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Interaction of epigenetics and SMAD signaling in stem cells and diseases

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Interaction of epigenetics and SMAD signaling in stem cells and diseases

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INTERACTION OF EPIGENETICS AND SMAD SIGNALING
IN STEM CELLS AND DISEASES

by

Cunye Qu

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CRANIO-FACIAL BIOLOGY)

December 2014

Copyright 2014 Cunye Qu

ii
Acknowledgements

Upon finishing my current study, I would like to express most sincere appreciation to
my Ph.D. advisor and dissertation committee chair, Dr. Songtao Shi for his continuous
support and tremendous endeavor in guiding my graduate study throughout all the
time. His altruistic effort was essential for my academic career development, including,
but not limited to, discovering important questions, developing scientific concept,
managing sophisticated research project, and preparing manuscript and proposal. I
will always be grateful for his role as a scientific model in my academic career.
I would also appreciate the enormous help from my Graduate Committee, Dr Yang
Chai, Dr. Qi-Long Ying, Dr. Michael Paine, and Dr Wei Shi. Your suggestions always
shaped the research as an impeccable study and your encouragement and support
were critical for me to explore untouched field and decipher exciting findings.
My more thanks are given to Dr. Ruili Yang, Dr. Haiyan Qin, Dr. Chider Chen, Dr.
Kentaro Akiyama, and Dr. Yi Liu for their excellent teamwork in the lab, providing
timely technical support, initiating knowledgeable discussion, and relaying scientific
enthusiasm. The experience of working with you is an invaluable asset in my future
academic life.
I particularly thank all the current and previous members of Dr. Shi’s lab and
colleagues in the Center for Craniofacial Molecular Biology for constructing such an
iii
inspiring scientific atmosphere, which always benefits me for achieving more and
more academic goals.
Special gratitude is given to my family in China and friends in the United States.
Thank you for unconditional support and encouragement, which is indispensable for
development of my academic career.

iv
Table of Contents
Acknowledgements ii
List of Tables vi
List of Figures vii
List of Symbols & Abbreviations ix
Abstract xii
Chapter 1: Introduction 1
1.1 TGFβ Signaling 1
1.2 TGFβ Signaling in Development and Stem Cells 2
1.3 TGFβ Signaling in Diseases
1.4 Interaction of Epigenetics and TGFβ Signaling 12
1.5 Summary of the Project 16

Chapter 2: BCOR Orchestrates Human ESC Pluripotency via
Regulating SMAD/Nanog Aixs 18
2.1 Introduction 18
2.2 Materials and methods 20
2.2.1 Cell culture 20
2.2.2 Immunofluorescence and immunohistochemistry 21
2.2.3 Microarray 22
2.2.4 Quantitative PCR 22
2.2.5 Western blot 22
2.2.6 Alkaline Phosphatase 23
2.2.7 DNA sequencing and RFLP 23
2.2.8 Teratoma 24
2.2.9 Gene knockdown and overexpression 24
2.2.10 BrdU/CFU Assay 25
2.2.11 Flow Cytometry 25
2.2.12 Luciferase Assay 25
2.2.13 ChIP-PCR 26
2.2.14 Statistical Analysis 27
2.3 Results 28
2.3.1 Generation of iPS cells from Bcor mutant cells 28
2.3.2 Bcor mutant iPSCs have altered self-renewal and differentiation 32
2.3.3 Bcor knockdown in normal pluripotent stem
cells mimics the phenotypes of OFCD iPSCs 37
2.3.4 OFCD iPSCs have elevated Nanog expression 40
2.3.5 BCOR regulates Nanog expression via variant PRC1 complex 44
v
2.3.6 SMAD signaling antagonizes BCOR and PRC1 complex
at Nanog promoter 48
2.4 Dicussion 52

Chapter 3: Epigenetic Regulation of TGF  Signaling in
Mesenchymal Stem Cells of Ossifying Fibroma 56
3.1 Introduction 56
3.2 Materials and methods 58
3.2.1 Animals 58
3.2.2 Reagents and antibodies 58
3.2.3 Primary cell cultures 59
3.2.4 Subcutaneous implantation of MSCs 60
3.2.5 Isolation of single colony clusters 60
3.2.6 Single-cell serial implantation 61
3.2.7 In vitro differentiation 62
3.2.8 Histological and immunohistochemical analysis 63
3.2.9 Flow cytometric analysis 64
3.2.10 Small interfering RNA 64
3.2.11 BrdU staining 65
3.2.12 Western blot 65
3.2.13 Microarray 65
3.2.14 Quantitative PCR 66
3.2.15 ELISA 66
3.2.16 Luciferase assay 67
3.2.17 ChIP-PCR 67
2.2.18 Statistical analysis 68
2.2.19 Accession number 68
3.3 Results 71
3.3.1 Benign ossifying fibroma (OF) contains
mesenchymal stem cells (OFMSCs) 71
3.3.2 TGFβ signaling is highly activated in OFMSCs 78
3.3.3 TGFβ inhibits BMP signaling to reduce bone formation and
activates Notch signaling to enhance stromal tissue growth 86
3.3.4 Upregulation of TSP1 contributes to activation of TGFβ
signaling in OFMSCs 91
3.3.5 Histone demethylase JHDM1D-mediated TSP1/TGFβ/SMAD3
autocrine loop contributes to TGFβ activation 93
3.3.6 Establishment of TSP1/TGFβ/SMAD3 autocrine loop
converts normal MSCs to OF-like MSCs 99
3.4 Discussion 102

Chaptor 4: Conclusions 107
Bibliography 110
vi
List of Tables

Table 1. Differentially expressed genes between OFMSCs and JMSCs 69
Table 2. PCR primers 70

vii
List of Figures

Figure 1.1 Canonical and non-canonical TGF  signaling 2
Figure 2.1 Generation of iPS cells from Bcor mutant cells 30
Figure S2.1 Generation of iPS cells from Bcor mutant cells 31
Figure 2.2 Bcor mutant iPSCs have altered self-renewal and differentiation 34
Figure S2.2 Bcor mutant iPSCs have altered self-renewal and dfferentiation 36
Figure 2.3 Bcor knockdown in normal pluripotent stem cells
mimics the phenotypes of OFCD iPSCs 38

Figure S2.3 Bcor knockdown in normal pluripotent stem cells
mimics the phenotypes of OFCD iPSCs 39
Figure 2.4 OFCD iPSCs have elevated Nanog expression 42
Figure S2.4 OFCD iPSCs have elevated Nanog expression 43
Figure 2.5 BCOR regulates Nanog expression via variant PRC1 complex 46
Figure S2.5 BCOR regulates Nanog expression via variant PRC1 complex 47
Figure 2.6 SMAD signaling antagonizes BCOR and PRC1
complex at Nanog promoter 50

Figure S2.6 SMAD signaling antagonizes BCOR and PRC1
complex at Nanog promoter 51

Figure 3.1 Benign ossifying fibroma (OF) contains mesenchymal
stem cells (OFMSCs) 74
Figure S3.1 Benign ossifying fibroma (OF) contains mesenchymal
stem cells (OFMSCs) 75

Figure 3.2 TGFβ signaling is highly activated in OFMSCs 81
Figure S3.2 TGFβ signaling is highly activated in OFMSCs 83
Figure 3.3 TGFβ inhibits BMP signaling to reduce bone formation and
viii
activates Notch signaling to enhance stromal tissue growth 88

Figure S3.3 TGFβ inhibits BMP signaling to reduce bone formation and
activates Notch signaling to enhance stromal tissue growth 84

Figure 3.4 Upregulation of TSP1 contributes to activation of TGFβ
signaling in OFMSCs 92

Figure S3.4 Upregulation of TSP1 contributes to activation of TGFβ
signaling in OFMSCs 90

Figure 3.5 Histone demethylase JHDM1D-mediated TSP1/TGFβ/SMAD3
autocrine loop contributes to TGFβ activation 96

Figure S3.5 Histone demethylase JHDM1D-mediated TSP1/TGFβ/SMAD3
autocrine loop contributes to TGFβ activation 98

Figure 3.6 Establishment of TSP1/TGFβ/SMAD3 autocrine loop converts
normal MSCs to OF-like MSCs 100

Figure S3.6 Establishment of TSP1/TGFβ/SMAD3 autocrine loop converts
normal MSCs to OF-like MSCs 101

ix

List of Symbols & Abbreviations

ASC Adult Stem Cell
BCOR BCL6 Corepressor
bFGF basic Fibroblast Growth Factor
BMP Bone Morhpogenic Protein
CDK Cyclin Dependent Kinase
CFU Colony Froming Unit
ChIP Chromatin Immunoprecipitation
DAPI 4',6-Diamidino-2-phenylindole
DMEM Dulbecco’s Modified Eagle’s Medium
DNMT DNA Methyltransferase
EB Embryoid Body
ECM Extracellular Matrix
EMT Epithelium to Mesenchyme Transition
ELISA Enzyme-Linked Immunosorbent Assay
ESC Embryonic Stem Cells
FBS Fetal Bovine Serum
HA/TCP Hydroxyapatite/Tricalcium Phosphate
HFSC Hair Follicle Stem Cell
HSC Hematopoietic Stem Cell
x
ICM Inner Cell Mass
IKK IκB Kinase
iPSC induced Pluripotent Stem Cell
JMSC Jawbone Mesenchymal Stem Cell
KSR Knockout Serum Replacement
LIF Leukemia Inhibitory Factor
LAP Latency Associated Peptide
LPL Lipoprotein Lipase
MAPK Mitogen-Activated Protein Kinase
MSC Mesenchymal Stem Cells
MuSC Muscle Stem Cell
-MEM alpha Minimum Essential Medium
NFκB Nuclear Factor kappa B
NK Natural Killer cell
OF Ossifying Fibroma
OFCD Oculofaciocardiodental Syndrome
OFMSC Ossifying Fibroma Mesenchymal Stem Cells
PAK2 P21 Activiated Kinase 2
PCR Polymerase Chain Reaction
PDCD4 Programmed Cell Death 4
PI3K Phopho-Inositide 3 Kinase
PPAR 2 Peroxisome Proliferator-Activated Receptor gamma 2
xi
SIP1 Smad Interacting Protein 1
TAK1 TGFβ Activated Kinease 1
TGFβ    Transforming Growth Factor beta
TIF1  Transcription Intermediate Factor 1 gamma 
TRAF6 TNF Receptor Associated Factor 6
TSP1 Thrombospondin 1
TRIM33 Tripartite Motif-containing 33

xii
Abstract

TGF  signaling is pleiotrophic pathway that plays crucial rols in both stem cell
behaviors and occurrence of diseases. Epigenetic modification of the components in
TGF  signaling affects the activity of the signaling, thereby influencing properties of
stem cells and contributing to the pathological process of diseases.
Loss-of-function mutation of BCL6 corepressor (BCOR) causes
oculofaciocardiodental (OFCD) syndrome in female patients, but males with the same
mutation are embryonic lethal, indicating the importance of BCOR in embryo
development. BCOR is reported to exert its suppressive function by association with
components of polycomb repressive complex1 (PRC1), but it is still largely unknow
that how BCOR-mediated epigenetic process regulates the stem cell fate and gives
rise to the symptoms in OFCD patients.
In Chapter 2 of this study, we investigated the interaction of epigenetics and TGF 
signaling by reprogramming Bcor mutant mesenchymal stem cells (MSCs) into
induced pluripotent stem cells (iPSCs). Both regulations by signaling and epigenetics
are essential for embryonic stem cell (ESC), but it remain elusive how the epigenetic
regulation and signaling molecules act in concert to maintain pluripotency. We
showed that iPSCs derived from OFCD patient and Bcor-knockdown ESCs have
altered self-renewal and differentiation due to elevated Nanog expression.
Mechanistically, Bcor, in conjunction with variant polycomb repressive complex 1
(PRC1), inhibited Nanog expression by H2AK119 monoubiquitination, which was
xiii
antagonized by the SMAD signaling to achieve equilibrium of NANOG heterogeneity.
In addition, upregulated NANOG resulted in deficiency of neural crest lineage
determination due to repressed Snail and Twist.
Collectively, this study demonstrated the role of BCOR in the lineage determination of
human pluripotent stem cells. The significance of the study is to decipher a
mechanism by which BCOR integrated environment cues of signaling and epigenetic
process to achieve orchestrated gene expression in pluripotency. The role of BCOR
showed epigenetic regulation of signaling pathway as an integral component of
network of ESC pluripotency.
Abnormal stem cell function makes a known contribution to many malignant tumors,
but the role of stem cells in benign tumors is not well understood. In Chaptor 3 of this
study, we utilized ossifying fibroma as a model to show the epigenetic regulation of
TGF  signaling in occurrence of disease. We found that ossifying fibroma (OF)
contains a stem cell population that resembled mesenchymal stem cells (OFMSCs)
and was capable of generating OF-like tumor xenografts. Mechanistically, OFMSCs
showed enhanced TGFβ signaling that induced aberrant proliferation and deficient
osteogenesis via Notch and BMP signaling pathways, respectively. The elevated
TGFβ activity was tightly controlled by JHDM1D-mediated epigenetic regulation of
thrombospondin-1 (TSP1), forming a JHDM1D/TSP1/TGFβ/SMAD3 autocrine loop.
Inhibition of TGFβ signaling in OFMSCs can rescue their abnormal osteogenic
differentiation and elevated proliferation rate. Furthermore, chronic activation of TGFβ
converted normal MSCs into OF-like MSCs via establishment of this
xiv
JHDM1D/TSP1/TGFβ/SMAD3 autocrine loop.
These results revealed that the role of epigenetic regulation of TGFβ signaling in
MSCs governed the benign tumor phenotype in OF. The significance of the study is to
identify a novel mechanism of conversion between normal stem cells and tumor stem
cells and highlight TGFβ signaling as a candidate therapeutic target for the disease.
In summary, this study demonstrated that interaction between epigenetic process and
signaling transduction is crucial in regulation of stem cell property and disease
progress, and deciphered the mechanism of how epigenetic modulation affected the
output of TGFβ/SMAD signaling, shedding light on development of targeted therapy.

1
Chapter 1: Introduction

1.1 TGFβ Signaling

Characterization and understanding of biological implication of transforming growth
factor-β (TGFβ) signaling has expanded since it was discovered in the early1980s.
TGFβ proteins are synthesized as propeptide precursors containing a prodomain,
latency-associated peptide (LAP), and the mature domain and are secreted in an
inactive form. The bioavailability of active TGFβ is dependent on the proteolytic
processing that releases cytokines in extracellular matrix (ECM). In canonical TGFβ
signaling, active TGFβ binds to cell surface type II serine/threonine kinase receptors
(TGFβRII) and the latter phosphorylates type I receptor (TGFβRI), forming
heteromeric complexes, which phosphorylates SMAD2 and SMAD3. Upon
phosphorylation, SMADs accumulate in the nucleus, form transcriptional complexes
with SMAD4 and other transcription factors, and regulate gene transcription. In
addition to the canonical SMAD pathway, TGFβ is capable of utilizing
SMAD-independent pathways for regulation of gene expression, such as pathways
involving mitogen-activated protein kinase (MAPK), nuclear factor κB (NFκB),
Rho-like GTPase and phophoinositide 3-kinase (PI3K)/Akt (Figure 1).
By adopting canonical or non-canonical pathways, TGFβ plays diverse roles in
development, stem cell hometostasis and diseases. Both pathways mediated by
TGFβ signaling are integral components for intercellular communication and cell
2
interaction with their microenvironement, contributing to normal development and
diseases.

Figure 1. Canonical and non-canonical TGFβ signaling. In canonical TGFβ
Signaling, the ligand transmits signaling by nuclear translocation of phosphorylated
SMAD2/3/4 complex, which drives downstream gene expression. In non-canonical
TGFβ Signaling, the ligand relay the signals through other factors, such as MAPK,
PI3K (Adapted from Akhurst R.J. et al., 2011).

1.2 TGFβ Signaling in Development and Stem Cells

TGFβ signaling made its debut with the evolution of multicellular organisms, indicating
its role in diverse and complex cellular environment in development and regulation of
3
homeostasis. TGFβ signaling can trigger a remarkable array of downstream signals
and cell response and therefore, TGFβ, as well as its superfamily members, is potent
morphogens during development. Both canonical and non-canonical TGFβ signalings
contribute to embryo development.
Loss of SMAD2, which mediates canonical TGFβ signaling, results in missing
mesoderm, fails to form organized egg cylinder and heterozygous embryos have
severe defects in left-right assymmetry and craniofacial development, which indicated
both the presence of active TGFβ signaling and dosage of SMAD2 is critical for
development (Nomura and Li, 1998).
Important development genes have SMAD binding elements at their promoter region
and activation of canonical TGFβ pathway can drive SMAD complex binding and
subsequent gene expression to initiate the lineage determination. In craniofacial
development, CTGF mediates SMAD-dependent TGFβ signaling to regulate
mesenchymal cell proliferation during palate formation (Parada et al., 2013).
SMAD-dependent TGFβ signaling further control myogenic differentiation and
myoblast fusion during tongue development by regulation of Fgf6 and Fgfr expression
(Han et al., 2012). In dental development, SMAD4 dependent signaling contribute to
odontoblast differentiation and dentin formation by maintaining expression of Dkk1
and Sfrp1, which regulate appropriate Wnt signaling (Grossmann et al., 2011).
Proliferation and maintenance of dental epithelium stem cells are also regulated by
TGFβ signaling driven expression of Fgf10 (Zhao et al., 2011).
4
During T cell development, activated SMAD2 by TGFβ signaling enhances
transcriptional factor E2A binding at Foxp3 promoter to drive the gene expression and
alternatively induce Id3 expression to compete GATA3 mediated transcriptional
inhibition of Foxp3 promoter (Maruyama et al., 2011). In addition, canonical activation
of TGFβ signaling transcriptionally repress IFN , thus suppressing NK cell function
(Laouar et al., 2005).
Furthermore, TGFβ signaling mediated epithelium to mesenchyme transition (EMT) is
essential to a vast array of developmental processes, which is exemplified by invasion
of heart cushion by endocardial cells, contributing to the formation of heart valves
(Mercado-Pimentel and Runyan, 2007). The ligand of TGFβ1,2,3 are expressed in a
time and space specific manner and application of neutralizing antibody demonstrated
that TGFβ2 is required for initiation of EMT, while TGFβ3 affect invasion and migration
in a sequential process, which is in line with the expression of ligands.
Although majority of TGFβ signaling route is mediated by SMAD complex comprising
SMAD2/3/4, non-canonical TGFβ pathway also plays important role in development.
Because SMAD4, as a common mediator, can interact with both phosphorylated
SMAD2/3 from TGFβ signaling, and phosphorylated SMAD1/5/8 from BMP signaling,
SMAD4 is also called co-SMAD. Conditional inactivation of SMAD4 in oral epithelium
gives rise to very milder phenotype and perturbation of p38 signaling imposes no
effect in these tissues either. However, when combined inactivation of SMAD4 and
p38 induces dramatic phenotype of cleft palate, similar to the receptor knockout,
demonstrating that ectodermal SMAD4 and p38/MAPK are functionally redundant in
5
TGFβ signaling during tooth and palate development (Xu et al., 2008b). Independent
of SMAD4, TGFβ signaling can drive SMAD2/3 to associate with IKK to induce
keratinocyte differentiation and epidermal differentiation (Descargues et al., 2008). In
the same vein, TGFβ signaling, in the absence of involvement of SMAD4, regulates
miR-21 maturation, which downreguates PDCD4 (programmed cell death4)(Davis et
al., 2008). Decreased PDCD4 will induce a contractile phenotype of human vascular
smooth muscle cells. In competition with SMAD4, TRIM33, also known as
TIF1  ), can interact with receptor activated
SMAD2/3 to regulate erythroid differentiation (He et al., 2006).
In TGFβ signaling, R-SMAD refers to SMAD2/3, which are directly phosphorylated by
TGFβ receptor. TGFβ signaling is also intimately associated with development by
R-SMAD independent pathway. SMAD4 can synergistically cooperate with IRF6 to
regulate the fate of medial edge epithelium (Iwata et al., 2013b). Perturbation of the
interaction results in compromised p21 expression and persistence of medial edge
epithelium. TRAF6 (TNF receptor-associated factor 6) can be directly activated by
TGFβ signaling and then activate TAK1 (TGFβ activated kinase-1) by lysine 63 linked
polyubiquitination, whereas phosphorylation of SMAD2 is not mediated by TRAF6
(Sorrentino et al., 2008). Activation of TAK1 induces a variety of cell context
dependent effects, including differentiation and apoptosis in heart epicardium(Takatsu
et al., 2000; Zhang et al., 2000). TRAF6 is also required for the SMAD-independent
activation of JNK and p38, which is important for inducing apoptosis during normal
physiological processes (Shim et al., 2005).
6
TGFβ signaling is well known for its growth inhibition on epithelial tissue and growth
promotion on mesenchymal tissue. A study indicated non-canonical TGFβ pathway
activated p21-activated kinase2 (PAK2) uniquely in mesenchymal cells, rather than
epithelial cells. Mechanistically, epithelial enriched ERBIN mediated complex
formation of ERBIN/MERLIN/PAK2 with inactivated PAK2. In this scenario, the cells
decide use non-canonical TGFβ pathway because of presence or absence of the
mediator protein ERBIN.
TGFβ even transduces the signaling without the involvement of TβRI. Activated T RII
by ligand can directly phosphorylate PAR6, which interact with E3 ubiquitin ligase
SMURF1 to downregulate RHOA1. In consequence, RHOA1 decrease results in loss
of tight junctions, leading to loss of epithelial cell polarity and initiation of epithelium to
mesenchyme transition (Ozdamar et al., 2005).
Given the critical role of TGFβ signaling in development, it is not surprising that
behaviors of stem cells are also subjected to regulation of the signaling. Stem cells
have the ability to self-renew and differentiate to maintain tissue remodeling and
wound repair, in which TGFβ signaling is an integral component to couple stem cell
function and environmental cues from niche. Regulation by TGFβ signaling is crucial
for both embryonic stem cells (ESCs) and adult stem cells (ASCs).
ESCs are pluripotent stem cells from inner cell mass (ICM) of blastocyst embryos with
the potential to give rise to a myriad of cell types. TGFβ signaling is intrinsically
involved in self-renewal and differentiation of ESCs, contributing to maintenance of
7
pluripotency or lineage determination dependent on the pathways and transcription
factors that interact with TGFβ signaling. Nodal/Activin signaling, a member of TGFβ
superfamily, was reported to contribute to nuclear translocation of phosphorylated
SMAD2/3 and subsequent transactivation of the key pluripotency gene Nanog (Xu et
al., 2008a). In culture with component defined medium, Activin and bFGF on a
fibronectin coated surface can support long term in vitro maintenance and
propagation of hESCs (Vallier et al., 2005). In analog, treatment of hESC by TGFβ
inhibitor SB431542 decreased the expression of Nanog (James et al., 2005). SMAD3,
in response to Nodal/Activin signaling, also co-occupy genome elements with OCT4,
forming physical complex with OCT4 and regulating the downstream targets
synergistically. Similarly, in the scenario of OCT4 deficiency, SMAD3 occupancy is
significantly reduced and loses response to Nodal/Activin stimulation (Mullen et al.,
2011).
Nodal/Activin signaling also promotes pluripotent stem cells differentiate to definitive
mesoderm and endoderm (Arnold and Robertson, 2009). On the contrary, dual
inhibition of SMAD signaling by SB431542 and Noggin, a BMP inhibitor, is capable of
sufficient induction of neuroectodermal lineage in hESCs (Chambers et al., 2009).
SMAD-interacting protein1 (SIP1) was identified as an important mediator of
Nodal/Activin induced differentiation (Chng et al., 2010). Elevated SIP1 expression,
upregulated upon treatment of SB431542, further suppresses residual Nodal/Activin
signaling, thereby dampening mesoderm inductive effect from Activin and BMP
signaling, favoring the “default” neuroectodermal pathway.
8
TGFβ signaling is also an integral component in regulation of behaviors of
tissue-specific stem cells. By interacting with specific master transcription factors,
SMAD complex demonstrated sequence specific binding. For example, SMAD3
occupies genome with OCT4 in embryonic stem cells, whereas it binds genome
elements with MYOD in myotubes, and PU.1 in pro-B cells, which complicates the
role of TGFβ signaling in regulation of development process (Mullen et al., 2011).
In hemaptopoietic stem cells (HSCs), TGFβ signaling enhances the transcription of
cyclin-dependent kinase (CDK) inhibitor P57 and suppresses PI3K/Akt pathway,
thereby preventing HSCs re-entry into cell cycle and maintaining quiescence
(Fortunel et al., 2000; Yamazaki et al., 2009). Interruption of TGFβ signaling by
deletion of TβRII results in decreased phosphorylation of SMAD2/3, increased cell
cycle and loss of long-term repopulation capacity of HSCs (Yamazaki et al., 2011). In
hair follicle stem cells (HFSCs), TGFβ signaling antagonized bone morphogenic
porotein (BMP) signaling to stimulate HFSC proliferation by counteracting quiescence
in the niche (Yamazaki et al., 2009). Consistently, blockage of TGFβ signaling by
deletion of TβRII shows elevated pSMAD1 and delayed hair cycle. With aging
progresses, signaling in muscle stem cells (MuSCs) demonstrates a shift form active
Notch to TGFβ signaling and inhibition of TGFβ signaling rescues the regenerative
capacity of MuSC by controlling CDK inhibitors (Carlson et al., 2008). It was also
demonstrated that TGFβ released from bone matrix coupled the bone formation and
resorption by orchestrating migration of mesenchymal stem cells (MSCs) (Tang et al.,
2009).
9
Collectively, TGFβ signaling contributes to homeostasis of embryo development and
regulates the harmony of stem cell self-renewal and differentiation.
1.3 TGFβ signaling in Disease
In light of the fact that TGFβ and its superfamily members regulate adult cells to
contribute to tissue homeostasis and regeneration, perturbation of the signaling can
make these processes go awry and lead to a staggering array of disease
development, such as cancers. Pleiotropic roles of TGFβ are involved in these
processes, including canonical and non-canonical pathways.
TGFβ signaling typically prevents incipient tumorigenesis by arresting proliferation,
differentiation, adhesion, as well as microenvironment, but malignant evolution of
tumor gradually eludes the suppression and abuses the pathway for their advantage
for tumor survival. Loss of SMAD3 has been indicated to contribute to gastric cancer
and T cell lymphoblastic leukemia (Levy and Hill, 2006). In contrast to SMAD2/3,
SMAD4 is a frequent mutation target in different cancer type, including at least of half
of pancreatic carcinomas, sporadic colorectal tumor with microsatellite instability, as
well as esophageal tumors, close to mutations in KRAS, p53 (Jaffee et al., 2002;
Sjoblom et al., 2006). Tumorigenesis upon loss of functional SMAD4 indicates the
tumor suppressive role of canonical TGFβ signaling, which is confirmed by
spontaneous tumor formation upon tissue-specific SMAD4 deletion in mouse liver or
pancreas (Bardeesy et al., 2006; Wang et al., 2005).
However, at advanced stage of tumor development, TGFβ signaling can uniquely
10
drive gene expression, transcriptionally or epigenetically, that is not inducible in
normal cells. By canonical pathway mediated SMAD complex, elevated TGFβ
signaling in human glioblastoma induced expression of leukemia inhibitory factor (LIF),
which activate JAK-STAT pathway and subsequent glioma initiation and recurrence
(Penuelas et al., 2009). Similarly, high elevation of canonical TGFβ signaling activity
confers poor prognosis of glioma patients by promoting cell proliferation as a result of
demethylation of Pdgf-b gene (Bruna et al., 2007). Similar to the role of canonical
TGFβ signaling in EMT during development, gene expression analysis of breast
cancer indicates a role of TGFβ induced EMT and treatment of the cells with TGFβ
signaling inhibitor confers the cells with a epithelial phenotype (Shipitsin et al., 2007).
Molecularly, canonical SMAD signaling induced HMGA2 (high mobility group A2)
expression, which drives downstream target of Snail, Slug, and Twist to contribute to
EMT (Thuault et al., 2006).
In addition to involvement in tumor development, canonical TGFβ signaling negatively
regulates epithelial growth, as indicated by faster skin wound healing with rapid rate of
keratinocyte proliferation and migration in SMAD3 null mice (Ashcroft et al., 1999).
Non-canonical TGFβ pathway is also involved in disease progress especially when
components of canonical TGFβ pathway are mutated, such as TGFβ receptor
mutation in Loeys-Dietz syndrome, which elicits cleft palate and other craniofacial
defects. Loss of Tgfbr2 in mouse neural crest cells gives rise to elevated TGFβ2 and
TGFβ receptor type III, resulting in SMAD independent TRAF6/TAK/p38 signaling
pathway. Haploinsufficiency of Alk5 or Tak1 rescues the craniofacial deformities,
11
suggesting the non-canonical TGFβ pathway contributes to the phenotypes in Tgfbr2
mutant mice (Iwata et al., 2012). Tissue restricted ablation of Tgfbr2 in cranial neural
crest gives rise to reduced lipolysis and spontaneous accumulation of lipid droplets in
palatal mesenchymal cells, which can be rescued by inhibition of p38 signaling,
indicating the role of noncanonical TGFβ signaling in lipid metabolism (Iwata et al.,
2014).
During development of Marfan syndrome, both ERK1/2 and SMAD2 are activated,
which can be inhibited by antagonist of TGFβ (Holm et al., 2011). However, only
selective inhibition of ERK1/2 ameliorates aortic growth, whereas SMAD4 deletion
exacerbates the phenotype with premature mouse death by inducing activation of
JNK. Application of JNK antagonist can rescue the aortic growth, indicating
noncanonical TGFβ signaling is a major driver for aortic phenotype in the disease.
Specific deletion of Tak1 in cranial neural crest tissues gives rise to failed palate
elevation and cleft palate, which further contributes to malformed tongue and
micrognathia. Mechanistically, loss of Tak1 releases the repression of Fgf10
expression by p38 (Song et al., 2013). Ablation of Tgfbr2 also cause microglossia due
to activation of cytoplasmic and nuclear ABL1 cascade, leading to failure of
CNC-derived cell proliferation and differentiation, as well as reduction of myogenic
cells (Iwata et al., 2013a).
In development of prostate cancers, long term function of TGFβ signaling induces
rearrangement of actin filament system and subsequent uncontrolled proliferation,
12
invasion and metastatic properties, which is mediated by activities of RHO GTPase
CDC42 and RHOA and their downstream regulation of P38 (Edlund et al., 2002).
Therefore, a plethora of pathological processes are associated with aberrant TGFβ
signaling and the interpretation of the role of the signaling in disease will provide basis
for developing targeted therapies.

1.4 Epigenetic Regulation of TGFβ Signaling

Epigenetic regulation of gene expression, the inheritance of changes without
alteration of DNA sequence, is critical for the hemeostasis of stem cells, including
DNA methylation, histone modification, nucleosome positioning, and microRNA
regulation. Disturbation of the processes gave rise to a variety of diseases, especially
when interacting with the components TGFβ signaling.
The methylation of the 5-carbon on cytosine residues (5mC) in CpG dinucleotides
was the first described covalent modification of DNA and is perhaps the most
extensively characterized modification of chromatin (Arnold and Robertson, 2009).
Three active DNA methyltransferases (DNMTs) have been identified in eukaryotes.
DNMT1 is a maintenance methyltransferase that recognizes hemimethylated DNA
generated during DNA replication and then methylates newly synthesized CpG
dinucleotides. Conversely, DNMT3a and DNMT3b, although also capable of
methylating hemimethylated DNA, function primarily as de novo methyltransferases to
13
establish DNA methylation (Sjoblom et al., 2006). DNA methylation of genes is
frequently associated with inhibited gene expression, especially at the promoter
region. For example, high elevation of canonical TGFβ signaling activity confers poor
prognosis of glioma patients and promotes proliferation of primary cells from glioma
patients due to induction of Pdgf-b gene (Bruna et al., 2007). Unmethylated status of
Pdgf-b gene renders the gene inducible by TGFβ signaling, dictating TGFβ as an
oncogenic factor.
A variety of histone modifications, including histone methylation, histone acetylation
and histone phosphorylation, also demonstrate functional influences on the regulation
of transcription. Histones are methylated on the side chains of arginine, lysine, and
histidine residues. Lysines may be mono-, di-, or tri-methylated, and arginine residues
may be symmetrically or asymmetrically methylated. The best-characterized sites of
histone methylation are those that occur on lysine residues including H3K4, H3K9,
H3K27, H3K36, H3K79, and H4K20. Some of these (H3K4, H3K36, and H3K79) are
often associated with active genes in euchromatin, whereas others (H3K9, H3K27,
and H4K20) are associated with heterochromatic regions of the genome (Barski et al.,
2007). Histone modification is involved in regulation of TGF  signaling in both
physical and pathological contexts.
In primary breast cancer cells, epigenetic-based gene suppression of TGF  pathway
including TGF 2, TGF R1, TGF R2 and TSP1 were observed, which was associated
with histone H3K9 methylation and deacetylation and loss of H3K27 trimethylation
rather than DNA hypermethylation (Hinshelwood et al., 2007). These changes
14
collectively lead to the disruption of coordinated networks on intercellular
communication and cause a fundamental change in cellular behavior that affects
processes such as proliferation, differentiation, and apoptosis, with progressive
dysregulation and acquisition of malignant phenotype.
The N-acetylation of lysine residues is a major histone modification involved in
transcription, chromatin structure, and DNA repair. Acetylation neutralizes lysine's
positive charge and may consequently weaken the electrostatic interaction between
histones and negatively charged DNA. For this reason, histone acetylation is often
associated with a looser chromatin conformation. Several viruses have been reported
to target the TGF  pathway, (Sjoblom et al., 2006), such as Kaposi sarcoma
herpesvirus (KSHV) encoded latency-associated nuclear antigen (LANA) leads to the
methylation of T RII promoter and deacetylation of proximal histones, which results in
silencing of the gene expression and blocking TGF  signaling.
TGFβ signaling also recognizes suppressive histone mark H3K9me3 and H3K18ac to
initiate expression of Gsc and Mixl1 in mesoderm differentiation (Xi et al., 2011).
Activation of SMAD signaling induces formation of TRIM33-SMAD2/3 complex, which
binds at H3K9me3 and dislocates the chromatin-compacting factor HP1γ, making the
gene accessible to SMAD2/3/4 complex and RNA polymerase recruitment.
Canonical activation of TGFβ signaling can either activate or repress gene
transcription dependent on recruited factors, such as histone acetylation transferase
to activate gene expression or corepressor such as histone deacetylase or SWI/SNF
15
chromatin remodeling complex to affect nucleosome positioning (Ross and Hill, 2008).
High density of nucleosome occupancy makes transcription starting site unaccessible
to RNA polymerase and therefore serves as an indicator of suppressed gene
expression. In response to TGFβ1 treatment, nucleosome landscape in HepG2 cells
shows dynamic change with many regions of depleted nucleosomes, which are
correlated with transcription of the targeted genes (Enroth et al., 2014)
MicroRNA is small non-coding RNA molecule that post-transcriptionally affects mRNA
stability and translation by targeting the 3′-untranslated region of various transcripts.
Dysregulation of miRNA influences a wide range of cellular processes including TGF 
signaling. microRNA target on many components in TGF  signaling, thereby
regulating the activity of the signaling (Butz et al., 2012). Downregulation of miR-30
and miR-200 in primary anaplastic thyroid carcinoma cells gives rise to the
upregulation of targeting genes, TGFβ receptor I and SMAD2, resulting in enhanced
TGF  signaling and EMT (Braun et al., 2010). miR-21 targets and decreases the level
of SMAD7, thus enhancing TGF  signaling and contributing to the development of
lung fibrosis (Liu et al., 2010). In addition, miR-204, miR-211, and miR-379 are
identified to inhibit induction of IL-11 in metastatic breast cancer cells by TGF ,
thereby decreasing the osteolysis by stimulated osteoclast function (Pollari et al.,
2012).
In sum, interaction between epigenetic process and TGF  signaling affectes the
outputs of the signaling, playing crucial roles in cell behaviors and the development of
diseases.
16
1.5 Summary of the Project

In this project, the interaction of epigenetic regulation and TGFβ signaling was
investigated in stem cells and diseases to answer the questions: 1) How TGFβ
signaling affects the lineage determination of pluripotent stem cells and adult stem
cells; 2) How epigenetic regulation impinges on the activity of TGFβ signaling; 3) How
interaction of epigenetic regulation and TGFβ signaling gives rise to the symptoms in
disease; 4) How epigenetic process contributes to the occurrence of stem cells in
disease.
In chapter 2, we showed that iPSCs from Bcor mutant MSCs had altered self-renewal
and differentiation, especially deficient neural crest lineage contribution, which is
related to the affected tissues of OFCD syndrome. Molecularly, loss of Bcor resulted
in failure of enrichement of componenets of variant PRC1 complex at Nanog promoter
and subsequent deposition of H2AK119 monoubiquitination. Importantly, we found
that SMAD signaling antagonized BCOR and variant PRC1 complex mediated
suppression of Nanog expression to achieve an equilibrium of NANOG heterogeneity
in pluripotent stem cells. Therefore, interaction of PRC1 mediated epigenetic process
and SMAD signaling contributed to the maintenance of ESC pluirpotency.
In chapter 3, we used ossifying fibroma (OF) as a disease model and found OF
contained a stem cell population that resembled mesenchymal stem cells (OFMSCs)
and was capable of generating OF-like tumor xenografts. Mechanistically, OFMSCs
17
showed enhanced TGFβ signaling that induced aberrant proliferation and deficient
osteogenesis via Notch and BMP signaling pathways, respectively. The elevated
TGFβ activity was tightly controlled by JHDM1D-mediated epigenetic regulation of
thrombospondin-1 (TSP1), forming a JHDM1D/TSP1/TGFβ/SMAD3 autocrine loop.
Inhibition of TGFβ signaling in OFMSCs can rescue their abnormal osteogenic
differentiation and elevated proliferation rate. Furthermore, chronic activation of TGFβ
can convert normal MSCs into OF-like MSCs via establishment of this
JHDM1D/TSP1/TGFβ/SMAD3 autocrine loop. These results revealed that epigenetic
regulation of TGFβ signaling in MSCs governed the benign tumor phenotype in OF
and highlighted TGFβ signaling as a candidate therapeutic target.
Collectively, the present study provided a systematic mechanism of the epigenetic
regulation TGFβ signaling in stem cells and diseases. Epigenetic regulation of TGFβ
signaling affects ouput of the pathway, which impinges on the lineage determination of
stem cells in a cell context dependent manner. The epigenetic regulation of TGFβ
signaling gives birth to the aberrant functions of stem cells in diseases, which
contributes to the patient symptoms. Therefore, this study has deciphered molecular
mechanism of epigenetic regulation of TGFβ signaling with experimental evidence,
shedding light on the drug discovery by targeting on aberrant TGFβ signaling.
18
Chaptor 2: BCOR Orchestrates Human ESC Pluripotency via
Regulating SMAD/Nanog Axis

2.1 Introduction

BCL6 corepressor (BCOR) is ubiquitously expressed in most tissues during
development (Wamstad and Bardwell, 2007). Loss-of-function mutation of Bcor results
in female Oculofaciocardiodental (OFCD) syndrome, characteristic of congenital
cataract, microphthalmia, and radiculomegaly (Ng et al., 2004). The same mutation in
males lead to embryonic lethality, implying critical role of Bcor in early embryonic
development (Gearhart et al., 2006). Bcor knockdown in zebrafish and frogs gave rise
to colobmas, perturbations in somite, skeletal and neural tube development (Hilton et
al., 2007; Ng et al., 2004). Although Bcor knockout lead to defects in mouse embryo
development, the model cannot faithfully recapitulate the human disease (Wamstad et
al., 2008). It remains largely unknown the functional role of Bcor in the early
development and human ESC differentiation.
BCOR was originally identified to interact with BCL6 and potentiate its repression,
which was mutually exclusive with other corepressors, such as SMRT and NCOR
(Huynh et al., 2000). Interaction of BCOR with BCL6 has critical implications in
multiple biological processes, including left-right (LR) asymmetry (Hilton et al., 2007;
Sakano et al., 2010), development of lymphoma (Polo et al., 2004). However, it
remains unknown how Bcor mutation molecularly regulates stem cell lineage
19
determination and gives rise to disease phenotypes.
BCOR regulates gene expression by associating with protein complex that mediate
epigenetic modification of chromatin. BCOR interacted with protein complex including
RING1B, RYBP, and KDM2B, components of polycomb group (PcG) proteins, which
are essential for early embryo development and stem cell lineage determination
(Gearhart et al., 2006; Sanchez et al., 2007). For the transcriptional repression by
polycomb repressive complex (PRC), initial deposition of H3K27me3 by PRC2 recruits
the components of PRC1 and subsequent monoubiquitination of H2AK119, which
reinforces the function of PRC2 (Cao et al., 2002; Min et al., 2003). However, recent
findings showed that in the variant PRC1, KDM2B binding at CpG island initiated the
gene repression by recruiting PCGF1/PRC1 complex, which including BCOR
interacting partners, such as RING1B and RYBP (Blackledge et al., 2014). Being
actively involved in the variant PcG complex, BCOR contributes to the deposition of
monoubquitination of H2A to repress gene expression, independent of PRC2 function
(Gearhart et al., 2006). BCOR also derails the output of signaling pathways, which is
exemplified by Notch signaling. BCL6/BCOR complex competed with Notch1
intracellular domain and restrict activation of gene subsets in a cell type dependent
manner, thereby achieving specificity of target gene transcription and LR asymmetry
(Sakano et al., 2010). Both PRC mediated epigenetic process and signaling
modulations play crucial roles in early embryo development and ES cell differentiation
and the function of BCOR is involved in both of the processes, however, it is still
unclear how BCOR couples the two modes of regulation to orchestrate human ES cell
20
pluripotency.
In this study, we found iPSCs generated from OFCD patient and Bcor knockdown
human ES cells had high self-renewal capacity and altered lineage determination.
Elevated TGFβ signaling in Bcor deficient pluripotent cells drove high Nanog
expression, which altered ES cell differentiation and impaired generation of neural
crest lineage. Importantly, SMAD signaling can reverse the repression of
monoubiquitination mediated by BCOR and PRC1 to elicit Nanog expression, but
further deposition of H3K27me3 by PRC2 blocked this effect. Collectively, BCOR
integrated both SMAD signaling and epigenetic processes of PRC1 to achieve
orchestrated gene expression in maintaining pluripotency.

2.2 Materials and methods

2.2.1 Cell culture

Human MSCs from tooth root of OFCD patients were cultured in α-MEM (Invitrogen)
plus 15% FBS, 100 mM L-ascorbic acid, 2 mM L-glutamine, 100U ml
-1
penicillin and
100 μg ml
-1
streptomycin. iPSCs from MSCs were generated by retroviral delivery of
the four conventional Yamanaka factors. After four weeks’ induction, individual
colonies that were morphologically similar to human ESCs were picked up and kept in
separate wells in human ESC culture media which contain knockout DMEM
(Invitrogen) plus 20% knockout serum replacement (KSR), 1% non-essential amino
21
acid, 55 mM β-mercaptoethnol, and 10 ng ml
-1
basic fibroblast growth factor (bFGF).
Clonal culture was established and the expanded cells were used for subsequent
experiment. Formation of embryoid bodies (EB) was induced by floating culture in
ultralow attachment plate (Corning) in iPSC culture media without bFGF. For neural
crest induction, Day 2 EB were transferred into neural induction medium (50%
DMEM/F12, 50% neural basal medium plus 1X insulin/transferring/selenite solution,
1X B27 supplement) for 5 days to form neurospheres. The neurospheres were then
plated for neural crest differentiation.

2.2.2 Immunofluorescence and immunohistochemistry

For immunofluorescence, cells were fixed in 4% paraformaldehyde for 30 min and
permeated by 0.1% TritonX-100. Subsequently, the cells were blocked with normal
serum matched secondary antibodies, incubated with the specific antibodies for 4h.
The target proteins were revealed by Alexa Fluor 488 or Alexa Fluor 546-conjugated
secondary antibodies (Invitrogen). 4',6-diamidino-2-phenylindole (DAPI) was used for
nuclear staining. Histostain-SP kit (Invitrogen) was used for immunohistochemistry
according the manufacturer’s protocol with using the following antibodies: anti-HNK1,
anti-βIII-TUBULIN, anti-SOX2, anti-α-SMA, anti-COLLAGEN II, and GATA6 (Sigma).

22
2.2.3 Microarray

Total RNA were extracted from Control iPSCs and OFCD iPSCs and microarray
assays were performed in the Genome Center of Children’s Hospital, Los Angeles,
using Genechip Human Gene 2.0 ST array (Affymetrix). The results were analyzed by
Partek Genomics Suite and Gene Set Enrichment Analysis (Broad Institute).

2.2.4 Quantitative PCR

RNA was extracted by using a QuickPrep mRNA Purification kit (GE) following the
manufacturer’s instructions. A QuantiTect Rev. Transcription kit (Qiagen) was used for
reverse transcription of 1 μg purified RNA from cells. Quantitative PCR was performed
on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad).

2.2.5 Western blot

Cells were lysed by RIPA buffer (25 mM Tris pH 7.6, 150 mM NaCl, 1% NP-40, 1%
sodium deoxycholate, 0.1% SDS) containing protease inhibitor cocktail (Sigma).
Proteins were quantified by BCA method (Bio-Rad). 20 mg proteins were loaded and
separated by SDS-PAGE (Invitrogen), transferred onto PVDF membrane (Bio-Rad)
23
incubated in blocking buffer with 5% non-fat dry milk (Santa Cruz) in TBS. The Protein
level was analyzed with the indicated antibodies and developed using a
chemiluminescence kit (Thermo scientific).

2.2.6 Alkaline Phosphatase

The activity of alkaline phosphatase (AP) of the cells was assayed by naphthol and
fast red violet method with an AP staining kit (Sigma) according to the manufacturer’s
protocol.

2.2.7 DNA sequencing and RFLP

Total DNA from the cells was isolated and purified by phenol: chloroform method and
primers that flanking the mutation sites were used in PCR amplification. The amplified
fragments were inserted into Topo cloning vector (Invitrogen) and sequenced by P7
primer. For Restrictive Fragment Length Polymorphism (RFLP), the amplified
fragments were digested by DraI enzyme overnight and separated in agarose gel.

24
2.2.8 Teratoma

Female non-obese diabetes/severe combined immunodeficient (NOD/SCID) mice
were purchased from Harlan. All animal experiments were performed under
institutionally approved protocols for the use of animal research at the University of
Southern California. Cultured cells were treated by 1 mg ml
-1
dispase and collected in
clumps, and 1 × 10
6
cells

were

injected into the mice intramuscularly. After six weeks,
the mice were sacrificed and the generated teratomas were harvested for subsequent
hematoxylin & eosin staining or immunohistochemistry.

2.2.9 Gene knockdown and overexpression

Lentiviral particles (Sigma) that express Bcor small interfering RNA were added in cell
culture and puromycin selection was performed. The survival colonies were expanded
and examined for Bcor gene expression. For overexpression, Bcor gene fragment was
inserted into a lentiviral backbone vector and packaged into lentiviral particles, which
were subsequently delivered into cells with puromycin selection. The survival colonies
were examined for Bcor gene expression.

25
2.2.10 BrdU/CFU Assay

For BrdU assay, cells were labeled with 10 μM BrdU overnight and the cell proliferation
was assayed using a BrdU staining kit (Invitrogen), according to the manufacturer’s
instructions. Positive cells were detected with a streptavidin-biotin system and
counterstained by hematoxylin. For CFU assay, cells were trypsinized into individual
cells and 1000 cells were seeded in to 6-well plate. The cells were stained with
toluidine blue after two weeks’ growth and the ratio of colony forming unit was
calculated.

2.2.11 Flow cytometry

For calculation of NANOG
+
cells, individual cells were treated by permeabilization
buffer (eBioscience) and then staining by anti-NANOG (Abcam). For examination of
HNK1, trypsinized cells from neural crest induction were directly stained by anit-HNK1
(Sigma). PE-conjugated secondary antibody was applied for revealing the signal. All
samples were analyzed with a FACS
calibur
machines (BD Bioscience).

26
2.2.12 Luciferase Assay

Plasmids used in the assay include pNanog-luc (Addgene), pSBE4-luc (Addgene) and
pCMV-RL (Promega). The indicated cells were starved in human ES cell culture media
containing 2% KSR and co-transfected with pCMV-RL and each of the other plasmids
at the ratio of 1:20 by lipofectamine LTX (Invitrogen). The cells were treated by 100 ng
ml
-1
Activin, or 10 ng ml
-1
bFGF, or 1 μM SB431542 for 12 h. Cell lysate was prepared
by a Dual Luciferase Reporter Assay System (Promega). Luminescence was
measured by a luminometer (Promega) and firefly luminescene was normalized to
renilla luminescence.

2.2.13 ChIP-PCR

Cultured cells were cross-linked by 1% formaldehyde for 10 min, which was quenched
by 125 mM Glycine. The collected cells were suspended in ChIP lysis buffer (1% SDS,
10 mM EDTA and 50mM Tris, pH8.1) and lysed by a Branson sonicator. An aliquot of
sheared chromatin was set aside as input. Immunoprecipitation was performed with
ChIP kit (Millipore), according to the manufacturer’s instructions. The indicated
antibodies were used with goat IgG as an isotype control. The precipitated DNA
fragments were purified by phenol: chloroform extraction for quantitative PCR analysis.
Percentage input was determined by the ratio of immunoprecipitated DNA to total.
27
2.2.14 Statistical Analysis

SPSS 18.0 was used for statistical analysis. P-values were analyzed by two-tailed
Student’s t test or one-way analysis of variance (ANOVA) followed by Student Neuman
Keuls test. A P-value less than 0.05 were considered significant.

28
2.3 Results

2.3.1 Generation of iPS cells from Bcor mutant cells

To explore the role of BCOR in early human ESC differentiation, we generated iPSCs
from tooth root mesenchymal stem cells of patient with OFCD syndrome (hereafter
referred as OFCD iPSCs). Compact colonies that were morphologically similar to
human iPSCs could be generated by introduction of four Yamanaka factors (Figure
2.1A), and iPSCs from normal counterparts, stem cells from apical papilla (control),
served as controls (hereafter referred as control iPSCs). OFCD iPSCs can be
maintained and passaged as conventional iPSCs, without obvious morphological
change. However, the reprogramming efficiency of OFCD MSCs was significantly
lower than control MSCs (Figure 2.1B). In line with previous findings, deficiency of
lysine-specific demethylase 2b (KDM2B), the interactive partner of BCOR in variant
PRC1 complex, dramatically decreased the reprogramming efficiency (Wang et al.,
2011). To confirm that the generated iPSCs were derived from OFCD mesenchymal
stem cells, sequencing of genomic DNA indicated that OFCD iPSCs still carried the
same mutation as parental MSCs (Figure 2.1C; Fan et al., 2009). The mutation
created the sequence of “TTTAAA”, which could be digested by DraI enzyme.
Restrictive fragment length polymorphism (RFLP) further confirmed that only the
fragment amplified from genome of OFCD MSCs and iPSCs showed the digestive
polymorphism, illustrating the reprogrammed cells still carried the same mutation
29
(Figure S2.1A). Bcor gene showed decreased expression in OFCD iPSCs, indicating
mutation induced mRNA decay (Grossmann et al., 2011). Similar to control iPSCs,
OFCD iPSCs expressed typical pluripotency marker genes, including alkaline
phosphatase (AP), OCT4, SOX2, SSEA4 and TRA-1-81 (Figure 2.1E). In addition, the
expression of pluripotency markers, Oct4 and Sox2, Rex1 and Cripto were further
confirmed by western blot or quantitative PCR (Figure 2.1F, S2.1B). Epigenetic
profiling demonstrated that promoter regions of Oct4 and Nanog underwent extensive
demethylation, characteristics of pluripotent stem cells (Figure S2.1C).
To determine the differentiation potential of OFCD iPSCs, we used in vitro embryoid
body (EB) formation and in vivo teratoma generation assays to examine the capacity
for lineage commitment. Both OFCD iPSCs and control iPSCs were able to form EBs
in floating culture (Figure S2.1D), giving rise to cells positive for βIII-TUBULIN, -SMA
and GATA6, makers of ectoderm, mesoderm and endoderm respectively (Figure 2.1G).
Injection of the iPSCs into NOD/SCID mice generated terotoma containing tissues of
neural epithelium, cartilage, and digestive duct, representing ectoderm, mesoderm
and endoderm, respectively (Figure 2.1G). Furthermore, teratoma from both control
iPSCs and OFCD iPSCs contained cells positive for βIII-TUBULIN, -SMA and GATA6,
indicating potentials of contributing to all somatic lineages (Figure S2.1E). These
results indicate that Bcor deficient cells can be reprogrammed into pluripotent stage
with multiple differentiating potentials.
30

Figure 2.1. Generation of iPS Cells from Bcor Mutant Cells. (A) Light microscopy
images showed that introduction of OCT4, SOX2, KLF4, and c-MYC into control cells
and OFCD cells generated human ESC-like iPSC colonies. (B) Staining of alkaline
phosphatase at day 30 indicated that reprogramming efficiency of OFCD MSCs were
much lower than that of control MSCs. (C) Sequencing of genomic DNA showed Bcor
gene locus from OFCD iPSCs had a mutation of missing cytosine (arrows) compared
to control iPSCs. (D) Quantitative-PCR indicated that Bcor gene expression was
significantly lower in OFCD iPSCs than control iPSCs. (E) Immunostaining showed
that OFCD and control iPSCs were positive for pluripotency markers alkaline
phosphatase, OCT4, SOX2, SSEA4, and TRA-1-81. (F) Western blot indicated that
after reprogramming, pluripotency-specific transcription factors OCT4, SOX2 and
REX1 were highly expressed in both control and OFCD iPSCs, which were not
detectable in MSCs. (G) Plating of day 9 embryoid bodies (EBs) from both control and
OFCD iPSCs gave rise to cells positive for βIII-TUBULIN, α-SMA, GATA6, markers for
ectoderm, mesoderm and endoderm, respectively (upper panel); subcutaneous
31
implantation of OFCD and control iPSCs in NOD/SCID mice for 30 days generated
teratomas contained tissues of neuroepithelium, cartilage and digestive ducts,
characteristic of ectoderm, mesoderm and endoderm respectively, indicating both
control and OFCD iPSCs have the potentials to develop to all somatic lineages
(bottom panel). Data were represented as results of three independent experiments.
(**P < 0.01). See also Figure S2.1. Scale bar, 50 μm.

Figure S2.1. Generation of iPS Cells from Bcor Mutant Cells. (A) Restriction
fragment length polymorphism (RFLP) experiment indicated that only DNA fragments
from OFCD MSCs and iPSCs, but not from normal control MSCs and iPSCs, can be
cut by DraI, verifying the presence of mutation. (B) Quantitative PCR showed after
reprogramming, expression of endogenous Oct4, endogenous Sox2, Rex1, and Cripto
were highly elevated in both control and OFCD iPSCs. (C) Bisulfite sequencing assay
of methylation status showed MSCs underwent extensive demethylation at Oct4 and
Nanog promoter during reprogramming process. (D) Floating culture of control and
OFCD iPSCs for 6 days generated spheres of EBs, mimicking the early development
process. (E) Immunohistochemistry of teratomas from both control and OFCD iPSCs
indicated presence of tissues positive for βIII-TUBULIN, α-SMA, GATA6, markers for
ectoderm, mesoderm and endoderm, respectively. Data were represented as the
mean ±SD of three independent experiments. (**P < 0.01). See also Figure 2.1. Scale
bar, 50 μm.
32
2.3.2 Bcor Mutant iPSCs Have Altered Self-renewal and
Differentiation

We next explored how loss of BCOR affected the self-renewal and differentiation of
iPSCs. Similar to their parental MSCs, OFCD iPSCs have higher proliferating rate
than control iPSCs, as assessed by BrdU assay (Figure 2.2A). Human iPSCs and ES
cells are sensitive to single cell passage with increased apoptosis and we found that
OFCD iPSCs, passaged in individual cells, repopulated more efficiently than that of
control iPSCs (Figure 2.2B). In parallel, Bcor mutant iPSCs showed reduced
expression of negative regulator of cell cycle, such as P15, P16, P21 (Figure 2.2C,
S2.2A). Cell cycle regulators that promote proliferation, including Cdc7, Cdc6,
Cdc25A, were highly expressed in OFCD iPSCs (Figure S2.2A). Increased
proliferation and self-renewal of stem cells are always associated with deficiency in
lineage determination. During the EB culture, control iPSCs formed the characteristic
cavity structure, indicating differentiation of iPSCs (Figure 2.2D), whereas most EBs
from OFCD iPSC only gave rise to compact spheres, as illustrated by hematoxyin &
eosin (HE) staining. The pluripotency markers, including OCT4, SOX2, and NANOG
were still maintained at high levels in the EBs of OFCD iPSCs (Figure 2.2E, S2.2B).
Markers of terminally differentiated cells have lower expression in EBs from OFCD
iPSCs, such as Krt9, Ncam, and Adam12 (Figure S2.2B). Profiling of marker genes of
different germ layers at day 6 EB demonstrated that, compared to control iPSCs,
OFCD iPSCs contributed more to ectoderm (Pax6, Nestin) and mesoderm (Gsc, T)
33
with decreased differentiation into endoderm (Afp, Gata4) and trophectoderm (Cdx2,
Hcg (Figure S2.2C). Patients with OFCD syndrome have characteristic
radiculomegaly and MSCs from the patient tooth root demonstrated enhanced
osteogenesis (Fan et al., 2009). We found the cartilage in OFCD iPSC teratoma
demonstrated bone-like structure, which were negative for toliudine blue staining
(Figure S2.2D). In addition, COLLAGEN II, as a marker of chondrogenesis, showed
decreased expression in OFCD iPSC teratoma, suggesting skewing against cartilage
development (Figure S2.2D). Consistently, MSCs differentiated from OFCD iPSCs
had high contribution to osteogenesis, as indicated by increased alizarin red staining
and western blot of higher RUNX2 and OCN expression (Figure S2.2E). Collectively,
Bcor loss in OFCD iPSCs conferred the cells with high self-renewal capacity and
altered differentiation potential, which may give rise to the development defects of
Bcor mutant embryos.
The affected tissues of OFCD syndrome patients were largely originated from neural
crest lineage. We then explored how loss-of-function mutation of Bcor may contribute
to the development of neural crest lineage. Teratoma generated from iPSCs contains
tissues of all lineages, so we first characterized neural crest tissues in teratoma of
OFCD iPSCs. Lineage of neural crest is generated form neural tube by an epithelial to
meshenchymal transition (EMT) (Duband et al., 1995). We found SOX2
+
neural tube
like structure in control iPSCs expressed BCOR, which was not detected in OFCD
iPSCs (Figure 2.2F). Large population of cells surrounding neural tube like structure
are positive for HNK1, an early neural crest marker, in teratoma generated from control
34
iPSCs, but not that from OFCD iPSCs (Figure 2.2F). Characterization of other neural
crest specific transcription factors during, including Snail, Slug, Twist, and Sox9 also
demonstrated decreased expression during OFCD iPSC differentiation (Figure 2.2G).
In in vitro induction of neural crest lineage, more cells migrate out of the colonies and
high percentage of cells are positive for HNK1 (Figure S2.2H, I). The results indicated
that OFCD iPSCs may be deficient in contributing to neural crest lineage. Neural crest
cells have remarkable migratory capacity with decreased E-cadherin expression.
Elevated E-Cadherin expression in the neural crest cells from OFCD iPSCs may
indicate that the cells have deficienct EMT (Figure 2.2J). In addition, the neural crest
cells from OFCD iPSCs had significantly decreased migration capacity compared to
that from control iPSCs (Figure 2.2K). Therefore, Bcor mutation in OFCD iPSCs gave
rise to the defect in neural crest lineage determination.

35
Figure 2.2. Bcor Mutant iPSCs Have Altered Self-renewal and Differentiation. (A)
BrdU assay showed OFCD iPSCs had higher proliferation rate, compared to control
iPSCs. (B) Colony forming unit (CFU) efficiency assay indicated single cells from
OFCD iPSCs had higher efficiency to form colonies compared to control iPSCs. (C)
Western blot assay indicated OFCD iPSCs had decreased expression of P21, P15,
and P16, compared to control iPSCs. (D) While day 6 EB from control iPSCs showed
cavity structure indicating differentiation, OFCD iPSCs only gave rise to compact
spheres, suggesting impaired differentiation (Left upper panel). HE staining of EBs
further confirmed the results (Left lower panel). Statistics of differentiated EBs
indicated that OFCD iPSCs had lower differentiation capacity (Right). (E) Western blot
showed day 6 EBs from OFCD iPSCs still maintained elevated expression of
pluripotent markers, OCT4, SOX2, and NANOG, compared to that from control iPSCs.
(F) Immunohistochemistry of teratoma showed that both control iPSC and OFCD
iPSCs gave rise to SOX2
+
neural epithelium (left panel), but Bcor expression was
largely undetectable in teratoma generated from OFCD iPSCs (middle panel). Around
neural tube of teratoma from control iPSCs, HNK1
+
cells were readily detected, which
was not seen in that from OFCD iPSCs (right panel). (G) Quantitative PCR indicated
that neural crest cells from OFCD iPSCs had lower expression of marker genes Snail,
Slug, Twist, and Sox9. (H) Light microscopy showed that in neural crest induction for 3
days, cells migrated from colonies of control iPSCs, rather than OFCD iPSCs. (I) Flow
cytometry of day 3 differentiated neural crest cells indicated that OFCD iPSCs
contributed less to HNK1
+
cells in neural crest induction. (J) Quantitative PCR
indicated that neural crest cells from OFCD iPSCs had higher expression of
E-Cadherin. (K) Migratory capacity assay indicated that neural crest (NC) cells form
OFCD iPSCs had lower migratory capacity. Data were represented as the mean ±SD
of three independent experiments. (*P < 0.05, **P < 0.01) See also Figure S 2.2. Scale
bar, 50 μm.
36

Figure S2.2. Bcor Mutant iPSCs Have Altered Self-renewal and Differentiation. (A)
Quantitative PCR indicated OFCD iPSCs expressed higher Cdc7, Cdc6 and Cdc25A,
positive regulator of cell cycle, and lower P21, P15 and P16, negative regulator of cell
cycle. (B) Quantitative PCR indicated day 6 EBs from OFCD iPSCs expressed higher
pluripotency marker Oct4, Sox2, Nanog and lower cell maturation marker Krt9, Ncam,
Adam12 compare to EBs from control iPSCs. (C) Quantitative PCR from day 9 EBs
showed OFCD iPSCs had lower expression of markers of endoderm (Afp, Gata4) and
trophoblast lineage (Cdx2, Hcg) and higher expression of markers of mesoderm (Gsc,
T) and ectoderm (Pax6, Nestin). (D) HE staining showed teratoma from OFCD iPSCs
had defect in cartilage development with bone like structure (arrow) in chondrogenesis
nodule (left panel), which was negative for Toliudine blue staining (arrow, middle
panel). Immunohistochemistry indicated lower expression of COLLAGEN II in the
cartilage from OFCD iPSCs (right panel). (E) Alizarin red staining showed MSCs from
OFCD iPSCs, similar to their parental MSCs, had elevated osteogenesis, as confirmed
by western blot assay of RUNX2 and OCN. Data were represented as the mean ±SD
of three independent experiments. (*P < 0.05, ** P < 0.01). See also Figure 2.2. Scale
bar, 50 μm.

37
2.3.3 Bcor Knockdown in Normal Pluripotent Stem Cells Mimics the
Phenotypes of OFCD iPSCs
In order to further confirm the role of Bcor in self-renewal and differentiation and avoid
the preset effect of Bcor loss in MSCs, we explored to mimic BCOR deficiency by
knocking down Bcor expression in normal human iPSCs and human ES cells.
Lentiviral delivery of siRNA of Bcor efficiently decreased the gene expression in both
human control iPSCs and H9 human ES cells (Figure S2.3). After puromycin selection,
compact colonies still survived with positive expression of AP, OCT4, SSEA4 (Figure
2.3A). Similar to OFCD iPSCs, pluripotent stem cells with decreased Bcor expression
have higher proliferation rate and repopulation capacity, as indicated by BrdU assay
and colony forming efficiency assay (Figure 2.3B, C). Bcor gene knockdown also
induced lower expression of P15, P16, P21, contributing to the high proliferation of the
cells (Figure 2.3D). Knockdown of Bcor further affected differentiation potential.
Pluripotent stem cells with lower Bcor expression generated more compact spheres
during EB formation, which demonstrated high expression of pluripotency markers,
OCT4, SOX2, and NANOG (Figure 2.3E, F). Analogous to OFCD iPSCs, Bcor
knockdown induced skewing lineage determination with lower contribution to neural
crest lineage, as indicated by the maker gene characterization, including Snail, Slug,
Twist, Sox9 (Figure 2.3G). In addition, Bcor knockdown induced elevated E-Cadherin
during differentiation (Figure 2.3H). Therefore, Bcor knockdown in pluripotent stem
cells recapitulated the phenotypes of OFCD iPSCs with altered self-renewal and
differentiation.
38

Figure 2.3. Bcor Knockdown in Normal Pluripotent Stem Cells Mimics the
Phenotypes of OFCD iPSCs. (A) AP staining and immunofluorescence indicated that,
after knockdown of Bcor, control iPSCs and human ESCs (H9 cells) still expressed AP ,
OCT4, and SSEA4. (B) BrdU assay showed that Bcor deficient pluripotent stem cells
had a higher proliferation rate. (C) CFU assay indicated single cells from Bcor deficient
pluripotent stem cells had higher efficiency to form colonies. (D) Western blot indicated
Bcor deficient pluripotent stem cells had decreased expression of P21, P15, and P16
compared to their normal counterparts. (E) Analysis of day 6 EBs indicated that Bcor
deficient pluripotent stem cells contributed less to EBs with differentiated cavities
compared to their normal counterparts. (F) Western blot showed EBs from Bcor
deficient control iPSCs and H9 cells had high expression of pluripotent markers OCT4,
SOX2, and NANOG. (G) Quantitative PCR results showed EBs from Bcor deficient
control iPSCs and H9 cells had lower expression of neural crest markers, including
Snail, Slug, Twist, and Sox9. (H) Quantitative PCR indicated that neural crest cells
from Bcor deficient iPSCs and human ESCs had higher expression of E-Cadherin.
Data were represented as the mean ±SD of three independent experiments. (** P <
0.01). See also Figure S 2.3. Scale bar, 50 μm.
39

Figure S2.3. Bcor Knockdown in Normal Pluripotent Stem Cells Mimics the
Phenotypes of OFCD iPSCs.Bcor siRNA efficiently decreased the gene expression.
Data were represented as the mean ±SD of three independent experiments. (** P <
0.01) See also Figure 2.3.

40
2.3.4 OFCD iPSCs Have Elevated Nanog Expression

To investigate how Bcor loss molecularly affected self-renewal and differentiation, we
analyzed the global gene expression pattern between OFCD iPSCs and control
iPSCs. Microarray results indicated that OFCD iPSCs had more genes elevated than
control iPSCs, suggesting release of the repression from BCOR may induce the gene
expression (Figure S2.4A). Gene set enrichment analysis (GSEA) of global gene
expression indicated enriched genes in OFCD iPSCs were positively correlated with
functions such as cell cycle and DNA replication (Figure S2.4B), consistent with high
proliferation rate of OFCD iPSCs. In parallel, enriched genes in OFCD iPSCs were
also negatively associated with development and differentiation (Figure S2.4B).
Among the differently expressed genes, we found higher level of Nanog expression in
OFCD iPSCs compared to control iPSCs, which was confirmed by both western blot
and PCR, whereas Oct4 and Sox2 were maintained at similar level to control iPSCs
(Figure 2.4A). The elevated Nanog expression of OFCD iPSCs and enhanced
self-renewal were consistent with the previous findings that overexpression of Nanog
enabled feeder-free maintenance of human ESCs (Darr et al., 2006). Transcriptional
fluctuation of Nanog expression are essential element of pluripotency and contributed
to the lineage determination of ES cells (Kalmar et al., 2009). Given the increased
NANOG expression and altered self-renewal and differentiation, we next investigated
how BCOR loss affect NANOG expression pattern.
Most pluripotency markers, such as TRA-1-81, were ubiquitously expressed in ESCs,
41
which was confirmed in control iPSCs (lower panel, Figure 2.4B), but NANOG uniquely
showed heterogeneous expression in control iPSCs (upper panel, Figure 2.4B).
Distinctive from control iPSCs, OFCD iPSCs demonstrated more homogeneous
expression of NANOG (lower panel, Figure 2.4B) and the percentage of NANOG
+
cells
in OFCD iPSCs was much higher than that in control iPSCs (Figure S2.4C). In
consistence, knockdown of Bcor in control iPSCs and H9 cells increased the
percentage of NANOG
+
cells (Figure S2.4D). To confirm whether BCOR affected the
NANOG expression pattern, we next tested whether reintroduction of Bcor expression
could rescue the homogeneous NANOG expression pattern. Elevated Bcor
expression level was detected after overexperssion (Figure S2.4E). The percentage of
NANOG
+
cells in OFCD iPSCs was significantly decreased by Bcor overexpression,
as showed by flow cytometry assay (Figure 2.4C). We then explored whether Bcor
overexpression affected the heterogeneity of NANOG expression. Although Bcor
overexpression did not affect the NANOG expression pattern in control iPSCs,
reintroduction of Bcor in OFCD iPSCs rescued the heterogeneous expression of
NANOG and decreased the percentage of NANOG
+
cells, as shown by overlay of
OCT4 and NANOG (Figure 2.4D). However, Bcor overexpression did not influence the
level of OCT4 and SOX2 in both control iPSCs and OFCD iPSCs despite of decreased
NANOG level (Figure 2.4E). These results indicated that BCOR was crucial for the
maintenance of NANOG heterogeneity in pluripotent stem cells.
42

Figure 2.4. OFCD iPSCs Have Elevated Nanog expression. (A) Western blot and
quantitative PCR demonstrated that NANOG, but not OCT4 and SOX2, had higher
expression in OFCD iPSCs compared to control iPSCs. (B) Immunofluorescence
showed NANOG had heterogeneous expression in control iPSCs with some cells
negative for NANOG, whereas OFCD iPSCs have ubiquitous NANOG expression. In
contrast, TRA-1-81, as a pluripotency marker, showed ubiquitous expression in both
control and OFCD iPSCs. (C) Flow cytometry indicated higher percentage of OFCD
iPSCs are NANOG positive than control iPSCs. (D) Immunofluorescence showed that
overexpression of Bcor significantly decreased the ratio of NANOG
+
cells to OCT4
+

cells in OFCD iPSCs, but not in control iPSCs. (E) Western blot showed
overexperssion of Bcor induced the downregulation of NANOG in OFCD iPSCs, not in
control iPSCs, whereas OCT4 and SOX2 levels were not affected. Data were
represented as the mean ±SD of three independent experiments. (*P < 0.05, ** P <
0.01). See also Figure S 2.4. Scale bar, 50 μm.
43

Figure S2.4. OFCD iPSCs Have Elevated Nanog expression. (A) Partek analysis of
microarray showed OFCD iPSCs had distinct gene expression pattern from control
iPSCs. (B) GSEA demonstrated gene expression pattern in OFCD iPSCs were
positively related to cell cycle and DNA replication and negatively related to
developmental maturation and cell maturation. The normalized enrichment score
(NES) were indicated. (C) Flow cytometry indicated that overexpression of Bcor in
OFCD iPSCs decreased the percentage of NANOG
+
cells. (D) Flow cytometry
indicated that percentage of NANOG
+
cells dramatically increased after knockdown of
Bcor expression in control iPSCs and H9 cells. (E) Quantitative PCR showed Bcor
level in OFCD iPSCs was elevated after overexpression. Data were represented as
the mean ±SD of three independent experiments. (***p < 0.001). See also Figure 2.4.
Scale bar, 50 μm.

44
2.3.5 BCOR Regulates Nanog Expression via variant PRC1
complex

BCOR is associated with members of polycomb group and regulates the repressive
function of PcG proteins. It is generally believed that PRC2 first deposits H3K27me3,
which is required for subsequent recruitment of RPC1 and H2AK119
monoubiquitination, but recent evidence indicates that that variant PRC1 function
independently to mediate H2AK119 monoubiquitination and then recruited PRC2 and
H3K27me3 (Tavares et al., 2012). One of the major mechanism of BCOR mediated
repression is by H2A ubiquitination and the repression is a crucial step for regulation
of gene expression in ES cells (Endoh et al., 2012). Monoubiquitination of Nanog
promoter was not affected in Eed
-/-
cells, indicating the repression was independent of
PRC2 (Tavares et al., 2012). We, therefore, hypothesized that BCOR may regulate
Nanog expression via variant PRC1 complex. We found that both control iPSCs and
OFCD iPSCs had similar expression level of components of variant PRC1 complex,
including RYBP, RING1B, and KDM2B, which were associated together (Figure 2.5A,
S2.5A). However, OFCD iPSCs showed significantly lower enrichment of RYBP,
RING1B, and KDM2B at Nanog promoter compared to control iPSCs, indicating at
BCOR loss may result in the deficiency of recruitment of variant PRC1 complex at
Nanog promoter and loss of the suppressive epigenetic modification (Figure 2.5B).
Consistently, we found that the global level of both PRC1 mediated H2AK119
monoubiquitination and PRC2 mediated H3K27me3 were decreased (Figure 2.5C).
45
Furthermore, monoubiquitinaiton of H2AK119 at Nanog promoter was much lower in
OFCD iPSCs than that of control iPSCs, consistent with increased Nanog promoter
activity and gene expression (Figure 2.5D), but the level of monoubiquitination was
not changed at Oct4 and Sox2 promoter (Figure S2.5B). Knockdown of the PRC1
components, including Rybp, Ring1b, and Kdm2b, induced elevated NANOG level
and decreased monoubiquitination at Nanog promoter (Figure 2.5E, F).
As NANOG was a repressor of neural crest lineage (Wang et al., 2012), we postulated
that increased NANOG expression in OFCD iPSCs may suppress generation of
neural crest cells and subsequently lead to the disease symptoms. Molecularly,
enriched NANOG binding at Snail and Twist promoters was detected, which may
repress the transcription of the genes and acquirement of neural crest cell identity
(Figure 2.5G). In consistence, Nanog knockdown in these cells rescued the
expression deficiency of Snail and Twist (Figure S2.5C, D). Thus, OFCD iPSCs had
deficiency in contributing to neural crest lineage due to repression of the transcription
of Snail and Twist by elevated NANOG expression, which may give rise to the male
lethality and female symptoms in OFCD syndrome.
Therefore, BCOR, in conjunction with variant PRC1 complex, mediated the epigenetic
suppression of Nanog promoter to regulate the gene expression and loss of BCOR
gave rise to the deficiency of binding of components of variant PRC1 complex at
Nanog promoter, thus the elevated gene expression (Figure S2.5E). The high NANOG
expression further resulted in the deficiency in generating neural crest lineage by
repressing Snail and Twist expression.
46

Figure 2.5. BCOR Regulates Nanog Expression via variant PRC1 complex. (A)
Western blot indicated control iPSCs and OFCD iPSCs expressed similar level of
RPC1 complex components, RYBP, RING1B, and KDM2B. (B) ChIP-PCR assay
showed enrichment of RYBP, RING1B, KDM2B at the Nanog promoter was
significantly lower in OFCD iPSCs than control iPSCs. (C) Western blot showed total
levels of both H2AK119 ub1 and H3K27me3 decreased in OFCD iPSCs compared to
control iPSCs. (D) ChIP-PCR assay indicated OFCD iPSCs had lower H2AK119 ub1
at Nanog promoter compared to control iPSCs. (E) Western blot indicated that
NANOG showed elevated expression after knockdown of RPC1 components, RYBP,
RING1B, and KDM2B. (F) ChIP-PCR assay showed that the enrichement of
ubiquitination at H2AK119 at Nanog promoter decreased significantly after knockdown
of RPC1 components, RYBP, RING1B, and KDM2B. (G) ChIP-PCR assay indicated
higher enrichment of NANOG binding at promoter region of Snail and Twist in neural
crest cells from OFCD iPSCs compared to that from control iPSCs. Data were
represented as the mean ±SD of three independent experiments. (*P < 0.05, ** P <
0.01). See also Figure S 2.5.
47

Figure S2.5. BCOR Regulates NANOG Expression via variant PRC1 complex. (A)
Co-immunoprecipitation by RYBP antibody in nuclear protein of control iPSCs indicated the
binding of variant PRC1 complex, RYBP, KDM2B, RING1B, rather than RPC2 component
EZH2. (B) ChIP-PCR assay showed that level of H2AK119 ub1 at Oct4 and Sox2 promoter
was similar between OFCD iPSCs and control iPSCs. (C) Western blot showed the efficacy
of Nanog siRNA. (E) Quantitative PCR indicated that Snail and Twist showed increased
expression after Nanog knockdown for 12 hours in neural crest cells from OFCD iPSCs. (E)
Scheme showed in control iPSCs, BCOR, in concert with RYBP, RING1B, and KDM2B,
suppressed Nanog expression by mediating epigenetic modification of H2AK119 ub1.
Deficient Bcor in OFCD iPSCs gave rise to failure of RYBP, RING1B, and KDM2B binding at
Nanog promter, thus the elevated gene expression. Data were represented as the mean ±SD
of three independent experiments. (*P < 0.05, ** P < 0.01). See also Figure 2.5.

48
2.3.6 SMAD Signaling Antagonizes Bcor and PRC1 Complex at
Nanog Promoter

NANOG is a direct downstream target of SMAD signaling in human ESCS and PRC1
mutant ES cells also demonstrated enhanced TGFβ signaling (Brookes et al., 2012;
Vallier et al., 2009; Xu et al., 2008). In mouse ES cells, the dynamic and orchestrated
equilibrium of NANOG heterogeneity as well as pluripotency is maintained by TGFβ
superfaimily signaling (Galvin-Burgess et al., 2013). Therefore, we postulated that
Nanog may be under the coordinated dual regulation of SMAD signaling and PRC1
complex.
Activation of SMAD signaling by Activin/Nodal, TGFβ family members, is required for
maintenance of human ES cell pluripotency and inhibition of the signaling precipitated
differentiation (Vallier et al., 2005). TGFβ signaling also plays crucial roles in lineage
commitment of human ES cells (Beattie et al., 2005; Besser, 2004). During
differentiation, SMAD signaling induced mesendoderm regulators Gsc and Mixl1 by
forming complex with TRIM33, which mediated active epigenetic modification (Xi et al.,
2011). We then explored how BCOR loss affected the SMAD signaling activity in
OFCD iPSCs. Activin induced phosphorylation of SMAD3 and increased expression
of Tgif2 and Pai1, which can be antagonized by TGFβ receptor inhibitor SB431542
(Figure S2.6A, B). In response to Activin treatment, control iPSCs showed increased
Nanog expression, indicating increased SMAD signaling activity, whereas OFCD
iPSCs demonstrated constantly elevated Nanog level without response to Activin
49
treatment (Figure 2.6A). Consistently, the activity of Nanog-luciferase in control iPSCs
demonstrated increase and decrease in response to Activin and SB431542,
respectively, whereas that OFCD iPSCs showed constant elevation without response
to the manipulation of the signaling (Figure 2.6B). In parallel, downstream target
genes of SMAD signaling, including Tgif2 and Pai1, showed increased expression in
OFCD iPSCs (Figure S2.6C). In consistence with the elevated SMAD signaling in
OFCD iPSCs, knockdown of Bcor expression in control iPSCs and H9 cells induced
elevated expression of Smad7 and Pai1 (Figure S2.6D).
As a repressor, we extrapolated that BCOR may transcriptionally regulate Nanog
expression. In response to Activin, the BCOR enrichment at Nanog promoter in
control iPSCs was decreased (Figure 2.6C), which was reversed by blockage of
SMAD signaling by Smad3 siRNA and SB431542 (Figure 2.6D, S2.6E), indicating the
function of BCOR is under the regulation of SMAD signaling. In response to activation
of SMAD signaling, the ubiquitination of Nanog promoter in control iPSCs
demonstrated significant decrease (Figure 2.6E). However, level of
monoubiquitination was not changed at Oct4 and Sox2 promoter in response to
Activin treatment (Figure S2.6F, G). In addition, the enrichment of RYBP, RING1B,
and KDM2B at Nanog promoter in control iPSCs showed significant decrease after
stimulation of Activin, indicating regulation of variant PRC1 complex by SMAD
signaling (Figure 2.6F).
Thus, BCOR suppressed Nanog expression by recruiting component of variant PRC1
and monoubiquitination of H2AK119, with which activation of SMAD signaling by
50
Activin competed (Figure S2.6H). Coordination between BCOR mediated epigenetic
process and SMAD signaling orchestrates dynamic NANOG heterogeneity and
equilibrium to maintain the pluripotency of ES cells.

Figure 2.6. SMAD signaling antagonizes BCOR and PRC1 Complex at Nanog
Promoter. (A) Quantitative PCR indicated Nanog was elevated after Activin treatment
for 6 hours in control iPSCs, whereas the Nanog expression was not responded in
OFCD iPSCs with constant high elevation. (B) Luciferase assay showed activity of
Nanog-luciferase was increased and decreased by 12 hour treatment of Activin and
SB431542, respectively, whereas OFCD iPSCs constantly showed elevated
Nanog-luciferase activity without response to Activin and SB431542. (C) ChIP-PCR
assay showed that BCOR binding at Nanog promoter in control iPSCs dramatically
decreased in response to Activin treatment for 3 hours. (D) ChIP-PCR assay showed
that blockage of Smad signaling by Smad3 siRNA or SB431542 increased the binding
of BCOR at Nanog promoter in control iPSCs. (E) ChIP-PCR assay showed control
iPSCs showed decreased H2AK119 ub1 at Nanog promoter in response to Activin
treatment for 3 hours. (F) ChIP-PCR assay indicated that after treatment by Activin for
3 hours, the enrichment of RYBP and RING1B at Nanog promoter in control iPSCs
showed significant decrease. Data were represented as the mean ±SD of three
independent experiments. (*P < 0.05, ** P < 0.01). See also Figure S 2.6.
51

Figure S2.6. SMAD signaling antagonizes BCOR and PRC1 Complex at Nanog
Promoter. (A) Quantitative PCR showed Activin treatment for 6 hours induced the
expression of Tgif2 and Pai1, whereas simultaneous SB431542 treatment blocked the
process. (B) Western blot indicated Activin treatment for 1 hour induced the
phosphorylation of SMAD3, whereas simultaneous SB431542 treatment blocked the
process. (C) Quantitative PCR indicated that downstream genes of SMAD signaling
Tgif2 and Pai1 were highly expressed in OFCD iPSCs compared to control iPSCs. (D)
Quantitative PCR showed after Bcor knockdown in control iPSCs and H9 cells had
elevated expression of Tgif2 and Pai1. (E) Western blot indicated that knockdown
efficacy of Smad3 siRNA. (F-G) ChIP-PCR assay showed that in control iPSCs the
level of H2AK119 ub1 at Oct4 and Sox2 promoter was not changed in response to
Activin treatment for 3 hours. (H) Scheme showed that SMAD signaling antagonized
with BCOR and variant PRC1 complex mediated suppression at Nanog promoter to
drive the gene expression. Data were represented as the mean ±SD of three
independent experiments. (*P < 0.05, ** P < 0.01, #P > 0.05). See also Figure 2.6.

52
2.4 Dicussion

In this study, we found that loss-of-function of BCOR in OFCD iPSCs resulted in
enhanced self-renewal and impaired differentiation of pluripotent stem cells. The
uncoordinated differentiation due to BCOR deficiency may contribute to the male
lethality and symptoms in female patients of OFCD syndrome. Mechanistically, Bcor
loss resulted in elevated Nanog expression. In synergy with PRC1 complex, BCOR
facilitated monoubiquitination of H2AK119 at Nanog promoter and suppressed the
gene expression, which was antagonized by activation of SMAD signaling. This
convergent regulation orchestrated the proper gene expression of Nanog in response
to environmental cues and coordinated the development process
Defect in this regulation may cause development abnormality and occurrence of
disease. BCOR loss gave rise to absence of components of variant PRC1 complex,
including RYBP, RING1B and KDM2B, and decreased suppressive
monoubiquitination of Nanog promoter, thus the elevated Nanog expression.
Disturbed Nanog expression further skewed the lineage determination of ESCs with
deficiency in endoderm and trophectoderm. In consistence, Nanog depleted
blastocystes differentiated into primitive endoderm in culture (Mitsui et al., 2003) and
Nanog knockdown also induced trophectoderm differentiation in addition to primitive
endoderm differentiation (Ivanova et al., 2006). We further found elevated NANOG
expression suppressed neural crest lineage contribution, which was associated with
the affected tissues in OFCD syndrome.
53
Fluctuation of specific gene expression in ES cells was found to contribute to the
pluripotency of ESCs. NANOG showed characteristic heterogeneous expression
pattern with reciprocal conversion between Nanog
low
and Nanog
high
states (Chambers
et al 2007). Disturbance of the equilibrium of NANOG heterogeneity with either
increased or decreased gene expression gave rise to the defect in self-renewal and
differentiation. Similar observations disclosed in the expression of Rex1 and Stella
have led to the proposition that ESCs experience dynamic heterogeneity and that
such metastability may be an essential component of pluripotent identity (Hayashi et
al., 2008). Our study identified a mechanism of the heterogeneity that BCOR
integrated signaling regulation from environment cues and intrinsic epigenetic
process to modulate the heterogeneous equilibrium of NANOG. It will be intriguing to
investigate the regulatory mechanism of other heterogeneously expressed genes and
decipher the elaborate network of pluirpotency.
Although we found BCOR and SMAD signaling coordinately regulated Nanog gene
expression, it remains elusive how activation of SMAD signaling compete with BCOR
binding at Nanog promoter to drive gene expression. SMAD showed cell type specific
binding pattern (Morikawa et al., 2011), which affected the lineage determination of
stem cells and conferred pleiotrophic function of TGFβ signaling (Fei et al., 2010).
TGFβ signaling can initiate differentiation by forming SMAD2/3-TRIM33 complex,
which recognized chromatin mark of H3K9me3 and displaces chromatin compacting
factor HP1γ for gene expression (Xi et al., 2011). It is highly possible that SMAD2/3
also recruit other partners to antagonize BCOR and PRC1-mediated repression.
54
Deciphering the role of BCOR also contribute to the understanding of exit of
pluripotency and stem cell lineage determination (Betschinger et al., 2013). TGFβ was
reported to both repress and activate gene expression, dependent on the interactive
partners. BRG1 was reported to activate downstream gene expression in a SMAD2
dependent manner (Ross et al., 2006). TGFβ signaling is a barrier to neural
differentiation and inhibition of SMAD signaling gave rise to high efficiency of neural
differentiation (Chambers et al., 2009). However, function of downstream interactive
partners alters the outputs of the singling. We found BCOR deficient pluripotent cells
have both high TGFβ signaling and high neural differentiation, which may result from
altered binding of variant PRC complex after BCOR loss. Similarly, TGFβ was well
known to promote chondrogenesis and inhibit osteogenesis (Qin et al., 2013), but we
detected both elevated TGFβ signaling and osteogenesis, which may also derive from
altered response of downstream target genes. This evidence indicates that alteration
of epigenetic component downstream of the transcription factors derails the outputs of
signaling modulation and subsequent stem cell lineage determination. BCOR
mediated molecular process is important for lineage commitment of both pluripotent
stem cells and MSCs, but the role of BCOR in other lineages is still unknown. Given
that both TGFβ signaling and BCL6 are critical for determination of hematopoietic cell
lineage, it will be interesting to investigate the role of BCOR in these lineages.
In summary, we deciphered a role of BCOR in mediating the regulation of Nanog
expression via ineracting with components of variant PRC1 and determining the
pluripotent stem cell lineage commitment and disease occurence. The study
55
contributed to the understanding of regulatory network of pluripotency, as well as early
ES cell differentiation.

56
Chapter 3: Epigenetic Regulation of TGFβ Signaling in
Mesenchymal Stem Cells of Ossifying Fibroma

3.1 Introduction

Ossifying fibroma (OF) is a common benign fibro-osseous neoplasm of orofacial
bones, which causes progressive enlargement of the affected jaw along with a
deficiency in bone formation (Gondivkar et al., 2011). Currently, complete surgical
removal of the affected bone is widely recommended in the management of OF.
However, patients often suffer difficult reconstruction with post-surgical disfigurement,
high and unpredictable recurrence rates and major loss of vital tissues
(MacDonald-Jankowski, 2009). Therefore, more appropriate treatments for OF are
needed.
A plethora of tumor stem cells have been identified in a vast array of tumors,
especially in malignancies (Xu et al., 2009; Zhang et al., 2009). This population of
cancer stem cells, usually accounting for a small percentage of bulk tumor cells, is
regarded as a driver of tumor growth, progress, metastasis and recurrence, implying
that effective therapy should be targeted at this population of cells (Visvader and
Lindeman, 2012). In addition, peripheral nerve progenitors have been shown to be
associated with benign neurofibroma tumorigenesis (Williams et al., 2008). However,
the detailed molecular mechanisms and regulatory networks that determine stem cell
function in most benign tumors, including OF, are largely unknown.
57
Mesenchymal stem cells (MSCs) are stromal progenitor cells capable of self-renewal,
multilineage differentiation, and immunomodulation (Pittenger et al., 1999; Uccelli et
al., 2007). MSCs have therefore been used in clinical settings for tissue regeneration
and immune therapies (Caplan, 2007; Qi et al., 2010). Additionally, multiple lines of
evidence indicate that stem cell properties of MSCs may affect cancer and benign
tumor behavior (Mani et al., 2008). However, it remains largely unknown how MSCs
participate in benign tumor development. Among the different signaling pathways
involved in MSC proliferation and differentiation, TGFβ signaling is of interest because
it has been reported to be associated with both stem cell function and tumor
development (Massague, 2008; Roelen and Dijke, 2003). TGFβ signaling enhances
MSC proliferation via nuclear translocation of β-catenin in a SMAD3-dependent
manner (Jian et al., 2006) and inhibits MSC differentiation via repression of RUNX2
function (Kang et al., 2005). It remains unknown whether TGFβ signaling is involved
in the development of mesenchymal cell-associated benign tumors. In this study, we
reveal that OF tumors contain mesenchymal stem cells (OFMSCs) capable of
recapitulating the parental tumor phenotype when implanted in vivo. TGFβ signaling is
highly activated in OFMSCs by the JHDM1D-mediated TSP1/TGFβ/SMAD3 autocrine
loop, contributing to OF phenotype characterized by suppressed bone formation and
aggressive stromal tissue growth.

58
3.2 Materials and Methods

3.2.1 Animals

Female immunocompromised mice (Beige nu/nu XIDIII) were purchased from Harlan.
All animal experiments were performed under institutionally approved protocols for
the use of animal research at the University of Southern California (USC#11141 and
11327).

3.2.2 Reagents and antibodies
TGFβ1, TSP1, and JAGGDED1 were purchased from R&D Systems. TβRI inhibitors
SB431542 and Notch pathway inhibitor DAPT were purchased from the
Sigma-Aldrich
®
Corporation. Block peptide LSKL and control peptide SLLK for TSP1
were obtained from AnaSpec. Anti-STRO-1 antibody was obtained from Caltag
Laboratories. Anti-CD146-PE, CD105-PE, CD90-PE, CD34-PE and CD45-PE
antibodies were purchased from BD Bioscience. Anti-PDGFRα antibody was
purchased from Abcam. Anti-human-specific mitochondria antibody was purchased
from Millipore. Anti-phosphorylated SMAD2, phosphorylated SMAD3, SMAD2,
SMAD3, SMAD4 and Notch3 were purchased from Cell Signaling. Anti-active TGFβ1
and TSP1 antibodies were purchased from R&D. Anti-JHDM1D, H3K9me1, H3K9me2,
H3K27me2 and H4K20me1 antibodies were purchased from Abcam. Anti-TGFβ
receptor I, TGFβ receptor II and JAGGED1 were purchased from Santa Cruz.
59
3.2.3 Primary cell cultures

Human ossifying fibroma tissues from surgical resection were obtained with written
informed consent and IRB approval from the University of Southern California
(HS-07-00701). Aseptically minced tumor samples were digested with 200 U/ml
collagenase I (Sigma) and 1mg ml
-1
dispase (Sigma) in PBS for 2 hr in a 37 ℃ water
bath with intermittent agitation. The cell suspension was filtered through a 70 μm cell
strainer (BD Falcon) to remove undigested tissue mass, centrifuged, and
resuspended in complete MSC culture medium (α-MEM containing 15% FBS, 100
mΜ L-ascorbic acid, 100 U/ml penicillin, 100 μg ml
-1
streptomycin and 2 mM
L-glutamine). The cells were cultured on a nontreated plastic dish (BD Falcon) and
rinsed twice with PBS to remove nonadherent cells. Confluent cells were passaged
with treatment of 0.05% trypsin and 1 mM EDTA (Invitrogen). Normal jaw bone MSC
(JMSC) culture was established from bone marrow of human jaw bone, according to a
previously reported MSC isolation protocol (Yamaza et al., 2011). Briefly, 2×10
6
whole
bone marrow cells were centrifuged and resuspended in MSC culture medium. The
cells were cultured on a nontreated plastic dish (BD Falcon) and rinsed twice with
PBS to remove nonadherent cells. For long-term TGFβ1 treatment, the cells were
treated with TGFβ1 for 5 days and then cultured in complete MSC medium. We
established primary OFMSC culture from four surgically resected OF samples, one
from each of four patients (#1-4), which were radiographically and histologically
diagnosed as OF lesions with no reactive normal bone around the lesions. JMSCs
60
were derived from a pool of normal JMSC samples (n=4) and served as the control
group.

3.2.4 Subcutaneous implantation of MSCs

For each subcutaneous implantation, 2×10
6
culture-expanded cells were mixed with
40 mg hydroxyapatite/tricalcium phosphate (HA, Berkeley Advanced Biomaterials) or
gelfoam in 0.5 ml medium and incubated at 37 ℃ for 90 min. After a brief centrifuge to
remove supernatant, cells and carrier were subcutaneously implanted into the dorsal
surface of female immunocompromised mice. For implants with long-term TGFβ1
treatment, the cells were treated with TGFβ1 for 5 days prior to implantation. Samples
were harvested at 8 weeks post-implantation for further analysis. The percentage of
bone volume out of the total volume of each implant was analyzed by NIH Image J as
described previously (Liu et al., 2011; Shi et al., 2002). Briefly, five representative
fields of hematoxylin and eosin (H&E) staining were selected, and newly generated
mineralized tissue area in each field was calculated as a percentage of total tissue
area.

61
3.2.5 Isolation of single colony clusters

10
4
cells in suspension were seeded in a 60 mm nontreated culture dish (BD Falcon)
in complete MSC medium and cultured for 2 weeks with a change of medium every
other day. Culture medium was removed and cells were stained by 1% toluidine blue
containing 2% PFA at room temperature overnight. Cells were then washed 3 times
with distilled water. A cell colony with more than 50 cells was counted as a Colony
Forming Unit-fibroblast (CFU-f). Each test was repeated 5 times.

3.2.6 Single-cell serial implantation

Single-cell cultures from OFMSCs or JMSCs were established by limited dilution and
seeded in a 96-well plate at the concentration of 1 cell/well. Microscopic observation
was used to confirm the presence of single cells in the wells. Cultures from single
cells were expanded to 2×10
6
cells and implanted with gelfoam into
immunocompromised mice subcutaneously. After 3 weeks of growth, implants (1
o

implant) were harvested and cells from the implants were isolated and cultured
according to primary cell culture protocol. Single-cell cultures were subsequently
established from these implanted cells and expanded for a second round of
implantation (2
o
implant). Implant tissues and cells were analyzed by
immunocytochemistry or single colony cluster isolation assays.
62
3.2.7 In vitro differentiation

For osteogenic differentiation, cells were grown to 100% confluence in MSC culture
medium followed by 4 weeks of exposure to osteogenic medium consisting of MSC
medium with 1.8 mM KH
2
PO
4
and 10
-8
M dexamethasone sodium phosphate. Cells
were subsequently fixed with 70% isopropanol and stained with 1% alizarin red
(Sigma) to assess mineralized nodule formation or osteogenic gene expression. For
inhibition of TGFβ signaling during osteogenic differentiation, 1 μM TGFβ receptor I
inhibitor SB431542 was added to the osteogenic medium. For osteogenic
differentiation after long-term TGFβ1 treatment, cells were treated with TGFβ1 for 5
days and then underwent osteogenic induction. For adipogenic differentiation, cells
were grown to 100% confluence in MSC culture medium followed by 4 weeks of
exposure to adipogenic medium consisting of MSC medium with 0.5 mM
isobutylmethylxanthin, 60 μM indomethacin, 60 μM Indomethacin and 10 μg ml-1
insulin. Cells were subsequently fixed with 60% isopropanol and stained with 0.18%
Oil red O. For chondrogenic differentiation, 1×10
6
cells were centrifuged in a 15ml
conical tube and cell pellets were cultured for 4 weeks in chondrogenic medium
containing regular MSC medium with 1% ITS, 100 nM dexamethasone sodium
phosphate, 2 mM sodium pyruvate and 10 ng ml-1 TGFβ3. Cell pellets were
embedded in paraffin for histological analysis.

63
3.2.8 Histological and immunohistochemical analysis

Tumor samples and mouse implantation samples were fixed in 4% PFA for 48 hours,
decalcified in 10% EDTA with 4% sucrose for 30 days, and subsequently embedded in
paraffin for H&E staining. For sirius red staining, tissue sections were stained with 0.1%
sirius red for 1 hour, and subsequently, hematoxylin was used for counterstaining.
Masson’s trichrome staining was performed according to the standard protocol. Briefly,
tissue sections were stained in accordance with the following procedures: 1) Bouin's
solution containing picric acid, formaldehyde, and Glacial acetic acid for 1 hour at 56
o
C; 2) Weigert's iron hematoxylin working solution containing ferric chloride,
hydrochloric acid, and hematoxylin for 10 minutes; 3) Biebrich scarlet-acid fuchsin
solution containing Biebrich scarlet-acid fuchsin and acetic acid for 10-15 minutes; 4)
Differentiate in phosphomolybdic-phosphotungstic acid solution containing
phosphomolybdic acid and phosphotungstic acid for 10-15 minutes; 5) Aniline blue
solution containing aniline blue and acetic acid for 5-10 minutes; 6) Differentiate in 1%
acetic acid solution for 2-5 minutes. Histostain-SP kit (Invitrogen) was used for
immunohistochemistry according to the manufacture’s instruction with using the
following antibodies: anti-PCNA (Santa Cruz), anti-SMAD2 (Cell Signaling),
anti-JAGGED1 (Santa Cruz), anti-Notch3 (Cell Signal), anti-HES1 (Sigma)
anti-Thrombospondin1 (Clone A6.1, Thermo Scientific), and anti-JHDM1D (Abcam).

64
3.2.9 Flow cytometric analysis

Culture-expanded cells were collected and stained with anti-STRO-1, anti-CD146,
anti-CD90, anti-CD105, anti-CD34, anti-CD45, and anti-PDGFRα antibodies. The
percentage of positive cells was analyzed by FACScaliber (BD Bioscience). For
isolation of CD146
+
STRO-1
+
cells, MSCs were incubated with rat anti-human
CD146-PE and mouse anti-human STRO-1 antibodies for 30 min, then cells were
incubated with goat anti-mouse IgG-FITC for 30 min, followed by sorting separation
using a BD SORP FACS Aria I Flow Cytometer to isolate CD146
+
STRO-1
+
and
CD146
-
STRO-1
-
subpopulations, according to manufacturer’s instructions.

3.2.10 Small interfering RNA (siRNA)

Cells in a 6-well plate were cultured until 70% confluent. Transfection cocktails were
prepared by mixing 6 μl 10μM siRNA (Santa Cruz) and 6 μl lipofectamine RNAiMAX
(Invitrogen) in 200 μl Opti-MEM (Invitrogen) and then incubated at room temperature
for 30min. Cells were washed twice with Opti-MEM and kept in 0.8 ml Opti-MEM, after
which the transfection cocktail was added drop by drop. After incubation at 37
o
C for 5
hours, the cells were added to 1 ml MSC culture medium. The knockdown efficiency
was verified by Western blot.
65

3.2.11 BrdU staining

Cells were labeled with 10 μM BrdU overnight and cell proliferation rate was assessed
using a BrdU staining kit (Invitrogen), according to the manufacturer’s instructions.
Positive cells were detected using a streptavidin-biotin system and counterstained by
hematoxylin.

3.2.12 Western blot

Cells in 6-well plates were lysed by RIPA buffer plus protease inhibitor cocktail (Sigma)
prepared according to the manufacturer’s instructions. 20 mg proteins were loaded
and separated by SDS-PAGE (Invitrogen), transferred onto a PVDF (Bio-Rad)
membrane incubated in blocking buffer and 5% non-fat dry milk (Santa Cruz) in TBS,
analyzed with the indicated antibodies and developed using a chemiluminescence kit
(Thermo scientific). Active TGFβ1 was detected under non-reducing conditions.

66
3.2.13 Microarray

Total RNAs were extracted from JMSCs and OFMSCs with an RNeasy kit (Qiagen).
Microarray assays were performed in the Genome Center of Children’s Hospital, Los
Angeles, using an Affymetrix Human Genome U133 Plus 2.0 Array. Partek Genomics
Suite and Ingenuity Pathway Analysis (IPA) software were used for data analysis.

3.2.14 Quantitative real-time PCR

RNA was extracted using an RNeasy mini kit (Qiagen) following the manufacturer’s
protocols. 1 μg of purified RNA from cells was used for reverse-transcription with a
QuantiTect Rev. Transcription kit (Qiagen). Real-time PCR was subsequently
performed on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Primer
sequences are listed in Table 2.

3.2.15 ELISA
A human TGFβ1 ELISA kit (R&D) was used for quantification of latent and active
TGFβ1 following manufacturer’s specifications. Latent TGFβ1 in cell supernatant was
determined by activation of hydrogen chloride. 1 ml cell supernatant was
concentrated to 50μl for active TGFβ1 quantification.
67
3.2.16 Luciferase assay

Plasmids used in the luciferase assay include pTsp1-luc (Addgene), pSBE4-luc
(Addgene) and pCMV-RL (Promega). JMSCs and OFMSCs were starved for 24 h in
medium containing 2% FBS and co-transfected with pCMV-RL and each of the other
plasmids at the ratio of 1:20 using lipofectamine LTX transfection reagent (Invitrogen),
according to the manufacturer’s instructions. Cells were treated with 2 ng ml-1 TGFβ1
or 1 M SB431542 for 16 h, and lysate was prepared using a Dual-Luciferase®
Reporter (DLR™) Assay System (Promega). Luminescence was measured by
luminometer (Promega). Firefly luminescence was normalized to renilla
luminescence.

3.2.17 ChIP-PCR

Cells were treated with a Branson sonicator to lyse nuclei and shear chromatin.
Immunoprecipitation was performed with a Millipore ChIP kit, according to the
manufacturer’s protocol. To precipitate Tsp1 promoter complexes, the indicated
antibodies were used, and goat IgG was used as an isotype control. The precipitated
Tsp1 promoter fragments were purified by phenol: chloroform extraction and used for
quantitative PCR analysis. Percentage input was determined by removing an aliquot
of sheared chromatin prior to immunoprecipitation.
68

3.2.18 Statistical analysis

P-values were obtained from two-tailed Student’s t test or one-way analysis of
variance (ANOVA) followed by Student Neuman Keuls test using SPSS 13.0.
Spearman correlation test was used to analyze relations between p-SMAD2 and
PCNA expression. A P-value less than 0.05 was considered significant.

3.2.19 Accession number

The raw expression results for microarray were deposited in Gene Expression
Omnibus under the study GSE49165.

69
Table 1. Differentially expressed genes between OFMSCs and JMSCs. Related to
Figure 3.2. Global gene expression between OFMSCs and JMSCs was compared by
Affymetrix Human Genome U133 Plus 2.0 Array analysis, followed by raw data
analysis using Partek Genomics Suite. P-value and fold change of gene expression of
TGF , BMP, and Notch signaling were shown.

70
Table 2. PCR Primers. Related to Figure 3.2,3.3,3.4,3.5

71
3.3 Results

3.3.1 Benign ossifying fibroma (OF) contains mesenchymal stem
cells (OFMSCs)

In order to determine whether OF tumors contain progenitor cells that are capable of
regenerating OF-like lesions, we established primary MSC culture from four surgically
resected OF samples, which were clinically diagnosed and histologically confirmed as
OF benign neoplasm. These OF-derived MSCs (OFMSCs) showed morphology
similar to normal jaw bone-derived MSCs (JMSCs) and expressed distinct MSC
markers STRO-1, CD90, CD146 and CD105, but not the hematopoietic lineage
markers CD34 and CD45 (Figure S3.1A). A small subset of OFMSCs also expressed
PDGFR-α, which is marker of multipotent MSCs known from murine bone marrow
(Figure S3.1A) (Morikawa et al., 2009). Compared to the control JMSCs, OFMSCs
are capable of generating more single colony clusters and have elevated rates of
proliferation and population doubling (Figures 3.1A, S3.1C); immunohistochemical
staining further confirmed that OF tumor samples contain more abundant
PCNA-labeled proliferative cells as than normal jaw bones (Figure 3.1B).
To evaluate their differentiation potential, we demonstrated that OFMSCs exhibited
decreased osteogenic differentiation capacity compared to control JMSCs, shown by
diminished mineralized nodule formation and reduced expression of osteogenic
72
markers runt-related transcription factor 2 (Runx2) and osteocalcin (Ocn) (Figures
3.1C, S3.1B, S3.1D, S3.1E). OFMSCs also exhibited decreased adipogenic
differentiation, as indicated by a reduced number of Oil red O-positive adipocytes and
downregulation of adipogenic markers peroxisome proliferator-activated receptor
gamma 2 (Ppar γ2) and lipoprotein lipase (Lpl) (Figures 3.1D, S3.1B, S3.1D, S3.1E).
In addition, OFMSCs showed a chondrogenic differentiation deficiency, shown by a
reduced number of toluidine blue-positive chondrocytes and suppressed expression
of Collagen II and Sox9 (Figures 3.1E, S3.1B, S3.1D, S3.1E). When OFMSCs were
subcutaneously implanted into immunocompromised mice with
hydroxyapatite-tricalcium phosphate (HA) as a carrier, OFMSCs regained
histo-pathological features of OF lesions, characterized by impaired bone formation
and increased growth of stromal tissue, while control JMSC implants did not (Figure
3.1F).
To demonstrate the specific role of OFMSCs in OF formation, we isolated cells based
on 2 widely used markers for human mesenchymal stem cells, STRO-1 and CD146
(Bianco et al., 2013; Sacchetti et al., 2007). When implanted into
immunocompromised mice subcutaneously, only STRO-1
+
/CD146
+
OFMSCs were
capable of generating OF-like lesions with scattered bone nodules and highly
proliferative stromal cells, as indicated by PCNA staining; implantation of
STRO-1
-
/CD146
-
cells failed to induce OF-like lesions or mineralized tissue (Figure
S3.1F).
Since MSCs have been recognized as a heterogeneous cell population containing
73
different sub-populations of stem cells with variable proliferation and differentiation
capacities, we further characterized single colony-derived OFMSCs. These OFMSC
colonies exhibited a wide range of enhanced population doubling and proliferation
rates, as well as suppressed in vitro osteogenic activity, similar to colonies derived
from normal MSCs (Figure S3.1G). Importantly, these single colony-derived OFMSCs
are also capable of regenerating OF-like lesions, as observed in OFMSC implantation
(Figure S3.1H). These results indicate that OFMSCs exhibit a multi-lineage
differentiation deficiency, a feature exhibited by tumor progenitor cells.
To characterize the in vivo self-renewal property of OFMSCs, we performed serial
implantation of single colony-derived OFMSCs (Figure 3.1G). We found that single
colony-derived OFMSCs expanded in culture were able to generate prominent
stromal tissue when subcutaneously implanted into immunocompromised mice using
gelfoam as a carrier vehicle (1
o
implant), while JMSC implants were not (Figure 3.1H).
Next, we isolated and expanded OFMSCs from the 1
o
implant and subcutaneously
implanted them into immunocompromised mice. The secondary OFMSCs were also
capable of regenerating stromal tissue (2
o
implant). OFMSCs isolated from both 1
o

and 2
o
implants were capable of forming single colony clusters (CFU-Fs) (Figure
3.1H), maintaining osteogenic and adipogenic differentiation potential (Figure 1I) and
displaying similar numbers of population doublings and proliferation rates to those
observed in primary OFMSCs (Figure 3.1J). To confirm that OFMSCs contributed to
stromal tissue growth in the implants, we showed that cells in both 1
o
and 2
o
implants
were positively stained for anti-human-specific mitochondrial antibody (Figure 3.1H).
74
These data imply that single colony-derived OFMSCs are capable of self-renewal and
differentiation in vivo.

Figure 3.1. OF lesions contain mesenchymal stem cells (OFMSCs). (A) OFMSCs
generated more single colony clusters (Colony-Forming Unit fibroblasts, CFU-f) than
control JMSCs when cultured at a low density. Culture-expanded OFMSCs showed
an elevated number of BrdU-positive cells compared to the control JMSCs. Continued
culture assay showed that OFMSCs have a higher rate of population doubling than
JMSCs. (B) Immunohistochemical staining showed that OF samples contain more
PCNA-positive cells than do normal jaw bone. (C) Alizarin red staining showed that
OFMSCs exhibited decreased capacity to form mineralized nodules under osteogenic
inductive culture conditions compared to JMSCs. In addition, OFMSCs showed
downregulation of the osteogenic markers RUNX2 and OCN, as assessed by
Western blot analysis. (D) OFMSCs exhibited decreased capacities to form Oil red
O-positive adipocytes under adipogenic inductive conditions and express the
adipogenic markers PPARγ2 and LPL, as shown by Western blot analysis. (E)
OFMSCs showed decreased capacity for chondrogenic differentiation, as indicated by
forming less toluidine blue-positive chondrocytes when cultured under chondrogenic
conditions and expressing lower levels of Sox9 and Collagen II as confirmed by
Western blot analysis. (F) Left panel: HE section of normal human jawbone and OF
tissue as indicated. OF lesion contained a reduced amount of bone (B) and increased
stromal connective tissue (CT) compared to the control jaw bone. Right panel: when
implanted into immunocompromised mice with hydroxyapatite/tricalcium phosphate
(HA), OFMSCs regenerated less bone and more stromal connective tissue than
75
JMSCs, recapitulating original OF characteristics. Quantitative analysis of bone
volume by ImageJ showed a significant reduction of bone volume in OFMSC implants.
(G) Schema of serial implantation of single colony-derived MSCs. Single
colony-derived MSCs were expanded to 2 ✕ 10
6
cells and subsequently implanted into
immunocompromised mice using gelfoam as a carrier (1
o
implant). After 3 weeks, 1
o

implants were harvested and enzymatically digested to release single cells for in vitro
expansion. 2×10
6
human cells from 1
o
implants were implanted into
immunocompromised mice to form 2
o
OFMSC implants. (H) Single colony derived
OFMSCs were capable of forming larger stromal tissue masses in gelfoam implants
and generating more single colony clusters (CFU-f) than control JMSCs. Human
mitochondria immunohistochemical staining confirmed that cells isolated from 1
o
and
2
o
MSC implants were of human origin. (I) OFMSCs derived from 1
o
and 2
o
implants
showed the same capacity as parental OFMSCs to form mineralized nodules, as
assessed by alizarin red staining, and adipocytes, as assessed by Oil red O staining.
(J) BrdU labeling assays showed that OFMSCs derived from 1
o
and 2
o
implants
possess proliferation capacities similar to parent OFSMCs. Continued culture assays
showed that OFMSCs derived from 1
o
and 2
o
implants had population doubling rates
similar to parent OFSMCs. Data are represented as mean±SD of five independent
experiments. (*, p<0.05, **, p<0.01). Scale bar, 100 μm (F) or 50 μm (H). See also
Figure S3.1 and Table 2.

76

Figure S3.1. OF lesions contain mesenchymal stem cells (OFMSCs). Related to
Figure 3.1. (A) Flow cytometric analysis showed that OFMSCs expressed MSC
surface molecules STRO-1, CD146, CD90, and CD105, but failed to express the
hematopoietic markers CD34 and CD45. A small percentage of OFMSCs also
expressed PDGFRα. (B) Quantitative PCR analysis showed that OFMSCs expressed
reduced levels of osteogenic markers Runx2 and Ocn, adopgenic markers Ppar γ2
and Lpl, and chondogenic markers Sox9 and Collagen II compared to control JMSCs.
(C) OFMSCs from the 3
rd
and 4
th
patients showed increased capacity to form single
colony clusters (CFU-f, left panel), increased proliferation rate, as assessed by BrdU
staining (middle panel), and increased population doubling rate (right panel)
compared to control JMSCs. (D) OFMSCs showed reduced capacities to form
mineralized nodules, as assessed by alizarin red staining (left panel), adipogenic
differentiation, as assessed by Oil red O staining (middle panel), and chondrogenic
differentiation, as assessed by toliudine blue staining (right panel). (E) Quantitative
PCR analysis indicated that OFMSCs expressed reduced levels of osteogneic
markers Runx2 and Ocn, reduced adipogenic markers Ppar γ2 and Lpl, and reduced
chondrogenic markers Sox9 and Collagen II compared to control JMSCs. (F) When
2×10
6
CD146
+
/STRO-1
+
OFMSCs were implanted into immunocompromised mice
subcutaneously using HA/TCP as a carrier, they generated OF-like lesions with less
bone formation and more PCNA-positive cells than were associated with JMSC
implants. However, implants of CD146
-
/STRO-1
-
cells using the same number of cells
failed to generate OF-like lesions or mineralized tissue. B: bone; HA:
hydroxyapatite/tricalcium phosphate; CT: connective tissue. (G) Single colony-derived
OFMSCs (n=10) showed variable rate of population doubling as evaluated by
continued culture assay, proliferation as evaluated by BrdU staining, and mineralized
nodule formation as evaluated by alizarin red staining, which were similar to those
observed in single colony-derived JMSCs. (H) H&E staining showed that implants of
2×10
6
single colony-derived OFMSCs (n=5) with HA/TCP as a carrier were capable of
77
forming OF-like structures with reduced amounts of bone and elevated amounts of
stromal tissue compared to the control JMSC implants. B: bone; HA:
hydroxyapatite/tricalcium phosphate; CT: connective tissue. Bone volume
quantification analyzed by ImageJ indicated that single colony-derived OFMSCs
generated much less bone than JMSCs. Data are represented as mean±SD of five
independent experiments. (*, p<0.05, **, p<0.01) Scale bar, 50 μm.

78
3.3.2 TGFβ signaling is highly activated in OFMSCs

We next determined the molecular mechanisms responsible for the increased
self-renewal and decreased osteogenic differentiation of OFMSCs. Although Hrpt2
mutation and haplo-insufficient expression have been reported in roughly 10% of
patients with OF lesions (Pimenta et al., 2006), the OF tumors from the four patients
recruited in this study did not harbor any Hrpt2 mutations. We compared the global
gene profile of OFMSCs and JMSCs using microarray and cluster analysis (Figure
S3.2A). Gene ontology by ingenuity pathway analysis (IPA) demonstrated that certain
genes in the TGFβ and Notch signaling pathways were highly differentially expressed,
being enriched in OFMSCs (Figure S3.2B). Given the fact that TGFβ signaling is
involved in mesenchymal cell proliferation and tumor development, we next
investigated the role of TGFβ in the signaling regulation of OFMSCs (Bruna et al.,
2007; Ikushima et al., 2009; Penuelas et al., 2009).
TGFβ1 is secreted into extracellular matrix in a latent form, which is subsequently
cleaved into an active form to serve its regulatory function (Pedrozo et al., 1999;
Pfeilschifter et al., 1990). As shown by ELISA, activated TGFβ1 was increased in the
culture supernatant of OFMSCs, compared to control JMSCs (Figures 3.2A, S3.2D).
Western blot analysis confirmed that activated TGFβ1, but not latent TGFβ1, was
upregulated in OFMSCs (Figures 3.2B, S3.2E). At the receptor level, expression of
TGFβ receptors I and II in OFMSCs was slightly decreased, as compared to control
79
JMSCs (Figure S3.2C). To confirm the up-regulated TGFβ1 activity in OFMSCs, we
evaluated downstream targets of the canonical TGFβ pathway, SMAD2/3/4.
Interestingly, OFMSCs showed elevated phosphorylated SMAD3 when compared to
JMSCs, while phosphorylated SMAD3 was not detected in JMSCs (Figures 3.2C,
S3.2F). In addition, expression of total SMAD3 and SMAD4 were consistently higher
in OFMSCs than in JMSCs (Figures 3.2C, S3.2F). When induced with TGFβ1, a
marked upregulation of phosphorylated SMAD3 and the TGFβ downstream target
genes Pai-1 and Smad7 was observed in OFMSCs, as compared to TGFβ1-treated
JMSCs (Figures 3.2C, 3.2D). Functional analysis showed higher SBE4-luciferase
activity in OFMSCs as compared to JMSCs, both at basal level and in response to
TGFβ1 treatment, providing further evidence for the upregulation of TGFβ signaling in
OFMSCs (Figure 3.2E). To further confirm the TGFβ signaling activity in OF lesions,
we examined a total of 11 archival OF pathologic samples with confirmed histological
diagnosis by pathologists. Immunohistochemical staining revealed upregulation of
TGFβ signaling in all these specimens, shown by a significant increase in
phosphorylated SMAD2 as compared to normal jawbone (Figures S3.2G and S3.2G’);
the cellular components also displayed abundant PCNA-positive cells, consistent with
an elevated proliferation rate. Statistical analysis using Spearman’s correlation test
showed a significant correlation between phosphorylated SMAD2 activity and the
number of PCNA-positive cells, indicating that elevated TGFβ signaling may
contribute to increased cell proliferation in OF neoplasm (Figure S3.2H). The elevated
level of phosphorylated SMAD2 was also confirmed in the in vivo OFMSC implants
80
generated from OFMSCs derived from all 4 patients (Figure S3.2I). Further studies
of the TGFβ1-mediated noncanonical pathways showed no significant changes in
ERK, p38, and JNK signaling in OFMSCs as compared to JMSCs (Figure S3.3A).
To further assess the functional role of TGFβ in OFMSCs, we blocked TGFβ signaling
using TGFβ receptor I (TβRI) inhibitor, SB431542, and observed suppression of
TGFβ1 downstream targets Pai-1 and Smad7 in OFMSCs (Figure S3.3B).
Interestingly, blockage of TGFβ signaling significantly enhanced osteogenic
differentiation of OFMSCs, as indicated by improved mineralized nodule formation,
upregulation of RUNX2 and OCN, and elevated in vivo bone formation, compared to
untreated control OFMSCs (Figures 3.2F, 3.2G). However, SB431542 treatment did
not improve osteogenic activity of JMSCs either in vitro or in vivo (Figures S3.3C and
S3.3D). More convincingly, we demonstrated that inhibition of TGFβ receptor I
rescued bone formation in the in vivo implants derived from STRO-1
+
/CD146
+

OFMSCs (Fig. S3.3E), but not those derived from STRO-1
-
/CD146
-
cells (Fig. S3.3E).
The enhanced osteogenic differentiation by SB431542 treatment was also observed
in OFMSCs from 1
o
OFMSC implants, suggesting that activation of TGFβ signaling is
inherited in the serial in vivo implantation (Figure 3.2H). Additionally, we showed that
efficient inhibition of TGFβ signaling by TGFβ receptor I ( T β r I) or Smad3 siRNA
(Figures S3.3F, S3.3G) led to a significant rescue of osteogenic deficiency in
OFMSCs, as evidenced by increased mineralized nodule formation and expression of
RUNX2 and OCN (Figures 3.2I, 3.2J, S3.3H). Moreover, inhibition of TGFβ signaling
by Smad3 siRNA also improved OFMSC-mediated bone formation when OFMSCs
81
were implanted into immunocompromised mice with HA/TCP as a carrier (Figure
S3.3I). In addition to osteogenic rescue, SB431542 treatment suppressed
TGFβ-mediated OFMSC proliferation, possibly regulating stromal cell growth in OF
lesions (Figure S3.3J). Collectively, these results show that TGFβ signaling is highly
activated in OFMSCs and responsible for their osteogenic deficiency and elevated
rate of proliferation (Figure S3.3K).

Figure 3.2. TGFβ signaling is upregulated in OFMSCs. (A) ELISA analysis showed
that the levels of active TGFβ1 were increased in OFMSC culture supernatant
compared to JMSC cultures. (B, C) Western blots showed that the expression levels
of activated TGFβ1, but not latent TGFβ1, were significantly increased in OFMSCs
(B). Compared to control JMSCs, OFMSCs expressed elevated levels of SMAD3,
SMAD4 and phosphorylated SMAD3. When treated with 2 ng ml
-1
TGFβ1, OFMSCs
showed more significant elevation of p-SMAD3, but the expression levels of SMAD3
and SMAD4 showed no significant change (C). (D) When treated with 2 ng ml
-1
TGFβ1, the expression levels of the TGFβ/SMAD3 downstream genes Pai-1 and
Smad7 were significantly upregulated in OFMSCs in comparison to the control
82
JMSCs, as determined by real-time PCR. (E) OFMSCs showed increased
SBE4-luciferase activity compared to JMSCs. 2 ng ml
-1
TGF-β1 treatment resulted in
significantly higher SBE4-luciferase activity in OFMSCs than in JMSCs. (F, G) In vitro
blockage of TGFβ signaling by 1 μM TβRI inhibitor SB431542 treatment resulted in a
marked upregualtion of the ostegenic markers RUNX2 and OCN in OFMSCs, along
with elevated capacity to form mineralized nodules in vitro, as assessed by alizarin
red staining (F). When implanted into immunocompromised mice with
hydroxyapatite/tricalcium phosphate (HA), SB431542-treated OFMSCs showed a
significantly increased capacity to form bone (B) in vivo, as shown by H&E staining
(G). (H) 1μM SB431542 treatment also improved osteogenic differentiation of
OFMSCs derived from 1
o
implants, as determined by alizarin red staining to show
increased mineralized nodule formation and Western blot to show upregulation of the
osteogenic genes RUNX2 and OCN. (I, J) Knockdown of T β r I or Smad3 by siRNA
elevated osteogenic differentiation of OFMSCs, as indicated by alizarin red staining
for mineralized nodule formation and Western blot for expression of the ostegenic
markers RUNX2 and OCN. Data are represented as mean±SD of five independent
experiments. (*, p<0.05, **, p<0.01, ***, p<0.001). Scale bar, 100 μm. See also Figure
S3.2 and Table1, 2.

83

Figure S3.2. TGFβ signaling is highly activated in OFMSCs. Related to Figure 3.2.
(A) Microarray clustering analysis was performed to identify the genes that were
differentially expressed in JMSCs and OFMSCs. Red area indicates genes with
84
elevated expression in JMSCs and green area represents genes with decreased
expression in JMSCs compared to OFMSCs. (B) Gene ontology analysis of
differentially expressed genes between JMSCs and OFMSCs as evaluated by
Ingenuity Pathway Analysis. Y axis represents related signaling pathways, lower X
axis represents the ratio of the molecules that have changed in their function or
pathway, and upper X axis represents p-value. The yellow line represents the
threshold of significant difference. (C) Western blot analysis showed that the
expression levels of TGFβ1 receptors I and II were not upregulated in OFMSCs. (D-F)
ELISA analysis indicated that culture media of OFMSCs from the 3
rd
and 4
th
OF
patients contained significantly increased levels of active TGFβ1 (D). Western blot
analysis showed that although latent TGFβ levels were similar in OFMSCs and
JMSCs, active TGFβ levels were dramatically enriched in OFMSCs (E). Western blot
analysis showed that OFMSCs expressed elevated levels of phosphorylated SMAD3
(F). TGFβ1 (2 ng ml
-1
) treatment significantly upregulated expression of
phosphorylated SMAD3 in OFMSCs (F). (G-H) H&E staining of OF tissue sections
from 11 archival patient samples showed decreased bone formation and increased
stromal cellular components compared to normal jaw bone (G). Immunohistochemical
staining showed the OF tissue sections had elevated expression of phosphorylated
SMAD2 and PCNA (G). A Spearman correlation study on adjacent slides indicated
phosphorylated SMAD2 was related to PCNA expression level (H). (I)
Immunohistochemical staining of OFMSC implants showed that their levels of
phosphorylated SMAD2 were upregulated compared to control JMSC implants. Data
are represented as mean±SD of five independent experiments. (**, p<0.01). Scale bar,
50 μm.

85

Figure S3.3. TGFβ signaling is highly activated in OFMSCs. Related to Figure 3.2.
(A) Western blot analysis showed that 2 ng ml
-1
TGFβ1 treatment affected
noncanonical TGFβ signaling similarly in OFMSCs and JMSCs. (B) Quantitative PCR
analysis showed that inhibiting TGFβ signaling with 1 μM TβRI inhibitor SB431542
downregulated expression of Pai-1 and Smad7 in OFMSCs. (C, D) Treatment with 1
μM SB431542 failed to increase JMSC-mediated mineralized nodule formation, as
assessed by Alizarin red staining (C); expression of RUNX2 and OCN, as assessed
by Western blot (L); and in vivo bone formation when implanted into
immunocompromised mice subcutaneously using HA/TCP as a carrier (n=5) (D). (E)
However, treatment with 1 μM SB431542 treatment resulted in an elevated bone
formation in implanted CD146
+
/STRO-1
+
OFMSCs, but not in implanted
CD146
-
/STRO-1
-
cells (n=5). (F) Western blot analysis showed the efficiency of
and Smad3 siRNA knockdown. (G, H) Quantitative PCR analysis showed that Smad3
siRNA treatment downregulated Pai-1 and Smad7 expression (G) and upregulated
Runx2 and Ocn expression (H). (I) Smad3 siRNA treatment increased
OFMSC-mediated bone formation in vivo (n=5). B: bone; HA:
hydroxyapatite/tricalcium phosphate; CT: connective tissue. (J) BrdU staining showed
that TGFβ1 treatment increased the proliferation rate of OFMSCs, which can be
abrogated by SB431542 treatment. (K) Schema of TGFβ signaling in OFMSCs. Data
are represented as mean±SD of five independent experiments. (*, p<0.05, **, p<0.01,
#, not significant). Scale bar, 50 μm.

86
3.3.3 TGFβ inhibits BMP signaling to reduce bone formation and
activates Notch signaling to enhance stromal tissue growth

Next, we examined the mechanism whereby activated TGFβ signaling in OFMSCs
results in an OF phenotype, characterized by osteogenic deficiency and stromal
overgrowth. It has been reported that activated TGFβ is able to inhibit BMP signaling
(Chen et al., 2012), which is one of the major pathways controlling osteogenic
differentiation. Therefore, we examined whether activated TGFβ signaling blocked the
BMP pathway to inhibit osteogenic differentiation of OFMSCs. Quantitative PCR
analysis showed that levels of BMP2, 4, 5, 6, 7 were all decreased in OFMSCs
(Figure S3.4A). Given the fact that loss of BMP4-7, but not BMP2, can be
compensated by other BMPs during osteogenic cell differentiation (Chen et al., 2012),
we selected BMP2 as a representative ligand to study how BMP signaling regulates
OFMSCs. OFMSCs showed downregulated expression of BMP2 as well as BMP
downstream genes Smad6 and Id1 (Figure 3.3A). In response to BMP2 treatment,
OFMSCs also showed decreased expression of phosphorylated SMAD1 (Figure
3.3B). In addition, the expression levels of Id1 and Smad6 were downregulated in
OFMSCs in the presence of BMP2 (Figure 3.3C). Blockage of TGFβ signaling by
Smad3 siRNA or SB431542 led to upregulation of phosphorylated SMAD1 and
downstream targets Id1 and Smad6 in OFMSCs (Figures 3.3D, 3.3E). These data
indicate that TGFβ signaling downregulates BMP/SMAD1 signaling to inhibit
osteogenic differentiation of OFMSCs.
87
In addition to their osteogenic deficiency, OFMSCs were capable of active
proliferation, along with formation of abundant stromal tissue when implanted in vivo.
We next examined whether activated TGFβ signaling in OFMSCs contributes to
elevated cell proliferation and stromal tissue growth. Notch signaling is responsible for
tissue growth in a variety of tumors (Ranganathan et al., 2011), and its interaction with
TGFβ signaling has been described in normal basal stem cell activity in the prostate
(Valdez et al., 2012). Using microarray analysis, we showed that Notch signaling
genes were upregulated in OFMSCs as compared to JMSCs. After confirming
elevated expression of specific Notch signaling genes Jagged1, Notch3, and Hes1 in
OFMSCs and OF samples (Figures 3.3F, S3.4B), we showed that inhibition of TGFβ
signaling by TβRI inhibitor or Smad3 siRNA resulted in downregulation of these Notch
genes (Figures 3.3G, 3.3H), suggesting that TGFβ governs Notch signaling in
OFMSCs. To verify that Notch signaling activation contributes to OFMSC proliferation
and stromal tissue growth, we treated cells with Jagged1 siRNA (Figure S3.4C) or
Notch inhibitor DAPT and found that inhibition of the Notch pathway reduced the
proliferation rate of OFMSCs (Figures 3.3I). As expected, JAGGED1 treatment
elevated the OFMSC proliferation rate (Figure 3.3I). We further demonstrated that
TGFβ treatment enhanced the proliferation of OFMSCs, which was abrogated by
Jagged1 siRNA or DAPT treatment (Figure 3.3J). More compellingly, we showed that
SB431542 and DAPT treatment suppressed OFMSC-mediated stromal tissue growth
of the in vivo implants in immunocompromised mice (Figure 3.3K). Notch signaling
has been reported to maintain the undifferentiated state of both MSCs and
88
osteoblasts; therefore, osteogenic deficiency of OFMSCs may be affected by altered
Notch signaling (Engin and Lee, 2010). However, knockdown of Jagged1 expression
by siRNA failed to affect OFMSC-mediated in vitro calcification, as assessed by
alizarin red staining, and osteogenesis, as assessed by Western blot to show the
expression levels of RUNX2 and OCN (Figures S3.4C and S3.4D). These findings
suggest that TGFβ promotes OFMSC proliferation and stromal tissue growth via
upregulation of Notch signaling (Figure S3.4E).

Figure 3.3. TGFβ inhibits BMP signaling to reduce bone formation and activates
Notch signaling to enhance stromal cell growth. (A) Western blot analysis showed
that OFMSCs expressed reduced levels of BMP2 and the BMP downstream genes
Id1 and Smad6 compared to the control JMSCs. (B) When treated with BMP2 at 5-50
ng ml
-1
, OFMSCs showed reduced expression of phosphorylated SMAD1 in
comparison to the control JMSCs. (C) Real-time PCR analysis revealed that the
expression levels of the BMP downstream genes Id1 and Smad6 decreased in
OFMSCs in the presence of 5 ng ml
-1
BMP2, compared to the control JMSCs. (D)
Western blot analysis showed that knockdown of TGFβ signaling by Smad3 siRNA
resulted in upregulation of p-SMAD1 in OFMSCs. (E) TβRI inhibitor SB431542
treatment also upregulated the BMP downstream genes Id1 and Smad6, as assessed
by real-time PCR. (F) Western blot analysis revealed that OFMSCs had upregulated
expression of the Notch signaling genes Jagged1, Notch3 and Hes1 in comparison to
JMSCs. (G, H) Western blot analysis showed that inhibition of TGFβ signaling by 1
89
μM TβRI inhibitor SB431542 or Smad3 siRNA treatment resulted in downregulation of
the Notch signaling genes Jagged1, Notch3, and Hes1. (I) Activating Notch signaling
by 100 ng ml
-1
JAGGED1 treatment could elevate OFMSC proliferation. However,
blockage of the Notch pathway by Jagged1 siRNA or treatment with 1μM Notch
signaling inhibitor DAPT reduced the OFMSC proliferation rate, as shown by BrdU
labeling assays. (J) BrdU labeling assays indicated that TGFβ increased the
proliferation rate of OFMSCs, whereas DAPT or Jagged1 siRNA abrogated the effect.
(K) Blockage of TGFβ signaling by SB43154 treatment or Notch pathway by DAPT
treatment resulted in a significantly reduced growth of stromal tissue in implanted
OFMSCs. The OF-like tissue volume was calculated by the following formula:
volume=length × width
2
× 0.52. Data are represented as mean±SD of five
independent experiments. (*, p<0.05, **, p<0.01). See also Figure S3.3 and Table 2.

90

Figure S3.4. TGFβ inhibits BMP signaling to reduce bone formation and
activates Notch signaling to enhance stromal cell growth. Related to Figure 3.3.
(A) Quantitative PCR analysis showed that expression of BMP2, 4, 5, 6, and 7 was
downregulated in OFMSCs compared to control JMSCs. (B) Immunohistochemical
staining showed that the Notch signaling genes Jagged1, Notch3, and Hes1 were
more highly expressed in OF samples than in normal jaw bone. (C) Western blot
analysis confirmed the efficacy of Jagged1 siRNA treatment. (D) Jagged1 siRNA
treatment failed to affect OFMSC-mediated mineralized nodule formation, as
assessed by Alizarin red staining, and expression of Runx2 and OCN, as assessed by
Western blot analysis. (E) Schema of TGFβ treatment-mediated inhibition of BMP
signaling and activation of Notch signaling. Data are represented as mean±SD of five
independent experiments. (*, p<0.05, **, p<0.01, #, not significant).Scale bar, 50 μm
for C and 25 μm for D.

91
3.3.4 Upregulation of TSP1 contributes to activation of TGFβ
signaling in OFMSCs

Our data showed that although there appeared to be no significant increase in
expression of TGFβ receptors or latent TGFβ in OFMSCs from our 4 studied patients
as compared to JMSCs, activated TGFβ1 signaling and mediated target gene
expression were significantly enriched in OFMSCs (Figures 3.2A, 3.2B, S3.2C).
Therefore, we hypothesized that activation of TGFβ signaling in OFMSCs may
originate upstream of TβRI, such as perhaps with increased activation of TGFβ ligand.
TSP1 is known to facilitate the conversion of latent TGFβ to active TGFβ
(Murphy-Ullrich and Poczatek, 2000) and our microarray data identified elevated
expression of Tsp1 in OFMSCs. We confirmed that TSP1 was highly expressed in
OFMSCs and OF tissues by Western blot, real-time PCR and immunohistochemical
staining (Figures 3.4A-C). When exposed to TSP1, significant upregulation of
activated TGFβ was observed in OFMSCs, as assessed by Western blot and ELISA
analysis (Figures 3.4D, 3.4E). These results suggest that TSP1 contributes to
activation of TGFβ signaling in OFMSCs.
Next, we examined whether elevated TSP1 affected osteogenic activity in OFMSCs.
As shown by alizarin red staining, Tsp1 knockdown by siRNA or TSP1 block peptide
treatment in OFMSCs effectively increased mineralized nodule formation in vitro,
whereas control peptide treatment showed no effect (Figure 3.4F). Moreover,
92
elevated RUNX2 and OCN expression were induced in Tsp1 siRNA and block
peptide-treated OFMSCs (Figures 3.4F, 3.4G). These results indicate that elevated
TSP1 in OFMSCs promoted TGFβ signaling to negatively regulate osteogenic
differentiation. Consistent with the role of TGFβ signaling in stromal growth, Tsp1
knockdown by siRNA also decreased the OFMSC proliferation rate (Figure 4H).

Figure 3.4. Upregulation of TGFβ in OFMSCs results from elevated TSP1
expression. (A, B) Western blot analysis and real-time PCR showed that OFMSCs
had elevated expression of TSP1 compared to the control JMSCs. (C)
Immunohistochemical staining showed that OF samples contained more
TSP1-positive cells than did normal jaw bone. (D, E) TSP1 treatment increased
expression levels of activated TGFβ1 in OFMSCs, as assessed by Western blot (D)
and ELISA analysis (E). (F) Knockdown of Tsp1 expression by Tsp1 siRNA or 5 μM
TSP1 block peptide LSKL treatment improved osteogenic differentiation of OFMSCs,
as indicated by elevated mineralized nodule formation assessed by alizarin red
staining, and upregulation of the osteogenic markers RUNX2 and OCN, assessed by
Western blot analysis. 5 μM SLLK was used as a control peptide. (G) Efficacy of Tsp1
siRAN assessed by Western blot. (H) Knockdown of Tsp1 by siRNA can inhibit the
proliferation of OFMSCs. Data are represented as mean±SD of five independent
experiments. (*, p<0.05, **, p<0.01). Scale bar, 100 μm (F) or 50 μm (C).

93
3.3.5 Histone demethylase JHDM1D-mediated
TSP1/TGFβ/SMAD3 autocrine loop contributes to TGFβ activation

To further investigate the mechanism underlying TGFβ-driven benign tumor growth in
OF, we examined the regulatory circuit of Tsp1. We found that TGFβ1 treatment
upregulated Tsp1 expression in OFMSCs in a time-dependent manner, a property not
detected in JMSCs (Figure 3.5A). As confirmed by Western blot, TGFβ1 treatment
increased TSP1 expression in OFMSCs, but not in JMSCs (Figure 3.5B). Inhibition of
TGFβ signaling by TβRI inhibitor SB431542, Tβ r I siRNA, or Smad3 siRNA
significantly reduced TSP1 expression in OFMSCs (Figures 3.5B, 3.5C). These data
indicate that inhibition of TGFβ/SMAD3 signaling reduced the level of TSP1.
Different downstream gene expression in response to TGFβ may result from promoter
methylation status, as indicated by Pdgf-b expression in gliomas (Bruna et al., 2007).
The methylation status of Tsp1 promoter, however, showed no difference between
OFMSCs and JMSCs (Figure S3.5A), indicating that other epigenetic mechanisms
may regulate expression of Tsp1. Histone methylation and demethylation are
antagonistic mechanisms employed by cells to modulate gene expression. Of the
genes with different expression levels in OFMSCs and JMSCs, we identified JHDM1D,
a histone demethylase, to be essentially elevated in OFMSCs, which was further
confirmed by Western blot and immunhistochemical staining (Figures 3.5D, S3.5B).
JHDM1D, also known as KIAA1718, is part of a histone demethylation subfamily
94
along with PHF2 and PHF8. JHDM1D performs a versatile array of histone
modification and plays a pivotal role in regulating craniofacial development (Qi et al.,
2010). Among the JHDM1D-mediated histone modifications, we found a marked
suppression of the repressive histone modification marks H3K9me1, H3K9me2,
H3K27me2 and H4K20me1 in OFMSCs when compared to JMSCs (Figure 3.5D).
Knockdown of Jhdm1d by siRNA in OFMSCs induced downregulation of TSP1 and
Tsp1-luciferase activity, as well as active TGFβ, suggesting that Tsp1 expression and
active TGFβ level may be regulated by JHDM1D activity (Figures 3.5E, 3.5F, S3.5C).
Moreover, JHDM1D knockdown by siRNA significantly increased methylation levels of
H3K9, H3K27, and H4K20 in the promoter of the Tsp1 gene, indicating that JHDM1D
may be essential for regulation of methylation status (Figure S3.5D).
To further investigate the role of JHDM1D in TGFβ-mediated gene expression, we
found Jhdm1d siRNA could rescue TGFβ1-mediated downregulation of H3K9me2 in
OFMSCs (Figure 3.5G). We showed that increased levels of H3K9me1 and
H4K20me1 induced by TGFβ1 treatment was also reduced by knockdown of Jhdm1d
(Figure 3.5G). However, in JMSCs, although TGFβ1 treatment affected the level of
some histone modification marks, knockdown of Jhdm1d failed to alter their levels.
These data indicate that JHDM1D specifically regulates histone modification in
OFMSCs. To determine how JHDM1D regulates Tsp1 expression in OFMSCs, we
examined the JHDM1D binding site at the Tsp1 promoter by ChIP-PCR assay. Among
the different tested regions around the transcriptional start site (TSS) of the Tsp1 gene,
JHDM1D binds at the region ~1kb from TSS when treated with TGFβ1 (Figure 3.5H),
95
whereas no JHDM1D binding was detected in JMSCs (Figure 3.5I). TGFβ1-mediated
JHDM1D binding to the Tsp1 promoter was abrogated by Smad3 knockdown,
suggesting that SMAD3 signaling mediates the binding (Figure 3.5J). Consistent with
the Tsp1 expression, OFMSCs showed reduced repressive histone marks H3K9me1,
H3K9me2, H3K27me2 and H4K20me1 at the Tsp1 promoter in comparison to JMSCs
(Figure 3.5K). Inhibition of TGFβ signaling by TβRI inhibitor SB431542 increased
these repressive histone marks at the Tsp1 promoter (Figure 3.5L), consistent with
the decreased expression of TSP1 after treatment with the TβRI inhibitor SB431542
(Figure 3.5B). Given the fact that JHDM1D expression is highly elevated in OFMSCs
compared to JMSCs, SMAD3 may enhance JHDM1D transcription by binding to the
JHDM1D promoter. We further showed that SMAD3 can bind to the JHDM1D
promoter region, suggestive of transcriptional regulation of JHDM1D by SMAD3
(Figure S3.5E). These findings collectively suggest that activated TGFβ/SMAD3
signaling upregulates Tsp1 expression by histone modification and that TSP1 can
regulate the conversion of the latent form of TGFβ to the active form, thus establishing
a positive JHDM1D/TSP1/TGFβ/SMAD3 feedback loop in OFMSCs (Figure S3.5F).
96

Figure 3.5. Histone demethylase JHDM1D-mediated
JHDM1D/TSP1/TGFβ/SMAD3 autocrine loop contributes to TGFβ activation. (A)
When treated with 2 ng ml
-1
TGFβ1 for 0, 3, 6, and 12 hours, OFMSCs showed a
significantly increased production of Tsp1 compared with control JMSCs, as shown by
real-time PCR. (B) Western blot analysis showed that TGFβ treatment elevated TSP1
expression level in OFMSCs, but not in JMSCs. Upregulation of TSP1 in OFMSCs
was reduced by 1 μM TβRI inhibitor SB431542 or Tβr I siRNA treatment. (C) Western
blot analysis showed that Smad3 siRNA treatment significantly reduced
TGFβ1-induced TSP1 expression. (D) Western blot analysis showed that OFMSCs
expressed elevated levels of JHDM1D and reduced H3K9me1, H3K9me2,
H3K27me2 and H4K20me1. (E, F) Knockdown of Jhdm1d by siRNA induced
downregulation of TSP1 and activated TGFβ1 in OFMSCs, as determined by Western
blot analysis (E) and Tsp1-luciferase activity (F). (G) Western blot analysis showed
that 2 ng ml
-1
TGFβ1 treatment upregulated H3K9me1 and H4K20me1 and
downregulated H3K9me2 with no effect on H3K27me2 in OFMSCs. However, TGFβ1
treatment upregulated H3K9me2, H3K27me2 and H4K20me1 with no effect on
H3K9me1 in JMSCs. Knockdown of Jhdm1d by siRNA significantly increased
H3K9me2 levels and decreased the H3K9me1 and H4K20me1 levels in
TGFβ1-treated OFMSCs. Knockdown of Jhdm1d appeared to have no singnifcant
effect on H3K9me2 or H4K20me1 levels in TGFβ1-treated JMSCs. (H-J) ChIP-PCR
97
analysis indicated that JHDM1D bound to the Tsp1 promoter in the presence of 2 ng
ml
-1
TGFβ1 in OFMSCs (H), but not in JMSCs (I). Smad3 siRNA treatment resulted in
significantly reduced binding of JHDM1D to the Tsp1 promoter (J). (K) ChIP-PCR
analysis indicated a lower level of the repressive histone modification marks
H3K9me1, H3K9me2, H3K27me2 and H4K20me1 in the Tsp1 promoter in OFMSCs
compared to JMSCs (L) ChIP-PCR analysis showed that the levels of H3K9me1,
H3K9me2, H3K27me2 and H4K20me1 in the Tsp1 promoter were increased in
OFMSCs when treated with 1 μM TβRI inhibitor SB431542. Data are represented as
mean±SD of five independent experiments. (*, p<0.05, **, p<0.01, ***, p<0.001, #, not
significant). See also Figure S3.4 and Table 2.

98

Figure S3.5. Histone demethylase JHDM1D-mediated TSP1/TGFβ/SMAD3
autocrine loop contributes to activation of TGFβ signaling. Related to Figure 3.5.
(A) Tsp1 promoter methylation assay by bisulfite sequencing analysis for comparison
between JMSCs and OFMSCs. (B) Immunohistochemical staining showed that
JHDM1D was more highly expressed in OF samples than in control jaw bones. (C)
Efficacy of Jhdm1d siRNA was assessed by Western blot. (D) ChIP-PCR assay
indicated that knockdown of JHDM1D in OFMSCs by siRNA downregulated the levels
of H3K9, H3K27, and H4K20 at the Tsp1 promoter. (E) ChIP-PCR assay showed that
SMAD3 could bind to the JHDM1D promoter region in OFMSCs, which was identified
by PCR primer pair 2 and 3 amplification under the condition of 2 ng ml
-1
TGFβ1
treatment. However, SMAD3 failed to bind to the JHDM1D promoter region in JMSCs
under the same conditions. (F) Schema of JHDM1D-mediated TSP1/TGFβ/SMAD3
autocrine loop. Data are represented as mean±SD of five independent experiments.
(*, p<0.05, **, p<0.01, ***, p<0.001).Scale bar, 50 μm.

99
3.3.6 Establishment of TSP1/TGFβ/SMAD3 autocrine loop
converts normal MSCs to OF-like MSCs

Since previous study suggests that chronic TGFβ treatment can establish epigenetic
regulation of cell function (Bechtel et al., 2010), we next tested whether long-term
activation of TGFβ signaling in JMSCs could establish the
JHDM1D/TSP1/TGFβ/SMAD3 autocrine loop. Indeed, JMSCs treated with TGFβ1 for
5 days showed an elevated proliferation rate and reduced osteogenesis, as observed
in OFMSCs (Figures 3.6A, S3.6A). Sirius red and trichrome staining further confirmed
that TGFβ1-treated JMSCs exhibited an in vivo differentiation pattern similar to that
observed in OFMSCs, characterized by increased stromal tissue formation (Figure
3.6B). When implanted into immunocompromised mice using gelfoam as a carrier,
TGFβ1 treated JMSCs acquired aberrant stromal tissue growth in vivo, as observed in
OFMSC/gelfoam implants (Figure 3.6C). These results indicate that TGFβ1 can
increase the in vivo self-renewal capacity of JMSCs, a feature lacking in normal
JMSCs.
To examine whether long-term TGFβ1 treatment induces JMSCs to display molecular
characteristics of OFMSCs, we showed that TGFβ1 treatment induced upregulation of
JHDM1D and TSP1 in JMSCs (Figures 3.6D, S3.6B). Moreover, TGFβ1-treated
JMSCs showed downregulation of the BMP signaling genes Id1 and Smad6, as well
as upregulation of the Notch pathway genes Jagged1, Notch3, and Hes1 (Figures
100
S3.6C, S3.6D). Similar to OFMSCs, these TGFβ1-treated JMSCs showed enrichment
of JHDM1D at the Tsp1 promoter (Figure 3.6E). Smad3 knockdown by siRNA during
the process of TGFβ1 treatment abrogated JHDM1D binding (Figure S3.6E).
Accordingly, TGFβ1-treated JMSCs showed reductions in the histone marks
H3K9me1, H3K9me2, H3K27me2 and H4K20me1 (Figure 3.6F) at the Tsp1 promoter.
These data suggest that establishment of epigenetic regulation of
JHDM1D/TSP1/TGFβ/SMAD3 is an inducible mechanism following TGFβ1 treatment
in JMSCs with characteristics similar to OFMSCs.

Figure 3.6. Establishment of JHDM1D/TSP1/TGFβ/SMAD3 autocrine loop
converts normal MSCs to OF-like MSCs. (A) Alizarin red staining showed that
long-term TGFβ1 treatment reduced the capacity of JMSCs to form mineralized
nodules in vitro and bone formation in vivo in HA/TCP implants. Decreased
osteogenic differentiation was confirmed by Western blot analysis showing
downregulation of RUNX2 and OCN. (B) Trichrome and Sirius red staining indicated
that long-term TGFβ1 treatment induced fibro-osseous tissue growth in JMSC
implants, similar to OFMSC implants. (C) When implanted into immuocompromised
mice using gelfoam as a carrier, long-term TGFβ1 treatment elevated stromal tissue
formation to produce tumor-like soft tissue. The tumor volume was calculated by the
101
following formula: volume=length × width
2
× 0.52. (D) Western blot analysis showed
that long-term TGFβ1 treatment induced upregulation of TSP1 and JHDM1D in
JMSCs. (E) ChIP-PCR analysis showed JHDM1D binding to the Tsp1 promoter in
long-term TGFβ1-treated JMSCs. (F) ChIP-PCR analysis indicated lower levels of the
repressive histone modification marks H3K9me1, H3K9me2, H3K27me2 and
H4K20me1 in the Tsp1 promoter in longterm TGFβ1-treated JMSCs compared to
nontreated JMSCs. Data are represented as mean±SD of five independent
experiments. (*, p<0.05, **, p<0.01, ***, p<0.001). Scale bar, 100 μm (A, B) or 50 μm
(C). See also Figure S3.5.

Figure S3.6. Establishment of TSP1/TGFβ/SMAD3 autocrine converts normal
MSCs to OF-like MSCs. Related to Figure 3.6. (A) Long-term TGFβ1 treatment
resulted in an increased proliferation, as assessed by BrdU-incorporation assay in
JMSCs. (B) Continued TGFβ1 treatment induced a significant upregulation of Tsp1,
as assessed by real time PCR. (C, D) Western blot showed that long-term TGFβ1
treatment induced downregulation of BMP signaling genes Id1 and Smad6 (C) and
upregulation of Notch signaling genes Jagged1, Notch3, and Hes1 (D) in JMSCs. (E)
Smad3 siRNA treatment abrogated TGFβ1-induced JHDM1D binding to Tsp1
promoter. Data are represented as mean±SD of five independent experiments. (*,
p<0.05).

102
3.4 Discussion

Due to their relatively “benign” nature, benign tumors are frequently managed with
surgical excision as a therapeutic approach, but post-surgical recovery and malignant
transformation remain challenging (Colmenero-Ruiz et al., 2011; Zama et al., 2004). A
major question that could transform this therapeutic approach is whether cells derived
from benign tumors can be induced to return to a normal state. Multiple lines of
evidence have suggested that cancer stem cells in different types of malignant tumors
are responsible for sustaining tumorigenesis and functional heterogeneity (Visvader
and Lindeman, 2012); however, it remains unclear whether a similar population of
stem-like cells exists in benign neoplasm or dysplasia and, if so, how their molecular
characteristics differ from those of normal counterparts. In this study, we found that
ossifying fibroma, a benign neoplasm, contains a unique population of mesenchymal
stem cells (OFMSCs) that can recapitulate the parental tumor phenotype when
implanted in vivo and identified TGFβ signaling as a key regulator for their benign
tumor stem-like cell phenotype. Furthermore, we showed that inhibition of TGFβ
signaling can induce OFMSCs to convert to being “normal” MSCs with enhanced
osteogenic activity and a reduced proliferation rate, suggesting a potential therapeutic
avenue for guiding benign tumor stem cells to function normally.
OFMSCs are capable of forming single colony clusters, expressing specific stem cell
surface markers, responding to differentiating induction cues, and generating tumors
when implanted in vivo, thus meeting the primary criteria for classification as cancer
103
stem cells. More importantly, transplantation of OFMSCs can recapitulate parental
tumor phenotypes with increased self-renewal and osteogenic deficiency, thus serving
as a platform for developing therapeutic approaches. Although OFMSCs share some
similarities with their normal counterparts (JMSCs), the difference is quite prominent.
High efficiency in CFU-f assay implied that OFMSCs, but not JMSCs, were capable of
increasing self-renewal, which was further confirmed in both 1
o
and 2
o
implantation of
single colony-derived OFMSCs. Furthermore, OFMSCs demonstrated significant
deficiency in osteogenic differentiation. Enhanced self-renewal and decreased
differentiation of OFMSCs may contribute to the OF phenotype: abnormal stromal cell
growth and osteogenic deficiency.
Understanding the difference in molecular regulation between normal and tumor stem
cells is fundamental for the development of targeted therapies to eradicate tumor cells
while minimizing damage to normal cells. By comparing global gene expression
patterns, we identified TGFβ signaling as a crucial regulator underlying the increased
self-renewal and osteogenic deficiency of OFMSCs. TGFβ1 generally enhances
proliferation of mesenchymal cells, but it elicited a particularly strong response in
OFMSCs, including SMAD phosphorylation and downstream gene expression. More
intriguingly, active TGFβ1 is specifically enriched in OFMSCs compared to JMSCs,
implicating the role of TGFβ signaling in OFMSCs and providing the rationale for
investigating potential therapies based on blocking TGFβ signaling. As expected,
knockdown of TGFβ signaling significantly enhanced osteogenesis of OFMSCs, as
indicated by elevated osteogenic gene expression and in vivo bone formation.
104
Elevated expression of other components of TGFβ signaling, such as SMAD3/4, may
also contribute to the activation of TGFβ signaling, but the effectiveness of TβRI
inhibitor SB431542 treatment in OFMSC osteogenesis implies that enrichment of
active TGFβ ligand may be still a dominant factor for the strong activation of the
pathway. Furthermore, in spite of enriched active TGFβ, the latent TGFβ1 levels in
JMSCs and OFMSCs are similar.
In a vast array of malignant tumors, TGFβ signaling activity is adversely associated
with prognosis via promoting proliferation, angiogenesis, metastasis and suppression
of immune response (Massague, 2008; Padua et al., 2008), but the role of TGFβ
signaling in benign tumor behavior is not well characterized. One effective strategy for
targeting tumor-initiating cells is to induce them to undergo differentiation and
apoptosis, which is exemplified by the application of retinoic acid in acute
promyelocytic leukemia (Wang and Chen, 2008). Distinct from malignant cells, which
hardly give rise to any functionally normal differentiated cells, cells in benign tumors,
without accumulated mutations or malignant transformation, have the potential to
differentiate normally. By the same token, OFMSCs can be induced to function as
normal MSCs by downregulating their TGFβ signaling. Therefore, molecular
manipulation of OFMSCs provides promising therapies for ossifying fibroma. Although
inhibition of TGFβ signaling dramatically improves osteogenesis of OFMSCs in vitro
and in vivo, providing a new paradigm for OF therapy, further questions regarding how
to implement blockage of TGFβ signaling as a therapy for OF lesions, such as how to
induce the functional pattern in jaw bones, still remain in need of further investigation.
105
The involvement of TGFβ in different tumor types is widely investigated, but the origin
of active TGFβ is rarely uncovered. In the present study, we revealed a
JHDM1D/TSP1-mediated epigenetic loop responsible for the enrichment of active
TGFβ1 and subsequent elevation of TGFβ signaling. TSP1, an extracellular matrix
glycoprotein, inhibits tumor cell growth and metastasis by angiogenesis suppression
(Boukamp et al., 1997; Volpert et al., 1998). Malignant tumors also hijacked
TSP1-activated TGFβ for efficient growth to bypass the TSP1-mediated suppression
(Filleur et al., 2001). In OF, TSP1-activated TGFβ promotes fibro-osseous stromal
cell growth and induces osteogenic deficiency. Intriguingly, Tsp1 expression in OF is
under the regulation of TGFβ signaling, forming a positive feedback loop that
strengthens the activity of TGFβ signaling, which is distinctive from normal JMSCs.
We determined that TGFβ-driven Tsp1 expression is regulated by the
JHDM1D-associated histone modification in OFMSCs. Upon activation of TGFβ
signaling, JHDM1D facilitates Tsp1 expression by removing inhibitory histone
modification. Expression of PHF8, a JHDM1D homologue, is detectable in the jaw of
zebrafish embryos at 3 d.p.f. and injection of PHF8 morpholino results in pronounced
orofacial defects, such as absence of the lower jaw (Qi et al., 2010). This evidence
suggests a conserved role of this histone demethylase family in the normal and
pathogenic osteogenesis of orofacial bones. Since expression levels of both JHDM1D
and SMAD3 are significantly elevated in OFMSCs as compared to JMSCs, SMAD3
may enhance JHDM1D transcription by binding to the JHDM1D promoter. Although
our data suggest that SMAD3 may enhance JHDM1D transcription by directly binding
106
to the JHDM1D promoter region, we cannot exclude the possibility that SMAD3
directly interacts with JHDM1D to regulate its activity. It remains elusive how exactly
SMAD3 regulates JHDM1D activity.
The JHDM1D/TSP1-mediated epigenetic loop strengthens signaling activity and
contributes to the cell proliferation and osteogenic deficiency of OFMSCs. More
interestingly, establishment of the epigenetic loop conferred normal JMSCs with
OF-like properties, including decreased osteogenic capacity, enhanced proliferation,
OF-like histone modification and increased Tsp1 expression. Prolonged TGFβ
treatment could establish a JHDM1D-mediated TSP1/TGFβ/SMAD3 autocrine loop,
resulting in positive feedback and changing JMSCs into OF tumor stem-like cells.
However, defining the component in the loop that functions as an initial inducer
remains to be explored. It has been recognized that cells can respond to transient
epigenetic modification to stabilize newly acquired phenotypic behavior (Ikegaki et al.,
2013); therefore, it is conceivable that the up-regulation of JHDM1D might be a key
factor in eliciting activation of the Tsp1 and TGFβ signaling cascade.
In conclusion, our work establishes a new human stem cell-based benign OF tumor
model with functional phenotype regulated by JHDM1D/TSP1/TGFβ/SMAD3
autocrine-mediated hyperactive TGFβ signaling. Blockage of TGFβ signaling and its
autocrine components in OFMSCs can rescue osteogenic deficiency and suppress
stromal growth, therefore providing a novel therapy for OF lesions.

107
Chaptor 4: Conclusions

This study demonstrated that interaction between epigenetic process and TGFβ
signaling transduction is crucial in regulation of stem cell properties and disease
progress, and deciphered the mechanism of how epigenetic modulation affected the
outputs of TGFβ signaling.
TGFβ signaling is pleiotrophic pathway that plays crucial rols in both stem cell
behaviors and occurrence of diseases. Defect in the signaling gave rise to skewed
differentiation of both ESCs and ASCs. In ESCs, defect in interaction between BCOR
and SMAD signaling resulted in increased differentiation of neuroectoderm and
mesoderm at the cost of endoderm and trophectoderm. In OF, elevated JHDM1D
elicited high activity of TGFβ signaling, which contributed to the osteogenesis
deficiency of OFMSCs.
Epigenetic process affected the function of the components of TGFβ signaling,
including ligand and downstream target genes, thereby regulating the outputs of the
signaling and stem cell behaviors. TGFβ/SMAD signaling drives Nanog expression
and contributes to maintenance of pluripotency by antagonizing variant PRC1
complex mediated epigenetic suppression. Release of the suppression due to loss of
BCOR resulted in constant elevated Nanog expression, losing the regulation by
SMAD signaling. In OFMSCs, elevated JHDM1D decreasd the suppressive histone
modification at Tsp1 promoter and Tsp1 expression was enhanced by SMAD signaling.
In return, increased Tsp1 proteolytically activated latent TGFβ, which induced the
108
osteogenesis deficiency of OFMSCs.
In Chapter 2 of this study, we investigated the interaction of epigenetics and TGFβ
signaling by reprogramming Bcor mutant mesenchymalstem cells (MSCs) into
induced pluripotent stem cells (iPSCs). Loss-of-function mutation of BCL6
corepressor (BCOR) causes oculofaciocardiodental (OFCD) syndrome in female
patients, but males with the same mutation are embryonic lethal, indicating the
importance of Bcor in embryo development. We found that iPSCs derived from OFCD
patient and Bcor-knockdown ESCs have altered self-renewal and differentiation due
to elevated Nanog expression. Mechanistically, BCOR, in conjunction with variant
polycomb repressive complex 1 (PRC1), inhibited Nanog expression by H2AK119
monoubiquitination, which was antagonized by the SMAD signaling to achieve
equilibrium of NANOG heterogeneity. In addition, upregulated NANOG resulted in
deficiency of neural crest lineage determination due to repressed Snail and Twist.
Collectively, this study demonstrated the role of BCOR in the lineage determination of
human pluripotent stem cells and deciphered a mechanism by which BCOR
integrated environment cues of signaling and intrinsic epigenetic process to achieve
orchestrated gene expression in pluripotency. The role of BCOR showed epigenetic
regulation of signaling pathway as an integral component of network of ESC
pluripotency.
Abnormal stem cell function makes a known contribution to many tumors, including
benign tumors. In Chapter 3 of this study, we found that ossifying fibroma (OF)
109
contained a stem cell population that resembled mesenchymal stem cells (OFMSCs)
and was capable of generating OF-like tumor xenografts. Mechanistically, OFMSCs
showed enhanced TGFβ signaling that induced aberrant proliferation and deficient
osteogenesis via Notch and BMP signaling pathways, respectively. The elevated
TGFβ activity was tightly regulated by JHDM1D-mediated epigenetic regulation of
thrombospondin-1 (TSP1), forming a JHDM1D/TSP1/TGFβ/SMAD3 autocrine loop.
Inhibition of TGFβ signaling in OFMSCs can rescue their abnormal osteogenic
differentiation and elevated proliferation rate. Furthermore, chronic activation of TGFβ
converted normal MSCs into OF-like MSCs via establishment of this
JHDM1D/TSP1/TGFβ/SMAD3 autocrine loop.
These results reveal that the role of epigenetic regulation of TGFβ signaling in MSCs
governs the benign tumor phenotype in OF and identify a novel mechanism of
conversion between normal stem cells and tumor stem cells, highlighting TGFβ
signaling as a candidate therapeutic target for the disease.

110
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Abstract (if available)

Abstract TGFβ signaling is pleiotrophic pathway that plays crucial roles in both stem cell behaviors and occurrence of diseases. Epigenetic modification of the components in TGFβ signaling affects the activity of the signaling, thereby influencing properties of stem cells and contributing to the pathological process of diseases. ❧ Loss-of-function mutation of BCL6 corepressor (BCOR) causes oculofaciocardiodental (OFCD) syndrome in female patients, but males with the same mutation are embryonic lethal, indicating the importance of BCOR in embryo development. BCOR is reported to exert its suppressive function by association with components of polycomb repressive complex1 (PRC1), but it is still largely unknown how BCOR-mediated epigenetic process regulates the stem cell fate and gives rise to the symptoms in OFCD patients. ❧ In Chapter 2 of this study, we investigated the interaction of epigenetics and TGFβ signaling by reprogramming Bcor mutant mesenchymal stem cells (MSCs) into induced pluripotent stem cells (iPSCs). Both regulations by signaling and epigenetics are essential for embryonic stem cell (ESC), but it remains elusive how the epigenetic regulation and signaling molecules act in concert to maintain pluripotency. We showed that iPSCs derived from OFCD patient and Bcor-knockdown ESCs have altered self-renewal and differentiation due to elevated Nanog expression. Mechanistically, Bcor, in conjunction with variant polycomb repressive complex 1 (PRC1), inhibited Nanog expression by H2AK119 monoubiquitination, which was antagonized by the SMAD signaling to achieve equilibrium of NANOG heterogeneity. In addition, upregulated NANOG resulted in deficiency of neural crest lineage determination due to repressed Snail and Twist. ❧ Collectively, this study demonstrated the role of BCOR in the lineage determination of human pluripotent stem cells. The significance of the study is to decipher a mechanism by which BCOR integrated environment cues of signaling and epigenetic process to achieve orchestrated gene expression in pluripotency. The role of BCOR showed epigenetic regulation of signaling pathway as an integral component of network of ESC pluripotency. ❧ Abnormal stem cell function makes a known contribution to many malignant tumors, but the role of stem cells in benign tumors is not well understood. In Chapter 3 of this study, we utilized ossifying fibroma as a model to show the epigenetic regulation of TGFβ signaling in occurrence of disease. We found that ossifying fibroma (OF) contains a stem cell population that resembled mesenchymal stem cells (OFMSCs) and was capable of generating OF-like tumor xenografts. Mechanistically, OFMSCs showed enhanced TGFβ signaling that induced aberrant proliferation and deficient osteogenesis via Notch and BMP signaling pathways, respectively. The elevated TGFβ activity was tightly controlled by JHDM1D-mediated epigenetic regulation of thrombospondin-1 (TSP1), forming a JHDM1D/TSP1/TGFβ/SMAD3 autocrine loop. Inhibition of TGFβ signaling in OFMSCs can rescue their abnormal osteogenic differentiation and elevated proliferation rate. Furthermore, chronic activation of TGFβ converted normal MSCs into OF-like MSCs via establishment of this JHDM1D/TSP1/TGFβ/SMAD3 autocrine loop. ❧ These results revealed that the role of epigenetic regulation of TGFβ signaling in MSCs governed the benign tumor phenotype in OF. The significance of the study is to identify a novel mechanism of conversion between normal stem cells and tumor stem cells and highlight TGFβ signaling as a candidate therapeutic target for the disease. ❧ In summary, this study demonstrated that interaction between epigenetic process and signaling transduction is crucial in regulation of stem cell property and disease progress, and deciphered the mechanism of how epigenetic modulation affected the output of TGFβ/SMAD signaling, shedding light on development of targeted therapy.

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Creator Qu, Cunye (author)

Core Title Interaction of epigenetics and SMAD signaling in stem cells and diseases

School School of Dentistry

Degree Doctor of Philosophy

Degree Program Craniofacial Biology

Publication Date 10/30/2016

Defense Date 10/15/2014

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

Tag disease model,histone modification,induced pluripotent stem cells,OAI-PMH Harvest,ossifying fibroma,SMAD signaling

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Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Shi, Songtao (committee chair), Chai, Yang (committee member), Paine, Michael L. (committee member), Shi, Wei (committee member), Ying, Qi-Long (committee member)

Creator Email cqu@usc.edu,qucunye@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-511330

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