Mysm1-/- BMDCs were impaired in inducing expression of MHCII and co-stimulatory molecules upon LPS stimulation. Mysm1-/- splenic DCs suppress allogeneic T cell reaction by inducing Treg differentiation. Moreover, we found that antigen specific immune stimulatory potency was reduced in Mysm1-/- DCs. Microarray data indicates that deletion of Mysm1 results in global changes of gene expression in BMDC many of which were related to cytokine/chemokine regulatory pathways. Collectively, this study suggests a novel mechanism for regulation of DC maturation by epigenetic control.
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Won, Hae Jung
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Mysm1, a histone de-ubiquitinase, is essential for dendritic cell development and function
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Keck School of Medicine
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Doctor of Philosophy
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Molecular Microbiology and Immunology
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11/18/2013
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10/15/2013
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dendritic cell,dendritic cell function,epigenetics,Hematopoiesis,histone H2A de-ubiquitinase,MYSM1,OAI-PMH Harvest
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Chen, Si-Yi (
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MYSM1