Roles for Myc in Self-Renewal of Pluripotent Stem Cells

ROLES FOR MYC IN SELF-RENEWAL OF PLURIPOTENT STEM CELLS

KERIAYN N. SMITH

(Under the Direction of Stephen Dalton)

ABSTRACT

Myc is a critical factor for embryonic stem cell maintenance. It also enhances the reprogramming of fibroblasts by effecting widespread changes in gene expression, effectively silencing the somatic cell gene expression program. Significant effort has been placed into identifying myc targets in embryonic stem cells as a step to define mechanisms of myc action. However, despite this, how myc regulates self-renewal and pluripotency remains unknown. To fill this gap, target genes and interacting proteins of c-myc in embryonic stem cells have been identified on a global scale. Key interacting proteins include epigenetic regulators Smarca4 and LSD1, which are important regulators of gene activation and repression in embryonic stem cells. Target genes of particular interest include the miR-17-92 cluster, through which myc acts to establish the cell cycle structure that is crucial for the maintenance of self-renewal. A second is the primitive endoderm specification factor Gata6. Myc binds to the promoter region of Gata6 in pluripotent cells and directly represses its transcription. In the absence of c- and N-myc, pluripotent cells differentiate to endoderm, concomitant with an increase in Gata6 transcription. The demonstration that myc represses Gata6 is a step toward defining mechanisms of pluripotent stem cell maintenance by myc. This mechanism of repression of lineage specific differentiation by myc was delineated by generating induced pluripotent cells, and inactivating c-myc and N-myc simultaneously. These experiments demonstrate that c- or N-myc is an absolute requirement for maintenance of the embryonic stem cell state, and one mechanism of sustaining self-renewal is repression of primitive endoderm differentiation.

INDEX WORDS: Embryonic stem cells, induced pluripotent stem cells, self- renewal, pluripotency, myc, Gata6, primitive endoderm

ROLES FOR MYC IN SELF-RENEWAL OF PLURIPOTENT STEM CELLS

KERIAYN N. SMITH

B.Sc, University of the West Indies, 2000

A Dissertation Submitted to the Graduate Faculty of The University of Georgia

in Partial Fulfillment of the Requirements for the Degree

DOCTOR OF PHILOSOPHY

ATHENS, GEORGIA

2009

Keriayn N. Smith

ROLES FOR MYC IN SELF-RENEWAL OF PLURIPOTENT STEM CELLS

KERIAYN N. SMITH

Major Professor: Stephen Dalton

Committee: David Puett Lance Wells Scott Dougan

Electronic Version Approved:

Maureen Grasso Dean of the Graduate School The University of Georgia August 2009

DEDICATION

This dissertation is dedicated to my mother Girdel Golding, whose struggles and sacrifices have brought me to this point, and to my daddy, Harry

Smith, for his heart.

ACKNOWLEDGEMENTS

I have learnt a lot on this journey. First, thanks to my Father, for a multitude of blessings, and providing me strength. I must also make special

mention of individuals whose patience, support and encouragement were

essential for me to achieve my goals. To Steve, thank you for your generous

assistance, advice and support. I learnt an incredible amount from you, and your

open door policy meant I was free to drop in at any time. I would also like to

express my gratitude to my committee members, Drs. David Puett, Lance Wells,

Scott Dougan as well as Brian Condie for always being available to me for

guidance, as it seems I am in constant need of advice. Special mention must

also be made of colleagues in the Dalton lab, past and present including Mandy,

Malini, Laura, Amar, Matt, Ian, Michael, Anne, Hope, Dave and Tim. To my

friends especially Kameka, Danielle, Natalie and Geneva who provided me with

never ending support and encouragement. I really appreciate you being there for

me. Finally, to my family, including my parents, aunts, uncles, brothers,

grandparents and cousins, I am truly blessed to have such a wonderful family.

To Andrew, thank you for being there for me, constantly.

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS...... v

LIST OF TABLES ...... viii

LIST OF FIGURES ...... ix

CHAPTER

1 Literature Review and Introduction...... 1

Derivation and Properties of Embryonic Stem Cells...... 1

Regulation of the Pluripotent State...... 7

Induced Pluripotent Stem Cells ...... 13

Myc Family of Transcription Factors...... 15

Perspective...... 23

2 Experimental Procedures ...... 29

3 Myc Target Genes in Embryonic Stem Cells ...... 47

Background ...... 47

Results ...... 48

Discussion ...... 69

4 An Absolute Requirement for Myc in the Maintenance of Pluripotent

Stem Cells ...... 74

Background ...... 74

Results ...... 75

Discussion ...... 107

5 Identification of Myc Interacting Proteins in Embryonic Stem Cells ...... 112

Background ...... 112

Results ...... 113

Discussion ...... 120

6 Final Discussion and Conclusions...... 123

REFERENCES...... 130

APPENDIX ...... 141

A Myc target genes identified in ChIP-Chip ...... 141

vii

LIST OF TABLES

Page

Table 1: Examination of the relationships between selected c-myc targets and c-

myc in differentiation and upon stimulation of c-myc activity...... 67

Table 2: c-myc interacting proteins identified by mass spectrometry of ESC extracts...... 119

viii

LIST OF FIGURES

Page

Figure 1.1: Selector genes that regulate lineage specification at the blastocyst

stage of the embryo ...... 25

Figure 1.2: Properties of ESCs...... 26

Figure 1.3: ESC transcriptional network ...... 27

Figure 1.4: Diagrammatic representation of the myc family members, and their

obligate binding partner max...... 28

Figure 3.1: Generation of cell lines expressing epitope tagged human c-myc...... 58

Figure 3.2: Accurate localization of c-myc in selected cell lines ...... 59

Figure 3.3: Selected cell lines differentiate upon removal of LIF ...... 60

Figure 3.4: Experimental Scheme for ChIP-Chip...... 61

Figure 3.5: Selected cell lines are appropriate for use in ChIP experiments ...... 62

Figure 3.6: Analysis of samples selected for ChIP-Chip...... 63

Figure 3.7: Functional annotation and grouping of a subset of c-myc target genes...... 64

Figure 3.8: Diagrammatic representation of c-myc binding sites in control

samples ...... 65

Figure 3.9: Diagrammatic representation of c-myc binding sites in the targets

Sall4 and MBD3...... 66

Figure 3.10: Validation of c-myc target genes identified in ChIP-Chip...... 68

Figure 4.1: iPSCs display characteristics of self-renewal and pluripotency ...... 87

Figure 4.2: iPSCs differentiate in vitro and in vivo ...... 88

Figure 4.3: Experimental scheme depicting inactivation of c- and N-myc in iPSCs...... 89

Figure 4.4: Cre excision facilitates deletion of c- and N-myc in iPSCs ...... 90

Figure 4.5: Deletion of c- and N-myc in iPSCs results in the loss of self-renewal...... 91

Figure 4.6: Deletion of c- and N-myc in iPSCs results in an increase in endoderm

marker transcript levels...... 92

Figure 4.7: Deletion of c- and N-myc in iPSCs results in the upregulation of

endoderm markers...... 93

Figure 4.8: Co-deletion of c- and N-myc results in slight increases in apoptosis...... 94

Figure 4.9: Loss of c- and N-myc results in decreased proliferation and

remodeling of the cell cycle...... 95

Figure 4.10: c-myc binds to the miR-17-92 cluster in ESCs ...... 96

Figure 4.11: c-myc regulates the miR-17-92 cluster in ESCs ...... 97

Figure 4.12: Rb2/p130 is upregulated upon loss of c- and N-myc...... 98

Figure 4.13: Activation of c-myc and N-myc but not L-myc is able to rescue the

self- renewal potential in dKO cells...... 99

Figure 4.14: Stimulation of c- and N-myc activity blocks the increase in endoderm

marker transcripts in dKO cells ...... 100

Figure 4.15: dKO embryoid bodies are reduced in size compared to embryoid

bodies derived from Flox iPSCs...... 101

Figure 4.16: dKO iPSCs are predisposed to form endoderm upon differentiation

in suspension culture ...... 102

Figure 4.17: dKO cells preferentially form endoderm in adherent differentiation

conditions...... 103

Figure 4.18: c-myc expression is reduced in primitive endoderm...... 104

Figure 4.19: The Gata6 promoter is bound by c-myc in ESCs ...... 105

Figure 4.20: c-myc represses Gata6 in ESCs...... 106

Figure 5.1: Experimental setup for the identification of c-myc interacting proteins

by co-immunoprecipitation/mass spectrometry analysis...... 116

Figure 5.2: Epitope tagged c-myc interacts with multiple proteins in ESCs ...... 117

Figure 5.3: c-myc interacts with components of the co-REST co-repressor

complex in ESCs ...... 118

Figure 6.1: Model illustrating the central role of myc in maintaining self-renewal

and pluripotency in ESCs and iPSCs...... 129

CHAPTER 1

LITERATURE REVIEW AND INTRODUCTION

Derivation and Properties of Embryonic Stem Cells

Early Embryonic Development

Mammalian embryogenesis begins after fertilization, when the zygote undergoes

a period of cellular divisions or cleavages progressing through the 2-, 4-, 8- and

16-cell stages. At approximately 3.0 days post coitum (d.p.c), murine totipotent

blastomeres of the cleavage stage embryo begin to compact due to the formation

of interconnected gap junctions. This embryonic stage is referred to as the

morula 1-3. The first differentiation steps then occur, and the embryo undergoes

cavitation to form the blastocyst.

At this peri-implantation stage (3.5 d.p.c), the embryo consists of an outer layer

of cells, the trophectoderm, that encapsulates a group of cells, the inner cell mass (ICM), within a fluid filled cavity known as the blastocoele (Figure 1.1).

Trophoblast cells that contribute to the fetal component of the placenta will arise from the trophectoderm 2, 3. At around the time of implantation (4.0 d.p.c), the

ICM will develop into the epiblast (primitive ectoderm) and the primitive

endoderm (PrE). It was initially thought that the PrE forms on the surface facing

the blastocoelic cavity, but Chazaud and colleagues 4 demonstrated that the ICM is already heterogeneous with specific cells expressing PrE and ICM markers as early as 3.5 d.p.c.

PrE provides a supporting role to the epiblast, which will become the embryo

proper. Differentiation of PrE will occur to form parietal endoderm and visceral

endoderm. The visceral endoderm, which remains in contact with the epiblast, is

involved in nutrient uptake and transport. It also provides signals that direct

morphogenesis and patterning of the developing epiblast 1-3. A recent study

expanded the prevailing model of visceral endoderm function even further, to

roles other than contribution to extraembryonic lineages 5. Using fate-mapping,

Kwon and colleagues demonstrated that both visceral endoderm and definitive

endoderm (epiblast derived) contribute to the developing gut tube of the embryo

Formation of the three embryonic germ lineages, ectoderm, mesoderm and

endoderm, occurs at gastrulation with the formation of the primitive streak at approximately 6.5 d.p.c. Prior to this stage, most cells of the developing embryo

maintain remarkable developmental potential. Pluripotent cells, those that retain

the ability to form any cell in the body, can be isolated from the morula, the ICM

of the blastocyst and the epiblast 6-11.

Embryonic Stem Cells

Pluripotent cells isolated from the ICM of blastocyst stage embryos are called

Embryonic Stem Cells (ESCs, Figure 1.2) 6-8. Aside from pluripotency, another key characteristic of ESCs is their ability to self-renew or continuously proliferate without undergoing cellular senescence. Thus with the right cues, they will maintain a normal karyotype indefinitely while being cultured in the laboratory 8,

12.

In vitro, ESCs can differentiate into derivatives of each of the three primary germ layers (Figure 1.2). Cells of the epidermal and nervous systems arise from ectodermal precursors. Cells derived from definitive endoderm include those of the respiratory system, the gut and its derivatives. Mesoderm precursors will differentiate to form muscle, blood, bone, other connective tissue, and those comprising organs of the urogenital system 1, 13, 14. Similar cell types arise from

ESCs in vivo, and they spontaneously form teratomas upon injection into immunodeficient mice. These tumors consist of cells of ectodermal, mesodermal and endodermal origin. Similarly, upon reintroduction to blastocysts, ESCs are able to contribute to the germline as well as differentiate into functional embryonic germ layer derivatives. These properties make ESCs an excellent model for early embryonic development and a promising resource for the replacement of damaged or degenerated tissue resultant from numerous diseases 1, 13, 14.

Mouse ESCs (mESCs) are typically maintained in vitro in fetal bovine serum

based tissue culture medium on a feeder layer of mouse embryonic fibroblasts

(mEFs). mEFs secrete the interleukin-6 family member cytokine, leukemia

inhibitory factor (LIF) that maintains mESC pluripotency 15, 16. Alternatively,

mESCs can be grown in medium supplemented with recombinant LIF in the

absence of a feeder layer 12. LIF signaling is mediated by the receptors LIFR and gp130. Their dimerization results in the signal being transduced by the

Janus kinase/Signal Transducers and Activators of Transcription (JAK/STAT)

pathway resulting in activation of STAT3 and downstream targets such as c-myc

17, 18.

Withdrawal of LIF results in the spontaneous differentiation into embryonic germ

layer derivatives 12. This spontaneous differentiation can be directed in a more

specific manner with the addition of various chemical inducers. For instance,

BMP4, a constituent of the fetal bovine serum component of ESC medium

normally cooperates with LIF to maintain mESC self-renewal. However, its

presence in the absence of LIF drives mesodermal differentiation and inhibits

neural differentiation 19. Likewise, supplementation of media with retinoic acid

induces differentiation into neurectoderm 12, 20.

Aggregation of mESCs in suspension culture under conditions of LIF removal

results in the formation of embryoid bodies. Embryoid bodies are routinely used

as an in vitro model for embryonic development as they recapitulate development

of the early embryo. An initial differentiation step occurs with the formation of a

layer of PrE on the external surface of developing embryoid bodies 21.

Subsequently, derivatives of the three primary germ layers form in a similar

temporal manner to the differentiating ICM, albeit lacking spatial information

present in the developing embryo 12.

Proliferation of Embryonic Stem Cells

The decision to enter the cell cycle is dependent on cell density and availability of nutrients. Cell division consists of a cycle through the S-phase of DNA synthesis, and M-phase of mitosis and cytokinesis. These phases are separated by two

GAP phases, G1 and G2 that monitor the readiness to divide and genomic integrity after DNA replication. The cell cycle is a tightly controlled process, with checkpoints ensuring accurate DNA replication and segregation of chromosomes at the end of mitosis 22.

ESCs proliferate at rates similar to pluripotent cells in the embryo, in the order of

one doubling every 8-12 hours 23, 24. Upon differentiation, there is significant

remodeling of the cell cycle, to a profile that is similar to what is observed in

somatic cell types 25.

In somatic cells, progression through the cell cycle is regulated by cyclin

dependent kinase (Cdk) activity, and inhibition of Cdk activity leads to cell cycle

arrest. Cdk activity is mediated primarily through binding of regulatory cyclins,

which bind Cdks to cause a catalytically activated conformational change. G1 to

S-phase transition and progression requires activation of cyclin D-Cdk4/6 and

cyclin E-Cdk2, and the G2 to M transition requires cyclin A/B-Cdk1/2. Activated

Cdks phosphorylate members of the pocket protein (Rb, p107, p130) family,

which releases sequestration of the E2F family of cell cycle regulators, permitting

the transcription of E2F target genes 22, 26.

Conversely, when anti-mitogenic signals activate the G1 checkpoint, Cdk activity

is inhibited resulting in hypo-phosphorylation of the pocket proteins and

inactivation of E2F transcription factors. This leads to repression of E2F targets

that are required for cell cycle progression. Signals inhibiting cell cycle

progression also induce the expression of Cip/Kip family of cell division inhibitors

that includes p21, p27 and p51, which bind to and inhibit cyclin/Cdk complexes

preventing the G1 to S phase transition. Additionally, the action of the Ink4

family members, consisting of p15, p16 and p18 causes G1 arrest 26.

In addition to progressing through the cell cycle in a shortened time frame, ESCs

maintain a unique cell cycle structure compared to somatic cells, with a truncated

G1 (approximately 2h; approximately 15-20% population) and the majority of

cells resident in S-phase (>50%) 24. There is a loss of periodicity due to constitutive expression of cyclins in ESCs, with a concomitant increase in associated Cdk activities. Furthermore, ESCs are able to cycle without restriction points as Rb is hyperphosphorylated and inactivated resulting in the

constitutive expression of E2F targets. Moreover, Cdk inhibitors are not expressed in ESCs 24, 25, 27.

Cell division times increase significantly upon differentiation. There is also significant remodeling of the cell cycle structure with an increase in the percentage of cells occupying G1 (>40%), and a reduction in the population in S- phase (to approximately 15%). This is due to a decrease in expression levels of regulatory cyclins, and upregulation of the cell cycle inhibitors p21 and p27, which results in decreased Cdk activity 24, 25, 28.

Regulation of the Pluripotent State

Transcriptional Regulation

Self-renewal and pluripotency of mESCs are governed by the actions of a regulatory network of transcription factors that inhibits differentiation while promoting continuous proliferation (Figure 1.3). The best studied members of this network include Oct4, Sox2 and Nanog, key transcriptional regulators that maintain expression levels of target genes required for maintenance of the ESC state 29, 30.

Oct4 is a member of the POU class of transcription factors and its expression is associated with stem cell populations. It is one of the earliest transcription factors to be expressed in the early embryo. Upon differentiation of the ICM and

trophectoderm, the expression of Oct4 becomes restricted to the pluripotent

population 31. Oct4 expression is maintained until formation of the epiblast,

followed by decreased expression upon germ layer specification. Thereafter,

primordial germ cells maintain Oct4 expression 32. Strict levels of Oct4 are

required for maintenance of mESC self-renewal. Loss of Oct4 expression

induces differentiation into trophectoderm, and overexpression leads to PrE

formation 33. Regulation of differentiation to trophectoderm by Oct4 is based on inhibition of the transcription factor Cdx2. Cdx2 is a trophectoderm specification factor, and the balance and mutual repression between Oct4 and Cdx2 influences the first differentiation step in the embryo 34-36 (Figure 1.1)

Oct4 heterodimerizes with Sox2, a SRY-related HMG box transcription factor that

works in conjunction with Oct4 to regulate its targets. The expression of Sox2

however, is not limited to pluripotent cells as it is also expressed in select neural

lineages 37, 38.

The expression of the NK-2 class homeodomain transcription factor, Nanog, is

also restricted to the pluripotent population of the ICM. Nanog overexpression

facilitates the maintenance of mESC self-renewal in the absence of the LIF 39, 40.

Nanog expression decreases on ESC differentiation, and its expression is

mutually exclusive with that of Gata6, a master PrE regulator 41 (Figure 1.1).

Nanog represses Gata6 transcription, and its loss results in PrE differentiation

with associated increases in Gata6 expression 40, 41. Not surprisingly, Gata6

overexpression resulted in the upregulation of PrE genes resulting in differentiation to PrE 42.

These transcription factors were the first defined components of the ESC core

transcriptional regulatory circuitry in genome-wide location analyses in both

human and mouse ESCs 29, 30, 43, 44 (Figure 1.3). These studies identified hundreds of target genes co-occupied by Oct4, Sox2 and Nanog that indicated the likelihood of coregulation of a large network of genes. Some target genes have roles in the maintenance of pathways that allow for maintenance of growth, proliferation and the metabolic state in the self-renewal of ESCs. For instance,

Boyer and colleagues 30 demonstrated their binding to promoters of active genes

important for maintenance of pluripotency such as Stat3, Lefty2, as well as

binding to their own promoters. Furthermore, promoters of repressed genes

required for lineage specification were also bound by Oct4, Sox2 and Nanog,

demonstrating both activatory and inhibitory potential of these genes. Some

examples include Otx1, Pax6, GSC and members of the Hox family of

developmental regulators 29, 30, 43, 44.

The expression of these components of the ESC regulatory network is also

strictly regulated, and feedforward loops facilitate the continuous expression of

these transcription factors, thus maintaining their own transcriptional status 29, 30,

43, 44. The regulatory effects of this transcriptional circuit are also further

expanded through protein-protein interactions 45.

miRNA regulation

This subclass of small single stranded RNAs of approximately 22 nucleotides modulates gene expression post-transcriptionally by base-pairing with target mRNAs. Binding results in degradation of the mRNA or repression of its translation. Base-pairing with target transcripts is dependent on homology of the mRNA to the seed region of the miRNA. A single target can thus be regulated by multiple miRNAs, and a single miRNA can regulate numerous mRNAs 46, 47.

Therefore, posttranscriptional regulation by miRNAs is rather complex.

Substantial evidence supports the importance of miRNAs in the regulation of

ESC biology. Expression analyses were carried out by Chen and colleagues to identify miRNAs that regulate cellular identity. Upon comparison of the expression levels of 302 mouse miRNAs in mESCs, embryoid bodies and somatic tissues, they identified 14 mESC miRNA coding sequences that were highly expressed in mESCs. These included miRNAs of the miR-290 and miR-

302 families 48.

Subsequently, the regulatory circuitry of ESCs was expanded to include miRNAs

with the identification of Oct4/Sox2/Nanog binding to regulatory regions of

several miRNAs. Two groups of miRNA promoters are directly bound by Oct4,

Sox2, or Nanog in ESCs. The first group includes ESCs specific miRNAs, such as members of the miR-290-295 and miR-302 cluster 49. Interestingly,

expression of members of this cluster was recently found to increase the

efficiency of reprogramming to induced pluripotent stem cells (iPSCs) 50. A

second miRNA group was also bound by members of the polycomb group proteins. These miRNAs were repressed in ESCs, and induced upon induction of differentiation 49.

Other clues to the importance of miRNAs in the maintenance of ESCs came from

an observation that elimination of Dgcr8 activity, an enzyme involved in miRNA

processing resulted in ESC differentiation defects. Additionally, Dgcr8 is

necessary for maintenance of the ESC cell cycle profile, as Dgcr8 null cells

accumulate in G1 51. Wang and colleagues then identified miRNAs that could rescue the cell-cycle defect, 27 demonstrated that members of the miR-290

cluster promoted the rapid proliferation of ESCs, and modulated the transition

from G1-S phase through suppression of the target p21. This cell cycle inhibitor

was identified as a target based on seed sequence homology, and introduction of

individual members of this cluster significantly decreased p21 protein expression

27.

Epigenetic Regulation

Various epigenetic regulatory mechanisms such as DNA methylation, histone

post-translational modifications, histone variant exchange and chromatin

remodeling can have a significant influence on gene transcription. As these

modifications dictate how tightly DNA associates with histones, and consequently

the accessibility of chromatin, they are important in the regulation of gene activity

52, 53.

That ESCs maintain vast differentiation potential is underscored by their unique

epigenetic status. Epigenetically, they exist in a poised state, with large regions

of transcriptionally active euchromatin relative to less active heterochromatin 52,

54. ESCs maintain an active epigenetic state with acetylated histones, such as

acetylated histone H4, highlighting active chromatin. Other activating histone marks including trimethylation of lysine 4 of histone H3 and histone H4

(H3K4me3 and H4K4me3 respectively) are mediated by the activity of histone methyltransferases 52-54. Such modifications are prevalent at transcriptionally

active regulatory regions and are common modifications at transcriptional start

sites. Additionally however, within regulatory regions of genes poised for

activation, significant portions contain bivalent domains that contain both active

and repressive histone marks thus keeping these developmentally important genes primed for activation when the appropriate differentiation signal is received

55, 56. Interestingly, a significant proportion of the targets regulated by the ESC core transcription regulators are those that contain bivalent domains, highlighting the multilayer control of the ESC state 30, 55.

Upon differentiation, as specific gene expression programs are established, there

are accompanying changes in histone modifications. These include global

decreases in acetylation, enacted by histone deacetylases (HDACs).

Hypoacetylation, a result of HDAC activity, results in more compact chromatin, a hallmark of transcriptionally inactive chromatin. Additionally other marks of repressed chromatin including H3-K9me3, and H3-K27me3 become established on pluripotency genes. Such marks are removed from genes that are required for lineage specification upon differentiation 54, 57, 58.

Induced Pluripotent Stem Cells

Reprogramming to the Pluripotent State

The identification of the transcriptional networks in ESCs paved the way for the efficient dedifferentiation of terminally differentiated cells to pluripotent cells. In this groundbreaking work, Takahashi and Yamanaka 59 retrovirally introduced a cocktail of four transcription factors, Oct4, Sox2, Klf4 and c-myc to fibroblasts of embryonic and adult origin, and were able to derive induced pluripotent stem cells (iPSCs). Subsequent to this study, other groups have been able use non- retroviral transduction methods and different cocktails to reprogram cells of other types into iPSCs 59-62, 63. Studies now, as a result, have begun to pave the way for use of these cells in cell therapy for a number of degenerative diseases.

iPSCs are morphologically similar to ESCs, express ESC markers, and undergo epigenetic modifications from that of a somatic cell, to the more permissive ESC state 64, 65. They differentiate in vitro into derivatives of the germ layers, and are

able to contribute to tissues of the developing embryo and the germline upon

blastocyst injection 65.

Mechanistically, it is likely that Oct4 and Sox2 facilitate reprogramming by

activating genes that induce self-renewal and pluripotency, while simultaneously

inhibiting genes of terminally differentiated fibroblasts. Klf4, a Kruppel-like

transcription factor coregulates some of these targets in conjunction with Oct4

and Sox2 44. It is likely that c-myc promotes proliferation of the iPSCs 59.

Furthermore, of the four reprogramming factors when introduced in isolation, c- myc primed somatic cells for the induction of pluripotency by causing extensive gene expression changes in fibroblasts 66. Takahashi and Yamanaka proposed

another role of c-myc was to facilitate access of the other transcription factors to

target genes due to its ability to recruit histone acetyltransferases (HATs) 59.

Indeed, numerous studies have shown the ability of myc to recruit epigenetic modifiers including histone methyltransferases and chromatin remodeling components 67-70. This suggests that myc may be responsible for the widespread

epigenetic modifications observed through the reprogramming process.

Not surprisingly, the other proto-oncogenic family members N- and L-myc have

also been substituted for c-myc in the reprogramming process 71. It is also likely

that the cell types that have successfully been reprogrammed express at least

one myc family member. Then, upon reprogramming, the expression level of c-

myc increases to facilitate the maintenance of self-renewal 59. It should be noted

that exogenous introduction of myc has since been found to be non-essential to reprogramming although it significantly increases efficiency 61, 71.

Myc family of Transcription Factors

Properties of the myc protein family

Members of the myc family of transcription factors (referred to as myc

throughout) are basic helix-loop-helix-leucine zipper proteins. c-myc, the

prototypical member of this proto-oncogenic family, is the cellular homolog to the

viral oncogene v-myc. v-myc was initially identified as the transforming factor of

the avian virus MC29 that was involved in a range of neoplasms in chickens 72.

Other family members, N- and L-myc were later identified based on sequence

homology. L-myc is typically associated with small cell lung cancer, while N-myc

was identified due to its high level of expression in childhood neuroblastomas. It

is also implicated in other cancers such as retinoblastoma, and embryonal

carcinomas 73-76.

The myc family members are structurally similar, and have a transactivation

domain located at the N-terminus (Figure 1.4). This region contains two myc

homology boxes, MBI and MBII that are highly homologous among the family

members. A nuclear localization signal located in a central region of myc

proteins mediates nuclear translocation of these transcription factors. The basic

helix-loop-helix leucine zipper domains are located towards the C-terminus.

These regions primarily mediate DNA binding to regulatory regions of target genes and dimerization with interacting proteins 77, 78.

Myc action can have diverse effects depending on the cellular context, and they regulate diverse cellular functions including growth, proliferation, cellular motility, differentiation, and apoptosis 77, 79-82. These diverse functions are partially dependent on the input signal, which can then be transduced by multiple signaling pathways. Activation of myc transcription can occur as the result of activation of various cellular pathways including sonic hedgehog (Shh), Wnt, mitogen activated protein kinase (MAPK), JAK/STAT and transforming growth factor β (TGF-β) pathways 80, 83-85.

A well established function for myc is regulation of cellular division. Exogenous c-myc expression promotes cell cycle entry of quiescent cells, and shortens G1 and accelerates S-phase entry in cultured fibroblasts 86. A number of cell cycle regulators, including cyclins D2, E and A are direct or indirect myc transcriptional targets. Cyclin D2 activity results in an increase in Cyclin E/Cdk2 activity by sequestration of p27. Furthermore, c- and N-myc interact with Rb 87, thus influencing the restriction point in G1.

Typically, there are only low levels of myc in resting cells, but protein levels rapidly increase upon stimulation by mitogenic factors. These higher levels persist as cells go through the cell cycle, and decline as cells undergo

quiescence 77. As a control mechanism against cellular transformation, ectopic expression of myc induces apoptosis through a p53-dependent pathway 88-90.

The correlation of the myc family members with tumorigenesis is well

documented, and deregulation of myc is a major underlying factor in a number of

cancers including Burkitt’s lymphoma, glioblastoma, neuroblastoma, and small

cell lung cancer 77, 79-82. This deregulation of myc activity may occur through a

number of different mechanisms including gene amplification, activating

mutations, chromosomal translocation, proviral insertion or retroviral transduction

83. Additionally, anomalies in signal transduction cascades, such as the

Ras/ERK pathway, that typically regulate c-myc activity, can also contribute to

oncogenesis, and its contribution with oncogenic Ras in transformation is well documented. N-myc can also cooperate with Ras in instances of Ras overexpression to transform embryonic fibroblasts 84,73. Furthermore, cells that

have aberrant myc activity are able to evade senescence due to sustained

telomerase activity, as the telomerase reverse transcriptase is an established

myc target 91.

Regulation of myc activity

Mitogenic stimuli increase myc protein stability through the activation of

Ras/MEK/ERK pathway resulting in the phosphorylation of Ser62 by ERK. This modification results in increased stability of myc protein. Conversely, phosphorylation of myc at Thr58 by glycogen synthase kinase 3β (GSK3β), a

modification that requires previous phosphorylation at Ser62, reduces the half-life of c-myc, targeting the protein for ubiquitin-mediated degradation 18, 92, 93.

Conservation of similar residues in N-myc facilitates similar regulation of N-myc

stability by GSK3β dependent mechanisms 94. Hence, stimuli may result in activation of some myc target genes, or repression of others through via the action of various cellular pathways.

Regulation of myc activity is also partially dependent on dimerization with the

obligate binding partner Max through their leucine zipper domains 95. The half-

life of the myc protein (approximately 30 min) is a fraction of that of Max, and therefore myc is the limiting component of such gene activation. However, Max availability is also important, as it can also bind to members of the antagonizing

Mad or Mnt family of proteins, resulting in the silencing of myc target genes 96.

Transactivation typically occurs through binding of the bHLH domain to E-box consensus sequences (5’-CACGTG-3’) of target genes. Upon binding, myc is then able to recruit coactivators and other proteins involved in transcription 96.

Modes of myc action c-myc has sweeping effects in cellular functions and it possibly regulates 10-15% of all cellular genes including cell cycle regulators, regulators of transcription and translation, and metabolic regulators 97, 98, 99. However, it is a relatively weak

transactivator, and substantial changes in target gene expression are rarely

observed 77, 99. Myc target genes also include those that regulate cell adhesion

and cytoskeletal structure, and rapid reduction in such transcripts occurs upon

activation of c-myc 100.

Myc also binds and regulates miRNA expression. Notably, the polycistronic miR-

17-92 cluster, comprised of miR-17, miR-18a, miR-19a, miR-20a, miR-19b, and

miR92-1 is bound by myc in some cancer cells 101, 102. This cluster is amplified

and overexpressed in multiple cancers including breast, colon and pancreatic

tumors 103, 104. In addition, exogenous expression of the miR-17-92 cluster

accelerated lymphoma progression when introduced into Eμ-myc mice.

Interestingly, this cluster exists in a regulatory feedback loop with the cell cycle

regulators E2F transcription factors, in that while the miRNA cluster is a transcriptional target of E2F1, E2F1 is also a validated target of the cluster 101,

105. The miR-17-92 cluster components also regulate other cell cycle regulators

such as p21 and Rb2/p130 in cancer cells, providing other mechanisms by which

this cluster regulates cell cycle progression 106, 107.

Myc is also able to modify the transcriptional status of genes through epigenetic

mechanisms. It modulates the permissibility of chromatin possibly by recruiting a

number of epigenetic modifiers including DNA methyltransferases, histone-

modifying factors, and chromatin remodeling machinery 68, 108. Active

transcription sites are marked by histone acetylation, which promotes an open

chromatin conformation, allowing entry of transcriptional machinery. Loss of N-

myc results in widespread gene silencing due to the disruption of acetylation in

neural progenitor cells and rat fibroblasts. This disruption resulted from loss of the HAT GCN5 and absence of its recruitment to target promoters in the absence of myc activity 70. In other cell types, histone acetylation can be modified by the

recruitment of TRRAP, a component of histone acetyltransferase complexes, by

its interaction with MycBoxII. TRRAP complexes also consist of TIP48/TIP49,

ATP dependent helicases implicating myc in chromatin remodeling 77, 96.

Myc target network gene repression occurs through less well-defined

mechanisms including recruitment of HDACs. Such recruitment results in gene

repression through chromatin remodeling to a more closed conformation, and

HDACs associated with Sin3 antagonize the action of Mad/Max or Mnt/Max

complexes. Another mechanism of myc repression is sequestration of other

transactivators. For instance, Myc inhibits the expression of Miz-1 targets such

as p21 by interacting with Miz-1. Interaction of Myc/Max with Miz-1 at initiator

regions of p21 impedes recruitment of the transcriptional coactivator p300, thus

preventing its access to target promoters 80, 84, 109.

Global proteomic analyses in colorectal cancer cells and human embryonic

kidney cells identified 221 c-myc associated proteins 110. Interacting proteins

were involved in many cellular processes including transcription; DNA replication;

protein synthesis, modification and degradation; metabolism, cellular structure

and cellular transport. This study underscored the widespread effects of myc via

interactions with other proteins.

Myc in Embryonic Development

N-myc and c-myc are expressed in pre-implantation embryos, with expression

becoming more restricted as development progresses. While c-myc remains

widely expressed in proliferating cells in development, N-myc expression

becomes more restricted to developing organs 111, 112. As described previously,

N- and c-myc are highly homologous, and Malynn and colleagues demonstrated

that N-myc sequence could functionally replace c-myc in murine development 113.

N-and c-myc are important for embryonic development and loss of N- or c-myc

function is lethal in mid-gestation. This lethality is due to the requirement of N-

and c-myc for cell proliferation particularly in the developing neural system and

vascular systems. Specifically, the c-myc null mutation results in death at 10.5

d.p.c due to decreased cellularity, insufficient vascularization of the yolk sac,

defective neural tube closure and defects on the developing heart 114. This lethality can be partially rescued by reintroduction of c-myc expression in extraembryonic tissues 115. N-myc null mice die at approximately 11.5 d.p.c due

to multiple defects in organ development 116, 117. L-myc is expressed in multiple

organs, particularly the lung, but this family member is generally less studied.

There is no demonstrable phenotype for L-myc null animals as embryos develop

normally, possibly due to compensation by other myc family members 118.

As expected, the phenotype of the Max knockout mice is more severe, with lethality occurring at an early post-implantation stage (approximately 5.5 – 6.5

d.p.c) after utilization and expiration of maternal stores 119. This suggests that c-

and/or N-myc are required for proper early embryonic development than their respective null phenotypes imply. To date, the precise function of myc during peri-implantation development has been difficult to elucidate due to their functional redundancy and overlapping expression patterns in the early embryo.

Adequate assessment of this would require characterization of double knockout embryos.

Myc Function in Stem Cell Populations

In adult stem cell populations, different groups have demonstrated that myc may

function by both promoting and inhibiting differentiation. For instance, c- myc

regulates differentiation of epidermal stem cells 120. c-myc also influences the

self-renewal or differentiation of hematopoietic stem cells (HSCs) by influencing interactions with the niche 121. Although c-myc is necessary for differentiation in

HSCs, it can also facilitate expansion of the progenitor cell population 121. Taking

these studies further to also address the role of N-myc, Trumpp’s laboratory used

a genetic approach to inactivate both c- and N-myc in hematopoietic stem cells

122. They found that these myc family members regulate the HSC population at

multiple levels including proliferation, survival, metabolic activity and

differentiation 122.

In global expression analyses, N-myc transcripts are at high levels in ESCs, and decrease approximately 7-fold in differentiation 123. N-myc knockout ESCs have

also been isolated, and are able to self-renew 116.

c-myc is important for maintenance of self-renewal in mouse ESCs, and it is a

target of LIF/Stat3 signaling. Its expression declines during embryoid body

differentiation due to GSK3β mediated phosphorylation on Thr58 and subsequent

degradation by the proteasome 18. Additionally, ectopic expression of a

stabilized form of c-myc (Thr58-Ala mutation) maintained self-renewal in the

absence of LIF. On the contrary, the expression of dominant negative mutant prevented mESC maintenance 18. However, c-myc null mESCs are able to self-

renew, but they proliferate more slowly and have defects in vascular and

hematopoietic differentiation 124. As mentioned previously, exogenous c-myc

only, results in widespread gene expression changes to facilitate reprogramming

66. In recent genome-wide location analyses, c-myc targets have been identified in ESCs, however, apart from regulation of proliferation and metabolic state, no functional mechanisms have been defined 44, 125, 126.

Perspective

The interplay between transcription factors, chromatin modifying components,

and remodeling factors and miRNAs are the basis of a complex yet dynamic

regulatory network for regulation of the properties of pluripotent stem cells.

The literature described previously indicates the likelihood of myc being a central

factor in the establishment of pluripotent cell identity. The precise mechanism of

myc function in the maintenance of self-renewal and pluripotency, while inhibiting

differentiation is not yet understood.

The hypothesis that myc is an ideal candidate for placement in such a central

role is based on its potential to globally regulate the ESC state due to its effect on

large numbers of target genes. Furthermore, its role as an epigenetic regulator

may affect the accessibility of chromatin, thus facilitating the recruitment of Oct4,

Nanog, Sox2 and other components of the transcriptional circuitry to target genes

including miRNAs. The abovementioned is further complicated by the possible

existence of multiple myc-containing complexes in ESCs. My studies address

significant gaps in our knowledge of ESC maintenance, as it pertains to the role of the myc family members.

Figure 1.1. Selector genes that regulate lineage specification at the blastocyst stage of the embryo. A. Oct4 and Nanog (maroon) specify pluripotent cell populations. Cdx2 (blue) is expressed in the outer layer of the late morula, and represses Oct4 and Nanog in the trophectoderm. Gata6 (green) is expressed in the PrE. B. Oct3 represses Cdx2, and Cdx2 represses Oct4 to result in the segregation of the ICM and trophectoderm. Nanog and Gata6 mutually repress each other in a similar manner to result in segregation of the epiblast and PrE from the ICM. (Adapted from 2).

A. B.

Nanog Blastocyst SSEA-1

Endoderm Ectoderm

Isolated inner cell mass Gata6 Otx2 FoxA2 Sox17

Mesoderm ES cells media+LIF Feeder layer T-Brachyury

Figure 1.2. Properties of ESCs. A. ESCs are derived from the ICM of the blastocyst, and are cultured in vitro on a feeder layer of mEFs. B. The hallmarks of ESCs, self-renewal and pluripotency are tested in the laboratory by monitoring expression of marker genes. Commonly used marker genes include Nanog and Oct4 (ESCs); Fgf5 (primitive ectoderm); Otx2 (ectoderm); T-Brachyury (mesoderm); and Gata6, FoxA2 and Sox17 (endoderm). Modified from 127.

Figure 1.3. ESC transcriptional network. Diagrammatic representation of major transcription factors regulating ESC self-renewal and pluripotency. Oct4, Sox2 and Nanog occupy the promoters of hundreds of genes in ESCs to sustain the expression of other transcription factors, signaling components and miRNAs, while inhibiting the expression of differentiation genes.

Figure 1.4. Diagrammatic representation of the myc family members, and their obligate binding partner max. The conserved myc homology boxes, phosphorylation sites, nuclear localization signal, and basic helix-loop-helix leucine zipper region that mediate DNA binding and some protein-protein interactions are indicated. Modified from 128.

CHAPTER 2

EXPERIMENTAL PROCEDURES

Cell Lines

The following mESC lines were used in these studies: R1 129, wild-type (WT)

AB2.1 and c-myc-/- AB2.1 (124, kindly provided by Dr. John Cleveland, Scripps,

Fl).

Culture of mESCs mESCs were cultured in the absence of feeders on gelatinized (0.2% in DPBS) tissue culture grade plastic. Cells were maintained at 37°C, 10% CO2 in

Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum

(FBS), 10% knockout serum replacement, 2mM L-glutamate, 1mM sodium pyruvate, 0.1mM β-mercaptoethanol, 100U/ml penicillin-streptomycin, and 1000

U/ml recombinant LIF (ESGRO, Chemicon). This medium was changed daily.

Cells were passaged every three or four days as needed using 0.05% trypsin-

EDTA (Invitrogen). After washing semi-confluent plates with phosphate buffered saline (PBS), trypsin was added until colonies began to detach from the culture dish. Cells were pipetted to obtain a single cell suspension, and added to an equal volume of mESC culture medium. After centrifugation at 200xg, mESCs

were resuspended in an appropriate volume of culture medium, and reseeded at

a density of approximately 1.5x104 cells/cm2.

Differentiation of mESCs

Differentiation was carried out by trypsinizing cells and replating in tissue culture

plates for adherent differentiation, or by replating in bacteriological dishes for suspension culture as embryoid bodies. Media used for differentiation consisted of Dulbecco’s Modified Eagle Medium supplemented with 10% FBS, 2mM L- glutamate, 1mM sodium pyruvate, 0.1mM β-mercaptoethanol, and 100U/ml penicillin-streptomycin. In some instances, this medium was supplemented with retinoic acid (4μM) to enhance differentiation. Medium was changed every second day.

To generate PrE, cells were aggregated in complete ESC medium at a

concentration of 2 x 105 cells/ml for 24 hours in the presence of sodium

orthovanadate (50 μM).

Generation of iPSCs

Conditional deletion of c-myc and N-myc was carried out using mice with the N-

mycfl/flc-mycfl/fl genotype (obtained from Dr. Paul Knoepfler, UC Davis). c-

mycfl/fl;N-mycfl/fl mice have loxP sites inserted at each 5’ of exon 2 and 3’ of exon

3 in the mouse c-myc and N-myc loci 130, 131. iPSCs were generated from mEFs

isolated from c-mycfl/fl;N-mycfl/fl embryos 59.

Culture of iPSCs

iPSCs were typically maintained on a feeder layer of mEFs on tissue culture

grade plastic. mEFs were seeded at a density of approximately 5x104/cm2 in

Dulbecco’s Modified Eagle Medium supplemented with 10% FBS, 2mM L- glutamate, 1mM sodium pyruvate, 0.1mM β-mercaptoethanol, and 100U/ml penicillin-streptomycin. iPSCs grown on mEFS were maintained at 37°C, 10%

CO2 in mESC complete medium as previously described. Alternatively, iPSCs

were cultured on tissue culture plates precoated with gelatin in mESC complete

medium. In both circumstances, fresh medium was added to the cells every

second day, and cells passaged as needed using trypsin as described

previously.

Differentiation of iPSCs

The pluripotency of generated iPSCs was tested in in vitro assays by performing

differentiation in suspension cultures as embryoid bodies, or in adherent cultures by removal of knockout serum replacement and LIF, in the presence or absence of retinoic acid as previously described.

Conditional Inactivation of c-myc and N-myc

c-myc-/-;N-myc-/- iPSCs were generated from c-mycfl/fl;N-mycfl/fl iPSCs by the

action of Cre recombinase, specifically using a pCAGiNeoCreGFP plasmid. This resulted in the simultaneous inactivation of c- and N-myc. Transfection efficiency was evaluated by microscopy, monitoring GFP expression.

Cloning and Expression vectors

The cloning vector pBluescript KS+, and the expression vectors pCAGiPURO or

pCAGiNEO 132 were used in these studies. Expression vectors conferred

resistance to puromycin or neomycin. The pCAG plasmid facilitated constitutive

expression under the control of the cytomegalovirus enhancer, chicken β–actin

promoter (CAG).

To generate epitope tagged c-myc, expression constructs with human c-myc

under the control of the CAG promoter were constructed. c-myc was either triple

hemagglutinin tagged (HA) or tagged with 6 concatamers of the myc tag (Myc) at

the N-terminus.

Expression constructs were prepared as follows: the plasmid pBS-h-myc

harboring the cDNA encoding human c-myc was used as the template in PCR.

PCR reactions consisted of 100ng DNA, PCR reaction buffer (Invitrogen), 250nM dNTP, 1mM MgCl2, 300nM primers, and 2.5 units Platinum Pfx (Invitrogen). DNA

was amplified in an amplification procedure consisting of 30 cycles of 94ºC for 30

sec, 55ºC for 30 sec, and 68ºC for 90 sec. Final extension was carried out at

68ºC for 10 min.

The forward primer, 5’- CAC ACA GAG CTC GAA TTC GCC ACC ATG AGC

GGC CGC CCC ATG CCC CTC AAC GTT AGC-3’ contained SacI and Not1

restriction sites, and the reverse primer 5’- CAC ACA CTC GAG GAA TTC TTA

CGC ACA AGA GTT CCG - 3’ contained an XhoI restriction site. Restriction

digestion with SacI and XhoI released the c-myc fragment. The plasmid pBSKS+ was similarly digested and removal of phosphate groups from the digested vector carried out by treatment with calf intestinal phosphatase. The desired fragments were purified by phenol-chloroform extraction, and ethanol precipitated after excising from a 1% agarose gel. The c-myc fragment was ligated into pBSKS+ using T4 DNA Ligase (New England Biolabs) at 16ºC for 2h to give pBS-c-myc1. pBS-c-myc1 and the plasmids pBS-3xHA and pBS-6xmyc that contained the

3XHA (HA) and 6xmyc (MYC) epitope tags cloned into Not1 sites were all digested with NotI. The HA and Myc tag fragments were excised after agarose gel electrophoresis and ligated in separate reactions into the Not1 site of pBS-c- myc1.

Ligations were carried out in a 10μl reaction volume consisting of T4 DNA ligase buffer, 10U of T4 DNA ligase (New England Biolabs), and insert and vector DNA in an approximate 3:1 ratio. The reaction was incubated at 16ºC for 2h, after which 5μl of the ligation reaction was transformed into competent E. coli (One

Shot Max Efficiency DH5α-T1, Invitrogen). Transformants were plated onto LB plates containing ampicillin (50μg/ml) and incubated overnight at 37°C. Colonies were cultured in LB media containing ampicillin (50μg/ml) and plasmid DNA extracted using the Qiagen Spin Miniprep or Midiprep kit (Qiagen) according to the manufacturers’ instructions. Insert integrity was verified by restriction digestion analysis or DNA sequencing to confirm correct insert orientation and plasmid integrity.

To generate expression constructs, restriction digests of the pCAGIPuro

expression vector, and the prepared plasmids were carried out with EcoRI. The

c-myc-HA and c-myc-Myc fragments were purified, ligated into purified

pCAGiPURO 132, and transformed. After transformation into E.coli and plasmid

DNA extractions using the QIAprep Spin Miniprep Kit (Qiagen) according to the

manufacturers’ instructions, plasmids were sequenced.

CreGFP expression was mediated through introduction of pCAGiNeoCreGFP.

The plasmid pCAGiNEOCreGFP was generated by restriction digestion of the pCAGCreGFP construct (Addgene 13776;133). The CreGFP fragment was

excised from pCAGCreGFP using XhoI and NotI, and introduced into pCAGiNeo.

The released fragment was ligated as previously described into pCAGiNEO and

construct integrity verified by restriction digestion analysis. MycER constructs

that can be activated by the addition of 4-hydroxytamoxifen (4-OHT) have been

previously described 18.

Transfection of Pluripotent Stem Cells

Approximately 2 x 105 cells were seeded into one well of a 6-well tissue culture

plate. Transfection was carried out on the following day with 10 μg DNA using

the Lipofectamine 2000 reagent (Invitrogen). Antibiotic selection begun 24h after transfection for 4 days, or clonal cell lines were generated after selection and expansion in the presence of antibiotic for 10-14 days.

Approximately 24h after transfection of pCAGCreGFP, iPSCs were isolated using fluorescence activated cell sorting according to the presence of GFP and cultured in the presence of neomycin (400 ng/ml for 72h).

Generation of stable iPS cell lines

Stable iPS cell lines expressing MycER were generated. Transfection and generation of stable cell lines were carried out as described previously.

Antibodies

Antibodies used in these studies were: rabbit anti-Cdk2 (Santa Cruz SC-6824), rabbit anti-c-myc (Cell Signaling Technologies #9402), mouse monoclonal anti-

HA tag (Cell Signaling Technologies #2367), rabbit anti-LSD1 (Cell Signaling

Technologies #2139), mouse monoclonal anti-myc tag (clone 9E10), mouse monoclonal anti-HA tag (Sigma H3662), rabbit anti-Oct4 (Santa Cruz SC-8628), rabbit anti-Nanog (Cosmo Bio REC-RCAB0004PF), rabbit anti-Max (Santa Cruz

SC-197), anti-HDAC1 (Santa Cruz SC-7872), anti-HDAC2 (Santa Cruz SC-

7899), rabbit anti-Sall4 (Abcam AB20112), c-myc (Santa Cruz N-262), and anti-

Nmyc1 (Upstate).

RNA isolation and Quantitative RT-PCR analysis

Total RNA was isolated from approximately 1x106 cells using the RNeasy kit

(Qiagen) according to the manufacturer’s instructions. cDNA was synthesized with approximately 1 μg RNA using the iScript cDNA synthesis kit (BioRad) using

the protocol recommended by the manufacturer. Real-time PCR was carried out

using gene specific TaqMan Gene Expression Assays (Applied Biosystems) and

the Universal PCR Master Mix (Applied Biosystems). In each 20μl amplification reaction, 5μl cDNA was added to 10μl Universal PCR mix and 1μl TaqMan. DNA was amplified in a 2-step amplification process consisting of an initial denaturation at 95ºC for 3 min, followed by 40 cycles of 95ºC for 15 sec and

60ºC for 1 min.

The mirVana miRNA isolation kit (Ambion) was used to isolate miRNAs, and mmu-miR20a was amplified using TaqMan MicroRNA assays (Applied

Biosystems) consisting of miRNA specific primers, primers and a Taqman probe

specific for miRNA20a. miRNA levels were normalized to GAPDH.

Cycling was carried out using the MyIQ Single-Color Real-Time PCR Detection

System (BioRad), and analyzed using BioRad iQ5 2.0 Standard Edition Optical

System Software. Transcript levels of target genes were assessed and expression normalized to GAPDH.

Immunofluorescence analysis

Cells were prepared for immunofluorescence analysis by plating on gelatinized

Lab-Tek chamber slides (Nunc) at a density of approximately 1.25 x 104 cells/ml.

Medium was aspirated, cells were washed with PBS, and fixed for 10 minutes at

room temperature with 4% paraformaldehyde. After washing to remove the

paraformaldehyde, cells were permeabilized for 5 minutes at room temperature with 0.2% Triton X-100 in PBS. Cells were then incubated in blocking solution

(10% goat or donkey serum in PBS) for 1 hour at room temperature. Cells were incubated with primary antibody at a 1:100-1:200 dilution in blocking solution overnight at 4°C. Cells were washed twice with PBS and incubated with secondary antibody (Molecular Probes, Invitrogen) at a 1:200 dilution in PBS for

45 min at room temperature in the dark. Nuclei were stained with 4’,6-diamidine-

2-phenylidole dihydrochloride (DAPI) at a 1:250 dilution in PBS. Slides were mounted with Prolong Gold mounting media and immunofluorescence visualized on a Leica DM 6000B microscope.

Western blot Analysis

Approximately 2 -3 x 107 cells were used to make cell extracts for immunoblot analysis. Cells were washed with ice cold PBS, and harvested using a cell scraper. Lysis was carried out with occasionally vortexing and pipetting cells in a modified RIPA buffer (50mM Tris pH 8.0, 150mM NaCl, 1% NP40, 0.5% deoxycholate, 0.1% SDS) containing protease (Roche) and phosphatase inhibitors (Calbiochem) for 30 min on ice. The lysate was frozen at -80°C until further use. After thawing, the lysate was passed through a 25-gauge needle, and centrifuged at 4°C at 20,800xg for 10 min. The supernatant was transferred to a clean tube, and protein concentration determined using the Bradford assay

(BioRad). Approximately 20-25μg protein per lane were electrophoresed on a

10% Tris-HCl gel at 20-30 mAmps. Electrophoresis was monitored by the

migration progress of the protein molecular weight standard (Dual Color

Molecular Weight Marker, BioRad). After electroblotting onto a nitrocellulose

membrane (BioRad), and blocking for 1h in 1% skim milk/PBST, membranes

were incubated in the appropriate primary antibody at a dilution of 1:1000-1:2000

in 0.5% skim milk/TBST at 4ºC overnight with gentle shaking. Excess antibody

was removed by washing with TBST, and the membrane incubated in HRP-

conjugated secondary antibody (DAKO) in 0.5% skim milk/TBST at a dilution of

1:2000 for 1 hour. Membranes were then washed and developed with ECLPlus

Western Blotting System Reagent (GE Healthcare) and detected by

chemiluminescence/autoradiography with X-ray film (GE Healthcare).

Chromatin Immunoprecipitation

Chip-on-Chip was carried out according to 30. Approximately 3x107 – 5x107 cells were used for each ChIP experiment. Chromatin was chemically crosslinked to cellular proteins by the addition of formaldehyde (1%) solution for 10 minutes at room temperature. Crosslinking was halted by the addition of glycine (125mM), and cells were rinsed twice with PBS and harvested. Cells were flash frozen in liquid nitrogen and stored at –80°C until required for use. Cells were thawed on ice and resuspended in lysis buffer 1 (50mM Hepes, 140mM NaCl, 1mM EDTA,

10% glycerol, 0.5% NP-40, 10% Triton X-100), centrifuged for 5 min at 1350xg, followed by resuspension in lysis buffer 2 (10mM Tris-HCl, 200mM NaCl, 1mM

EDTA, 0.5mM EGTA). Nuclei were pelleted by centrifugation at 1350xg, resuspended in 1-3ml of lysis buffer 3 (10mM Tris-HCl, 100mM NaCl, 1mM

EDTA. 0.5mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine), and sonicated to shear the crosslinked chromatin to an average size of 300-1000 bp.

Samples were immersed in an ice-water bath and sonicated using a Sonicator

3000 (Misonix) at power 7 for 10 x 30 second pulses with a 90 second pause in between pulses. Triton X-100 (final concentration, 2%) was then added and the mixture centrifuged at 20,800xg for 10 min at 4ºC. Shearing was verified by reversing the crosslinks of 50µl cell lysate in elution buffer (50mM Tris-HCl,

10mM EDTA, 1% SDS) and incubating at 65°C overnight. Contaminating RNA and protein were removed using RNase (10mg/ml for 30 min at room temperature) and proteinase K (20mg/ml for 30 min at 55°C). DNA was purified using a QIAquick kit according to the manufacturer’s instructions. DNA shearing was assessed after electrophoresis on a 1.5% agarose gel.

Magnetic beads were used in the isolation of c-myc bound chromatin. They were previously prepared by adding 100μl Dynabeads Protein G to 1ml Block Solution

(0.5% BSA in PBS). After mixing, the tube was placed on a magnetic device

(Dynal MPC), and the supernatant removed. Dynabeads were then washed twice with 1.5 ml Block Solution, and finally resuspended in 250μl Block Solution with 10μg of the respective antibody. This was placed on a rotating platform overnight at 4ºC. The following day, Dynabeads were washed three times with

Block Solution.

After verification of DNA shearing, a 50µl aliquot was removed as the input

control, and the whole cell extract was incubated overnight at 4°C with 100µl of

Dynabeads Protein G that had been precoated with 10 µg of the appropriate

antibody. Beads were washed 7 times with RIPA buffer and once with TE buffer

containing 50 mM NaCl at 4°C. Antibody-bound complexes were eluted from the

beads by adding 150µl elution buffer (1% SDS, 50mM Tris-HCl, 10mM EDTA)

and heating at 65°C for 15 min with occasional vortexing. Crosslinking was

subsequently reversed by overnight incubation at 65°C. Whole cell extract DNA

that was previously reserved as the input control was also treated for crosslink

reversal. Whole cell extract and immunoprecipitated DNA were purified with

RNaseA for 2h and proteinase K treatment as described before. DNA was precipitated after phenol:chloroform extraction and ethanol precipitation. After

resuspension in 10 mM Tris-HCl, purified DNA was used in real-time PCR as

described above with primers for target genes. PCR was carried out in triplicate

using primers as follows, Ncl forward 5’-TTGGCTATGATGCGAGTCC-3’, reverse

5’-TGAACCTGGAGTTATACCTACC-3’, Sall4 forward 5’-

CTGTAAGCCCCTTCTCCTTGAC-3’, reverse 5’-

TCCCTCTTGGTCGGTCTGTAG-3’, Smarcad1 forward 5’-

GTGGACTGGATGGCTTTGAAC-3’, reverse 5’-

GCAATCACTGGAGAAGGTAAGG-3’, Ezh2 forward 5’-

CTTAACCGCCCTCTTCTCC-3’, reverse 5’-TGCCTACTGGTGCTCTCC-3’, Set

forward 5’-GGTAAGTTCCTCCATTGC-3’, reverse 5’-

CCACATCACCTTCATTCC-3’, Mybbp1a forward 5’-

CTGTGTGTCTGGCAAGGG-3’, 5’ reverse GCATCTCGGATTTCTTCTTACC-3’,

MBD3 forward 5’-GGCTCACGAACCTATTTAGAAC-3’, reverse 5’-

TCCTCCTTCCTCCACTATCC-3’ Mel18 forward 5’-

CAAGGGAAGGGAAAGCAAGG-3’, reverse 5’- AGACACTGACCGAGGATAGC-

3’ ODC1 forward 5’-CCCGGAAACCCTACTCAGAATC-3’, reverse 5’-

TCAAAGTCTGCCCGATCTTAGG-3’ Stat3 forward 5’-

GATACGGCTCGCTTCTGC-3’, reverse 5’-GTCTCCACAAACCCAAATGC-3’

Zfp207 forward 5’-GGTTCTTCGGGCGAGAGG-3’, reverse 5’-

AACGGAGACAGGAGTTCTTAGG-3’, Gata6 forward 5’-

TTGGCTGGCGTTCCGAATGG-3’, reverse 5’-

GATACGGGTCACCTTGGCTTCC-3’, miRNA-17-92 cluster forward 5’-

CACGAGGATCACAGCAGTTGG-3’, reverse 5’-

GCAAGCCTGAACTCTACTGTCC-3’

Amplification was carried out using 5μl DNA, 500nM each primer and 10μl Sybr

Green Supermix (BioRad) in each 20μl reaction. Cycling was carried out in 2-

step amplification with cycles consisting of an initial denaturation at 95ºC for 3

min followed by 40 cycles of 95ºC for 15 sec and 60ºC for 1min. The MyIQ

Single-Color Real-Time PCR Detection System (BioRad) was used and data

analyzed using BioRad iQ5 2.0 Standard Edition Optical System Software.

Expression values for immunoprecipitated DNA were normalized to whole cell

extract DNA.

For genome-wide location analyses, immunoprecipitated chromatin and whole

cell extract DNA were amplified, fluorescently labeled with Cy3 and Cy5 dyes

respectively, and hybridized to Mouse Expanded Promoter ChIP-on-Chip Set

(Whitehead Institute, Agilent Technologies). Each set contains microarrays

printed with 60-mer oligonucleotide probes that cover the mouse genome

spanning positions -5.5kb to +2kb relative to the transcription start site for each

gene. Processing of the data including normalization, and combination of each

set were carried out using the DNA Analytics software (Agilent) to give a list of

statistically significant bound target genes.

Preparation of Antibody Crosslinked Tosylactivated Magnetic Beads

Dynabeads MyOneTM Tosylactivated beads were used to immunoprecipitate

Myc-tagged c-myc complexes from AB2.1c-mycMyc mESCs. Dynabeads were

prepared by washing 50mg three times in coating buffer (0.1M sodium borate

buffer). Dynabeads were resuspended in 795μl coating buffer, and 40μg anti-

Myc tag (clone 9E10 antibody) added. Ammonium sulphate stock solution (415

μl; 3M ammonium sulphate in 0.1M sodium borate buffer pH 9.5) was added, and

the mixture incubated for 16-24h at 37ºC with slow tilt rotation. The tube was

placed on a magnet (Dynal MPC) for 2min, the supernatant removed, and replaced with blocking solution (0.5%BSA, 0.05% Tween 20) overnight at 37ºC.

After removal of blocking solution, the coated Dynabeads were washed three

times with 0.1% BSA, 0.05% Tween 20 and stored in this solution until ready for

use.

Coomassie Blue Staining

Efficiency of the coating reaction was determined by staining with Coomassie

Blue. The staining of IgG bands in the antibody preparation was compared to that in the supernatant removed from the beads after the coating reaction.

Equivalent volumes of each sample were loaded onto a polyacrylamide gel, and electrophoresis carried out as previously described. Gels were washed three times for 5min, and 50ml Bio-Safe Coomassie Stain (BioRad) added. This was shaken gently on a rotating platform for 1h, rinsed with water for 30min, and viewed.

Immunoprecipitation Analysis

Native complexes were isolated from whole cell extracts using mammalian cell lysis buffer (50mM Hepes, 250mM KCl, 0.1mM EDTA, 0.1mM EGTA, 10% glycerol, 0.1% NP-40, 1mM dithiothreitol) supplemented with protease inhibitors.

Alternatively, nuclear extracts were prepared by washing cells in tris buffered saline (TBS), and swelling in lysis buffer A (10mM Hepes, 10mM KCl, 0.1 mM

EDTA, 0.1mM EGTA, 1mM dithiothreitol) with protease inhibitors. NP-40 (1.6%) was added, and the sample vortexed to disrupt the cellular membrane. After centrifugation at 1350xg and removal of the cytoplasmic supernatant, nuclear proteins were extracted with 20mM Hepes, 0.4M NaCl, 1mM EDTA, 1mM EGTA,

1mM dithiothrietol, protease inhibitors while shaking at 4ºC for 15 min 134.

Extracts were subjected to immunoprecipitation with the antibody (1μg) coupled to tosylactivated magnetic beads. Protein extracts were incubated with magnetic beads for 3h under slow rotation. The bead-antibody-protein complexes were separated by placing the tube on the magnet (Dynal MPC) for 2 min. Beads were then washed with cold PBS three times, and proteins eluted with 200μl glycine pH2.5. Eluate was neutralized by Tris-HCl pH8.8, and immunoprecipitated extracts were subjected to SDS-PAGE as described previously, or subjected to mass spectrometry analysis. Mass Spectrometry was carried out in collaboration with the Wells Laboratory (University of Georgia).

Silver Staining Analysis

Silver staining was carried out by fixing polyacrylamide gels in 50% ethanol, 10% acetic acid for 30 min, followed by a 10 min incubation in 10% ethanol and three times washes in distilled water. Sodium thiosulfate (0.2g/L) sensitization was done for 90 sec, followed by several washes. Silver nitrate (2mg/ml) was added, and the gel incubated for approximately 30 min, and washed three times.

Following development (sodium carbonate, 600mg/ml; formaldehyde, 0.02%; sodium thiosulfate) for 10 min, the reaction was stopped using 6% acetic acid, washed and stored.

Alkaline Phosphatase Staining

Cells were washed with PBS, and alkaline phosphatase staining was carried out using a Leukocyte Alkaline Phosphatase Kit (Sigma) according to the manufacturers’ instructions. A positive score was assigned to brightly stained

colonies with a tightly packed morphology. A negative score was given to

flattened faintly stained or unstained colonies with no defined colony boundary.

At least 150 colonies were scored, and the percentage of alkaline phosphatase

positive cells calculated.

Terminal Deoxynucleotide Transferase dUTP Nick-End Labeling (TUNEL)

Cells were washed with PBS and fixed with 4% PFA for 1 hour at room temperature. Fixed cells were washed PBS and apoptotic cells detected with an

In Situ Cell Death detection kit according to manufacuturer instructions (Roche).

Cell Cycle Analysis

Cells were trypsinized, washed with PBS, and fixed in 70% ethanol at -20°C

overnight. After washing with PBS, cells were incubated in a staining solution of

propidium iodide (50 ug/ml), RNase A (200 ug/ml), BSA (100 ug/ml) in PBS for

30 min at 37°C in the dark. Cells were then subjected to flow cytometry and analyzed by FloJoTM software.

To analyze the number of cells in S-phase, 5-Bromo-2`-deoxyuridine (BrdU,

50um) was added to culture medium for 2 or 24h. Cells were fixed with 4% PFA

in PBS for 10 min, washed with PBS, and treated with 2N HCl for 20 min.

Immunocytochemistry was performed as previously described using a rat anti-

BrdU antibody (1:100, Abcam).

Fluorescence activated cell sorting (FACS)

FACS was used for the isolation of GFP positive and negative cells from

AFPGFP ESCs, and after CreGFP transfections. This was performed on the

MoFlo XDP (Beckman Coulter) and data analyzed by FloJO TM. For subsequent experiments, cells were collected, and replated in ESC or differentiation medium as described above.

CHAPTER 3

MYC TARGET GENES IN EMBRYONIC STEM CELLS

Background

Previous studies have demonstrated that c-myc and N-myc are important for

embryonic development but not until mid-gestation stages 114, 116. Roles for the myc transcription factors in the pluripotent cells of peri-implantation embryos have therefore not yet been demonstrated. Demonstrating roles for myc transcription factors in early embryonic development is challenging because they are functionally redundant and display overlapping expression patterns at this developmental stage 111, 113. Therefore, no lethality would be anticipated for peri-

implantation embryos with the inactivation of a single family member. In support

of this, null c-myc and null N-myc mESCs have been generated and they retain the potential to self-renew 116, 124. While there is strong evidence linking myc to

the establishment and maintenance of pluripotency 18, 66, the functional

redundancy issue has clouded the interpretation and certainty of these findings.

An overarching goal of my studies was to unequivocally establish roles for myc in

pluripotent cells and also to define mechanisms of action. A number of

approaches were taken to address these issues. These include genetic

inactivation of c- and N-myc, identification of c-myc target genes, and identification of c-myc interacting proteins. Together, data from my studies will contribute to defining mechanisms by which myc regulates gene expression to maintain pluripotent stem cells.

Results

Identification of myc target genes in ESCs

c-myc target genes were identified on a global scale using ChIP-Chip. Since

DNA binding proteins are crosslinked to DNA in vivo, ChIP facilitates the mapping of transcription factor binding sites. In genome-wide analysis, hybridization to tiled promoter arrays spanning regulatory regions of the entire genome facilitates the global identification of myc binding sites. Ideally, to garner useful information from such microarray-based techniques, there should be high

signal with minimal background.

To achieve this, chromatin bound to epitope tagged c-myc was

immunoprecipitated using monoclonal antibodies against the epitope tags. A

large number of polyclonal antibodies against c-myc are commercially available,

but reactivity and specificity in other techniques does not indicate specificity and

suitability for use in ChIP. In addition, such antibodies that react with multiple

epitopes sometimes yield high background in ChIP experiments, requiring careful

testing and validation. The approach utilizing monoclonal anti-epitope tag

antibodies was used to eliminate the background signal obtained upon immunoprecipitation with some polyclonal antibodies. The epitope tagged c-myc constructs were introduced into null c-myc mESCs to maintain physiological levels of c-myc protein.

Generation and Assessment of Cell Lines for ChIP-Chip

The c-myc null (AB2.1c-myc-/-) and WT AB2.1 mESCs were obtained from Dr. John

Cleveland (Scripps, Fl). The presence and absence of c-myc in WT and c-myc null mESCs respectively was verified by immunoblot analysis (Figure 3.1A). N- myc expression increased in c-myc null mESCs compared to WT (Figure 3.1A), as expected due to autoregulation between the myc family members 135. This increase in N-myc expression is a possible compensation mechanism by which these mESCs maintain self-renewal in the absence of c-myc.

For ChIP experiments, stable cell lines were generated by transfecting epitope tagged human c-myc into the AB2.1c-myc-/- cell line. Two versions of epitope tagged c-myc were used. The first consisted of c-myc fused to three concatamers of the HA tag (c-mycHA), and the second of c-myc fused to six concatamers of the myc tag (c-mycmyc). Both epitope tags were inserted at the

N-terminus of the c-myc protein. A negative control cell line was generated by transfection with the pCAGiPuro vector 132. This control cell line, expressing no c-myc, should display negligible background binding in ChIP analyses and was used as a control.

One clonal cell line stably expressing each of these c-myc constructs (c-mycHA,

c-mycMYC) or empty vector was selected for use in ChIP-Chip. Each cell line displayed the morphology of compact colonies with distinct boundaries that is characteristic of mESCs when cultured on gelatin coated tissue culture dishes

(Figure 3.1B). These mESC lines were examined for expression of c-myc by immunoblot analysis. To eliminate the non-specific effects due to transgene overexpression in further studies, cell lines were selected that expressed levels of c-myc protein similar to levels expressed by the WT cell line. Thus, both cell

lines (c-mycHA, c-mycMYC ) chosen for further studies exhibited protein levels

similar to, or lower than the WT cell line (Figure 3.1C).

Selected cell lines were examined by immunostaining to verify correct nuclear

localization of epitope tagged c-myc. As expected c-mycHA, c-mycMYC cell lines

displayed nuclear staining using antibodies raised against the HA-tag and the

Myc-tag respectively (Figure 3.2). Nuclear staining was absent from the negative

control (empty vector) cell line. Furthermore, immunocytochemical analysis also

indicated that each cell line expressed the pluripotency marker, Nanog (Figure

3.2).

The ability of the c-mycHA and control cell lines to differentiate was investigated

using quantitative RT-PCR (qRT-PCR). During embryoid body differentiation, the

c-myc and Nanog transcript levels decreased, while mRNA levels for the

mesendoderm marker, Brachyury increased (Figure 3.3). This indicated that cell

lines expressing epitope tagged c-myc differentiated when aggregated in

suspension culture similar to WT mESCs.

Selected Cell Lines are appropriate for use in ChIP-Chip

After validating the selected cell lines, they were then used to identify c-myc

targets in ChIP experiments (Figure 3.4). As a first step, pan-c-myc, 9e10 (myc

tag) or anti-HA antibodies were used in comparative analyses to establish

relative efficiencies in chromatin immunoprecipitations. This was done in WT, c-

myc null, control (vector) cell lines, as well as cell lines expressing the epitope tagged c-myc (c-mycHA, c-mycMYC).

For initial investigations into whether the selected cell lines were appropriate for use in ChIP experiments, enrichment of the E-box containing c-myc target,

nucleolin 68 was assayed in each cell line. Muscarinic acetylcholine receptor

(AchR), a non-myc target 68 was used as a negative control. These experiments

were initially carried out with the pan c-myc antibody so that comparisons could

be made simultaneously for WT, c-myc-/-, the c-mycHA, c-mycMYC and negative

control cell lines.

In studies using the pan c-myc antibody, a 9-fold enrichment of chromatin in the

regulatory region of the nucleolin gene was obtained relative to AchR chromatin

in the WT cell line. In contrast, the enrichment of nucleolin over AchR in the c-

myc null ESC line was 0.5-fold (Figure 3.5A). The negative control cell line

(vector only) showed similar baseline enrichment to the c-myc null ESC line.

Both cell lines expressing epitope tagged c-myc (c-mycMYC, c-mycHA) showed

enrichment of 31 and 7-fold (Nucleolin:AchR) respectively, above and slightly

below the 9-fold enrichment observed for the WT cell line (Figure 3.5A). This

indicates that the selected cell lines behave similar to the WT cell line that

expresses endogenous c-myc and they are appropriate for use in ChIP-Chip

studies.

Although the pan c-myc antibody immunoprecipitated myc-bound chromatin with

relatively low background, and has since been used in ChIP-Chip studies 126, antibodies against the epitope tags were also assessed. The antibodies that are specific for the epitope tags were used in ChIP experiments to determine if they could immunoprecipitate myc-bound chromatin. The antibody raised against the myc tag was used as the immunoprecipitating antibody in ChIP experiments with the c-mycMYC cell line, and the vector cell line used as the negative control.

Eleven-fold enrichment was observed for the cell line expressing myc tagged c-

myc for nucleolin:AchR (Figure 3.5B). Similarly, 25-fold enrichment was

observed in experiments using the anti-HA antibody as the immunoprecipitating

antibody with the c-mycHA cell line (Figure 3.5B). This demonstrated that these

antibodies were capable of immunoprecipitating c-myc bound chromatin in ChIP

assays, giving high specificity with low background. Use of these antibodies in

ChIP-Chip analyses would thus yield specific results with minimal non-target

interactions.

To reduce background variation upon hybridization to promoter arrays, technical

replicates were prepared for use in ChIP-Chip experiments. This was done by

immunoprecipitating c-myc bound chromatin from a single cell lysate sample

from the appropriate cell line. This was done in triplicate using antibodies against the respective epitope tag.

Significant enrichment was observed in each sample when the nucleolin:AchR

ratios were compared. There was at least a 10-fold increase in enrichment ratios

for nucleolin:AchR for each replicate sample from each cell line expressing

epitope tagged c-myc. Specifically, enrichment ratios were 21-fold, 18-fold and

60-fold for c-mycHA replicates, and 10-fold, 34-fold and 34-fold for c-mycmyc replicates. Conversely, the ratios observed for replicates for the vector only control cell line were not significantly different for nucleolin:AchR, with a value between 1.2 and 1.8 (Figure 3.6). These enrichment values indicated that the samples were appropriate for use in ChIP-Chip studies, and thus chromatin was hybridized to whole genome promoter arrays (Agilent).

c-myc binds to genes implicated in diverse processes in mESCs

Approximately 1,562 genes were found to be c-myc targets based on enrichment of the immunoprecipitated chromatin relative to chromatin from whole cell extract.

Target genes were identified by bound probes in the whole genome promoter arrays, and were detected in c-mycHA, c-mycMYC cell lines but were not detected in the controls (Figure 3.7A). Specifically, 724 targets were found at least once in both cell lines, 675 only in c-mycHA, and 163 only in the c-mycMYC cell line (Figure

3.7A, Appendix A). These targets are associated with diverse cellular processes including gene expression (23%), cell cycle (21.7%), cell growth (18%), protein synthesis (13.6%), RNA processing (7%), nucleic acid metabolism (5.9%), cell death (4.7%), embryonic development (4%), and cell signaling (2.1%) (Figure

3.7B, Appendix A).

ChIP-Chip data were analyzed for validation purposes by examining chromosomal plots of target genes identified by DNA Analytics (Agilent). This software selects target genes by calculating the enrichment at specific tiled probes of the array, and comparing values of the immunoprecipitated chromatin to the whole cell extract. Probes that are considered bound are p<0.001.

As expected, c-myc was found to bind in the regulatory region of the positive control nucleolin, and there were no regions of binding in the negative control

AchR (Figure 3.8). Similarly, two targets identified in this study Sall4 and MBD3, that are used as case studies also showed c-myc binding regions upstream of the transcriptional start site (Figure 3.9).

A number of targets including Sall4, MBD3, Mybbp1a, Set, Smarcad1, Zfp207,

Ezh2, Otx2, Gata6 and the miR17-92 cluster (Table 1) were selected for further

study. These candidate targets were selected because they have been

previously implicated in transcriptional or epigenetic regulation in early

development, or maintenance of ESC self-renewal or pluripotency. Sall4 is a

member of the ESC core regulatory circuitry that regulates Oct4 and Nanog

transcription 136, 137. MBD3 is a member of repressor complexes such as NURD,

and recruits HDACs and DNA methyltransferases to promote gene silencing 138.

Mybbp1a, originally identified based on its ability to regulate the transcriptional status of c-myb 139, may act in concert with other transcription factors to modulate gene expression. Set is a multifunctional protein that regulates histone accessibility through histone methylation 140. Smarcad1 is an ATP-dependent

helicase member of the SWI/SNF family of the chromatin remodeling factors 141.

The function of Zfp207 remains undefined, but as a zinc finger protein, likely acts as a transcription factor. Ezh2 is a member of the polycomb group proteins that are important for inhibiting lineage specification genes in early development 142.

Otx2 is important for specification and development of neurectoderm 143. Gata6

is important for PrE differentiation 42, and the miR17-92 cluster has been

implicated in cell cycle control of other cell types 101. Further studies with Gata6

and miR-17-92 will be described in Chapter 4.

Sall4, MBD3, Mybbp1a, Set, Smarcad1, Zfp207, Ezh2, Otx2 were validated in

independent ChIP experiments using different cell extracts. Validation of c-myc

binding to these targets was successfully verified in independent ChIP

experiments (Figure 3.10). This representative sample indicated that targets

identified by ChIP-Chip using the c-mycHA, c-mycMYC cell lines were likely to be

true myc targets. However, further studies with specific targets that were identified herein should be validated on a case-by-case basis.

To establish if these genes were regulated in a manner consistent with them being myc targets, corresponding transcript levels were evaluated during differentiation. These data are summarized in Table 1. An expectation was that as ESCs differentiate and downregulate c-myc transcripts, mRNAs for the potential target genes should also decrease if they are regulated by c-myc.

Similar to the established c-myc targets nucleolin and ornithine decarboxylase

(ODC), there was a reduction in Mybbp1a, Set, Sall4, Smarcad1 and Zfp207

transcripts in early differentiation. These genes remain candidate targets for myc regulation. There was no significant change in Ezh2, and Otx2 transcripts

increased on differentiation (Table 1), indicating that Ezh2 may not be an

authentic myc target, and Otx2 might be a target repressed by myc.

To evaluate this by a different approach, expression of candidate genes was

examined in a 4-OHT regulated c-mycER cell line 18. The c-mycER fusion

protein consists of c-myc fused to the ligand binding domain of the estrogen

receptor and following addition of 4-OHT, the labile c-mycER fusion protein

translocates to the nucleus where it can activate and repress target genes. Upon stimulation of c-mycER, the expression of Nucleolin, Mybbp1a, Set, Sall4 and

Smarcad1 increased. Similar to the relationship observed with c-myc levels in differentiation studies, Ezh2 transcript showed no significant change, and Otx2 transcript levels decreased (Table 1). These data confirm earlier results and indicate that c-myc positively regulates Nucleolin, Mybbp1a, Set, Sall4 and

Smarcad1, has no effect on Ezh2 transcription, and negatively regulates Otx2 in mESCs.

B. c-mycMYC c-mycHA c-myc-/-V

C. myc HA -/-V WT c-myc c-myc c-myc -/- c-myc

CDK2

Figure 3.1. Generation of cell lines expressing epitope tagged human c- myc. A. Immunoblot analysis of AB2.1 WT and c-myc null cell lines showing the absence of c-myc expression in the c-myc null cell line, compared to WT, and an upregulation of N-myc in the c-myc null cell line. B. Phase contrast images (20x mag.) of selected cell lines showing typical ESC morphology. C. Immunoblot analysis of selected cell lines demonstrating similar protein levels or levels lower than the WT cell line.

Figure 3. 2. Accurate localization of c-myc in selected cell lines. Selected cell lines were subjected to immunocytochemistry to examine nuclear localization of c-myc and expression of the pluripotency marker, Nanog.

1.20 1.40 c-myc Nanog 1.00 1.20 1.00 0.80 0.80 0.60 0.60 0.40 0.40

0.20 Relative Expression Relative Expression 0.20 0.00 0.00 myc -/-;myc -/- -/-; -/-

Differentiation Differentiation

12.00 Brachyury 10.00

8.00

6.00

4.00

Relative Expression 2.00

0.00 myc -/-; -/-

Differentiation

Figure 3.3. Selected cell lines differentiate upon removal of LIF. qRT-PCR analysis of the c-mycHA cell line and negative control cell line examining transcript levels of c-myc, the pluripotency marker, Nanog, and mesendoderm marker, Brachyury.

Sample Control

Crosslink protein to DNA

Lyse cells and sonicate to fragment DNA

Immunopreciptate WCE

Purify and label DNA

Cy5 Cy3

Hybridize to microarray

Figure 3.4. Experimental Scheme for ChIP-Chip. Output is given as plots showing targets enriched in the immunoprecipitated samples relative to the WCE.

Figure 3.5. Selected cell lines are appropriate for use in ChIP experiments. A. ChIP data comparing enrichment for the WT cell line with selected cell lines expressing epitope tagged c-myc. Enrichment ratios for nucleolin:AchR are 9- fold for WT, 7-fold for the c-mycmyc cell line, 31-fold for the c-mycHA cell line, 1 for the control cell line with vector alone and 0.5-fold for the -/- cell line. B. ChIP experiments using the anti-myc tag antibody for immunoprecipitations indicated an 11-fold enrichment for nucleolin:AchR while background was observed for the control cell line. C. ChIP experiments using the anti-HA tag antibody for immunoprecipitations indicated a 25-fold enrichment for nucleolin:AchR, and no significant enrichment was observed for the negative control.

100

10 Nucleolin AchR 1

Relative Enrichment

0.1 -1 -2 -3 -1 -2 -3 -1 -2 -3 A A A yc yc yc or or or H H H M M M ct ct ct Ve Ve Ve Sample

Figure 3.6. Analysis of samples selected for ChIP-Chip. ChIP data showing significant enrichment for the myc target nucleolin compared to AcHR in samples from the c-mycHA and the c-mycmyc cell lines, and baseline ratios for the control cell line transfected with vector only.

A. c-mycHA c-mycmyc

675 724 163

Cell Signaling Embryonic Development Cell Death Gene Expression Nucleic Acid Metabolism

RNA Processing

Protein Synthesis Cell Cycle

Cellular Growth

Figure 3.7. Functional annotation and grouping of c-myc target genes. A. Number of targets obtained after ChIP-Chip using cell lines expressing epitope tagged c-myc. B.Target genes of c-myc in mESCs are implicated in diverse cellular processes.

Ncl Normalized Log Ratio

4 0.18 Is In Bound Region

0.16 P[Xbar]

3 0.14 0.12 0.1 2 0.08 0.06 1 0.04 0.02 0 0

5 0 4 6 79 7 191 933 467 7 7 9 1 1 4 8 9 1 1 81892558 819 8 819 8 8 8 -0 -0 -0 5-08 0-08 8-088194 9 8 0 6 8 7 9 1146 1 4408 4 8 9 9 9 1 1 1 1 8 8 8 8 881891968 8 8 8 0 :0 :0 :0 :0 r1: 1 r1:088190755-088190799r1 1 r1:088192131-088192190r1 r1:088195724-088195 hr1:088188039-088188098hr hr hr1:08819 c ch c ch ch c ch ch c ch

AchR Normalized Log Ratio 1.8 0.45 Is In BoundRegion 1.6 0.4 P[Xbar] 1.4 0.35 1.2 0.3 1 0.25 0.8 0.2 0.6 0.15 0.4 0.1 0.2 0.05 0 0

34 97 261 286 7 7 -008730197-008730438 075-008 644-008 6 8 0153 0388 3 3 872 872 08729707-008729751 :00 :00 0 r19 hr19 hr19: c ch chr19:008728799-008728856chr19:008729592-008729639c chr19:0087chr19:0087

Figure 3.8. Diagrammatic representation of c-myc binding sites in control samples. This is shown for the positive control nucleolin and an absence of such regions in the negative control AchR. Bound regions are identified by bound probes with a log ratio greater than approximately 2 and p <0.005. Bound regions (given a score of 1) and peaks are in red. Validation was carried out according to 68.

Sall4

4 0.18 0.16

3 0.14 0.12 Normalized Log Ratio 0.1 2 Is In Bound Region 0.08

0.06 P[Xbar] 1 0.04 0.02 0 0

5594556232575015801058656590025936059751

1684 1684 1684 1684 1684 1684 1684 1684 87-

4558 168 168456176-168457442-168457966-168458601-168458948-168459301-168459696- r r2: r2: r2: h h h chr2: ch 2: chr2: chr2: chr2: c c c chr2:168460002-168460061chr2:168460817-168460867chr2:168463281-168463340

MBD3 Normalized Log Ratio 4 0.12 Is In Bound Region P[Xbar] 0.1 3 0.08 2 0.06

0.04 1 0.02 0 0

9 8 4 056 751 558 00664 01241 03127 06046 9800 98 98009469801 98 98013979801 98 98032969803 98 7 -07 -07 -0 -07 -07 -07 -07 -07 -07 2 1338 3237 00436 801006 801699 803500 9 9 9 79800620 7 79801195 7 7 :0798 :0 :07980090:0 :0 :07980 :0 :079803083-07:07980 :0 :079805991-07 0 0 0 0 0 0 0 0 0 0 0 r1 r1 1 r1 r1 r1 r1 r1 r1 r1 r1 ch ch chr ch ch ch ch ch ch ch ch

Figure 3.9. Diagrammatic representation of c-myc binding sites in the targets Sall4 and MBD3. Bound regions are identified by bound probes with a log ratio greater than approximately 2, and p <0.005. Bound regions (given a score of 1) and peaks are in red. Validation was carried out using primers indicated by arrowheads.

Table 1. Examination of the relationships between selected c-myc targets and c- myc in differentiation and upon stimulation of c-myc activity. Occurrence Response to Regulation by Target 1 Comments in ChIP-Chip differentiation c-myc Ncl 6 Decrease Positive Established myc target ODC 4 Decrease Not determined Established myc target Mybbp1a 4 Decrease Positive Transcriptional regulator Ezh2 2 No Change No Change Polycomb component Mel18 2 Not determined Not determined Polycomb component Stat3 3 Not determined Not determined Transcription factor Set 4 Decrease Positive Transcriptional regulator MBD3 4 Not determined Not determined Transcriptional corepressor Sall4 4 Decrease Positive Transcription factor, regulates Oct4, Nanog Smarcad1 3 Decrease Positive Chromatin remodeling, SWI/SNF Zfp207 5 Decrease Not determined Zinc finger protein, function not well established Otx2 2 Increase Negative Transcription factor Gata6 2 1 Increase Negative Transcription factor miR17-922 6 No change Positive miRNA 1 Total number of replicate samples from which the particular target was identified. 2 See Chapter 4 for validation and further study.

25 c-mycHA 20 c-mycVector

Relative Enrichment 10

0 t 7 ll4 0 2 Se 2 zh2 l18 DC Ncl hR Sa e p1a O Otx E M Stat3 Ac MBD3 Zfp ybb M

Figure 3.10. Validation of c-myc target genes identified in ChIP-Chip. Relative Enrichment for select c-myc targets after ChIP assays comparing background enrichment obtained in the control cell line with enrichment after immunoprecipitation with the anti-HA antibody in the c-mycHA cell line. Nucleolin is the positive control and AchR, the negative control.

Discussion

Myc regulates diverse targets in ESCs

The identification of c-myc target genes was one approach used to gain insight

into myc function in mESCs. This was done on a global scale using ChIP-Chip.

The ChIP-Chip methodology permits identification of transcription factor target

genes. DNA is crosslinked to interacting proteins in vivo, permitting the spatial

and temporal mapping of factors bound to regulatory regions within the genome.

One drawback to this approach however is a high background signal that can

confound identification of true targets. This is partially due to non-specific interactions with the immunoprecipitating antibody, particularly with polyclonal

antibodies. To overcome this, the strategy utilized in my studies involved the use

of epitope tagged human c-myc expressed in a c-myc-/- mESC background. This

was a successful approach since binding of the tandemly arranged epitope tags

by their respective monoclonal antibodies resulted in only negligible background.

Immunoprecipitation with anti-HA tag and anti-myc tag antibodies therefore

facilitated high-resolution analysis of c-myc bound regions.

Another frequent challenge with the identification of true c-myc targets is the non-

specific binding frequently observed in overexpression studies 80. Since overexpression of proteins frequently results in off-target effects, such non-

specific interactions were kept to a minimum by selecting cell lines for use that

express c-myc at approximate physiological levels. Furthermore, use of two experimental cell lines, each expressing a different epitope tagged c-myc construct, provided added confidence in data generated, as transcriptional

targets found in both groups are less likely to be false positives. For an

additional layer of control, the negative control cell line that was transfected with

empty vector was tested in comparison with the c-myc-/- cell line, and only gave

basal background signals, similar to the c-myc-/- cell line.

The Myc Cancer Gene hubsite (www.myccancergene.org) lists greater than

1,600 genes as c-myc targets in processes ranging from cell cycle regulation,

metabolism, chromatin maintenance, RNA and protein synthesis and processing,

to cellular structure and adhesion. A similar number (1,562) and diverse array of

total c-myc targets were identified in this study. However, both these numbers

are still likely to be an underestimation of the true numbers of c-myc target genes

144.

This is a possibility because some targets that are weakly bound by myc may not be efficiently immunoprecipitated in ChIP experiments. Also, chromatin

immunoprecipitation may be inefficient in situations where myc interacts indirectly

with a particular target gene. This may be the case if myc is a component of

multi-protein complexes, where it is not directly contacting DNA. In such

scenarios, myc may be tethered to DNA via another protein 109, and chromatin

might not be efficiently crosslinked via the other protein to myc. This would prevent successful immunoprecipitation with myc.

One surprising finding in my study on c-myc targets in mESCs (Appendix A), compared to those identifying targets of the other reprogramming factors, Oct4,

Sox2, Klf4 29, 30, 44, was the scarce appearance of other members of the ESC core circuitry, as well as lineage specification genes. While Oct4, Sox2, Klf4 and

Nanog co-occupy their promoters, other transcription factors that have been implicated in ESC maintenance, as well as those that could induce differentiation

29, 30, 44, the myc targets identified primarily have roles in metabolism and cellular proliferation. This was similar to findings in other genome-wide location analyses that have since been performed by other research groups 44, 66.

Although the majority of targets identified in global identification of myc target genes 44, 66, 126 show significant overlap, each data sent contains unique target genes. For instance of the 724 targets that were common to both cell lines used in my studies, more than 200 were not identified in the study by Kim and colleagues 44. Furthermore, there was substantial variation in the number of targets identified. The number of targets identified by other research groups were 1172 66, 1459 126 and 3542 44 compared to the 1562 targets identified in my studies. This could be attributed to variations in technique to affect stringency that would result in dissimilar immunoprecipitation efficiencies. Additionally, alterations in the reagents used could also contribute to this disparity. While

Orkin’s research group used an affinity based approach (and verified with the

pan c-myc antibody) 44, Sridharan and colleagues 66, and Kidder and colleagues

126 used the pan-myc antibody described previously to immunoprecipitate c-myc

bound chromatin. Furthermore, Affymetrix tiling arrays spanning 28,000 mouse

promoter regions (approximately 18,000 mRNAs) were utilized by Kim and

Kidder and associates 44, 126, while Agilent arrays with 17,000 of the best defined mouse transcripts were used in my studies and by Sridharan and colleagues 66.

Additionally, binding of target genes is dynamic and is partially dependent on

input signals in a particular cell. Therefore, targets bound by myc in a given cell

population may fluctuate. Moreover, at a particular promoter epitopes not may

be exposed for antibody recognition due to masking by protein-protein

interactions within complexes. Furthermore, transcription factors scan DNA for

binding sites, and if the proportion of time spent at a given target site may be transient, this may preclude detection by ChIP analyses.

These factors could contribute to the variation in myc target genes between the

two experimental cell lines used in my studies, and among targets identified to

date by various research groups. This also increases the likelihood of

identification of unique targets in each study, as is the case with one of the

targets identified in my studies (Gata6). Such targets require strict validation

standards to verify it is a true target. Therefore, confirmation in independent

samples in separate ChIP experiments was carried out for Gata6 (Chapter 4).

The identification of Gata6 as a target in my study and not others, does suggest however that some targets might have been overlooked. This also underscores the importance of investigating functional control of discovered targets (by myc and other transcriptional regulators), to couple genome wide screens to functional analyses.

Select targets detected in both cell lines that were absent in the negative control cell line were individually validated in separate ChIP analyses. Examination of these targets relative to c-myc activity indicated that as shown in previous studies in other cell types, c-myc differentially regulates target genes in ESCs, acting as both a transcriptional activator of Sall4, Smarcad1 and Mybbp1a for instance, and repressor of Otx2.

Sall4 is a particularly interesting finding as it is a regulator of Oct4 and Nanog transcription. Thus, c-myc regulation of Sall4 potentially places it atop the ESC regulatory hierarchy. Repression of Otx2 is one possible mechanism by which c- myc maintains self-renewal through repression of neural specification.

CHAPTER 4

AN ABSOLUTE REQUIREMENT FOR MYC IN THE MAINTENANCE OF PLURIPOTENT CELLS

Background

The demonstration that c-myc was a regulator of ESC maintenance by Cartwright

and colleagues led to its inclusion as one of the 24 factors originally evaluated by

Yamanaka and colleagues in the reprogramming of mouse fibroblasts to iPSCs

59. As mentioned previously, c-myc is one of the final four factors used for

successful reprogramming in the initial studies by Yamanaka. At this time, the

authors proposed that the purpose of c-myc was to facilitate binding of other

reprogramming factors to target genes by increasing global acetylation 59.

Although subsequent reports have since revealed that exogenous myc is not an

absolute requirement, withdrawal of myc from the reprogramming cocktail

significantly reduces the efficiency of the process 71. Since cell lines that

reprogram efficiently without exogenous myc tend to express endogenous myc, it

remains likely that myc is essential for reprogramming.

Another cocktail utilized by Thomson’s research group used a different set of

factors, Oct4, Sox2, Nanog and Lin28 to achieve reprogramming 62. One point of

interest pertaining to this mixture however, is that Lin28 was identified as a c-myc

target in my study (Appendix A), as well as others that have recently been carried out 44. This raises the possibility that Myc promotes reprogramming, at least partially, through regulation of Lin28.

Such studies performed in the reprogramming field, together with the

identification of myc target genes in pluripotent cells on a genome-wide scale by

others have suggested that the primary role of myc in pluripotency is to promote cellular proliferation and to maintain metabolic activity 44, 66, 126. Sridharan and

colleagues also implicated c-myc as being important for silencing the somatic cell

gene expression program. They used microarray analysis to demonstrate that

introduction of exogenous c-myc induces repression of fibroblast specific genes,

and promotes an ESC gene expression program 66. Although, these data provide added information into the role of myc in establishment of the pluripotent state, specific roles in the regulation of self-renewal and pluripotency remain undefined, and functional validations of the proposed roles have yet to done. An investigation into these questions was another objective of my studies.

Results

Mechanisms of myc regulation of self-renewal and pluripotency

c- and N-myc are functionally redundant, and an absence of functional copies of

both genes is lethal in mid-gestation, while L-myc is not required. To adequately

address the requirement for myc in mESC maintenance, it is necessary to

inactivate both c- and N-myc. Mice containing alleles with exons 2 and 3 of both

c-myc and N-myc flanked by LoxP sites (N-mycfl/fl; c-mycfl/fl) were obtained for

this purpose. Cre mediated excision would then result in the inactivation of both

c- and N-myc.

iPSCs were generated from embryos from these mice to determine the effect of

co-deletion of c- and N-myc in pluripotent stem cells. To do this, mEFS isolated

from N-mycfl/fl; c-mycfl/fl embryos were reprogrammed to the pluripotent state by

retroviral transduction of Oct4, Sox2 and Klf4 71. Introduction of this cocktail of

factors results in the generation of iPSC lines that were characterized on the

basis of pluripotent marker expression and differentiation potential. These iPSCs

were then utilized to determine the effect of co-deletion of c- and N-myc on self-

renewal and pluripotency. Furthermore, N-mycfl/fl; c-mycfl/fl iPSCs were used to elucidate mechanisms by which c- and N-myc regulate target genes to sustain pluripotent cells.

The self-renewal and pluripotency of iPSCs was assessed by evaluating

expression of marker genes and by their ability to differentiate into derivatives of

the embryonic germ layers.

N-mycfl/fl; c-mycfl/fl iPSCs displayed morphology similar to that observed for

ESCs, that is, formation of compact colonies, with distinct boundaries in culture

(Figure 4.1A). These colonies were alkaline phosphatase positive, another

characteristic of pluripotent cell types. Furthermore, they expressed markers of

pluripotency, including Nanog and SSEA-1, as judged by immunofluorescence

analyses. Immunostaining was also used to determine that N-myc and c-myc

expression was intact (Figure 4.1B).

The differentiation potential of iPSCs was then examined. In vitro, N-mycfl/fl; c- mycfl/fl iPSCs differentiated into derivatives of the three germ layers, as monitored by qRT-PCR of Gata6 (endoderm); Brachyury (mesoderm); (ectoderm) Otx2 and

Fgf5 (primitive ectoderm) (Figure 4.2A). To further examine the differentiation potential of N-mycfl/fl; c-mycfl/fl iPSCs, their ability to form teratomas after

intramuscular injection into the hind limb of non-obese diabetic/severe combined

immunodeficient (NOD/SCID) mice was determined. N-mycfl/fl; c-mycfl/fl iPSCs

formed teratomas in vivo within 6 weeks of injection into recipient mice.

Dissections were carried out to determine if representative cellular derivatives of

the primary germ layers were present. Cells of ectodermal, endodermal and

mesodermal origin were observed in teratoma sections after

immunohistochemical analyses as indicated in Figure 4.2B.

Therefore N-mycfl/fl; c-mycfl/fl iPSCs displayed characteristics of self-renewal in

that they morphologically resembled pluripotent cells, and expressed markers of

pluripotency. They also had the potential to differentiate in vitro and in vivo. This indicated that they were successfully reprogrammed, and were appropriate for further studies to inactivate c- and N-myc.

Loss of pluripotency and differentiation to PrE following inactivation of c-

and N-MYC

The excision of floxed c- and N-myc alleles was mediated by transfection of a

CreGFP fusion construct into N-mycfl/fl; c-mycfl/fl iPSCs. Cre-mediated

recombination would result in excision of the floxed alleles, to result in effective

null phenotypes. FACS was used to separate GFP positive and GFP negative

cells, and expression of the CreGFP transgene was monitored by GFP

fluorescence after FACS. GFP negative cells (Flox) served as the control

population, and GFP positive cells (dKO) had deleted exons 2 and 3 of c-myc

and N-myc alleles upon excision by Cre (Figure 4.3, 4.4A).

To ensure that excision was efficient, agarose gel electrophoresis was used to visualize products amplified from genomic DNA by PCR. Primers were used to amplify floxed alleles to verify the presence of the CreGFP construct, and to amplify the products after excision. Floxed alleles of c-myc and N-myc were 500 and 260 bp respectively. The introduction of Cre was verified by the presence of an 880 bp band, and the presence of this band corresponded to amplification of alleles after c- and N-myc deletion by dKO specific primers. These amplification products were 650 and 350 bp for c- and N-myc respectively (Figure 4.4B).

Flox iPSCs maintained characteristics of self-renewal such as producing a

compact colony with a well-defined boundary when cultured on gelatin coated

tissue culture dishes in ESC medium. They also exhibited alkaline phosphatase

activity. In contrast, dKO cells exhibited a loss of self-renewal potential as determined by striking morphological changes when cultured in the presence or absence of mEF feeders. dKO cells were larger, did not grow in a well-defined colony, and displayed a more flattened morphology. This was accompanied by a loss of alkaline-phosphatase activity (Figure 4.5). These changes indicated that c- or N-myc is required to sustain self-renewal in iPSCs.

To determine changes at the molecular level that result in this loss of self- renewal, qRT-PCR analysis of marker transcripts of the three embryonic germ layers was carried out. There were notable increases in the expression of the endoderm marker genes Gata6 and FoxA2, showing 4-fold and 6-fold increases in transcripts respectively. On the contrary, much smaller changes were observed for the mesendoderm/mesoderm marker Brachyury, and the primitive ectoderm marker, Fgf5 (Figure 4.6).

Immunofluorescence analysis confirmed these observations. Loss of c- and N- myc expression was accompanied by a loss of Nanog and SSEA-1 expression, with increased expression observed for the endoderm markers Foxa2 and Gata4

(Figure 4.7). This suggested that lineage specific differentiation, primarily to endoderm occurred upon co-deletion of c- and N-myc.

An increase in apoptosis has been reported upon co-deletion of c- and N-myc in hematopoietic stem cells 122. TUNEL analysis was carried out to determine if

there was a similar increase in apoptosis upon deletion of c- and N-myc in

iPSCs. However, there were only slight increases in the level of apoptosis

between Flox and dKO cells. This indicated that at the timepoint assayed, loss of c- and N-myc did not cause gross changes in programmed cell death (Figure

4.8).

Loss of N- and c-myc results in remodeling of the cell cycle

Myc is also an established regulator of the cell cycle, and is likely to play a

central role in the characteristic cell cycle structure of pluripotent cells described

previously. Therefore, the cell cycle profile of dKO and Flox cells was examined.

Flox cells displayed the expected pluripotent cell cycle structure with the majority

of cells (>60%) in S-phase. There was significant restructuring of the cell cycle

profile of dKO cells compared to Flox cells with decreases in the percentage of

cells occupying S-phase, and increases in those occupying G1 in dKO cells

(Figure 4.9A). BrdU analyses confirmed these data, as dKO cells had reduced

uptake of BrdU compared to Flox iPSCs following a 2h pulse. However, dKO

cells still proliferate, and >90% incorporated BrdU after labeling for 24h ,

indicating that they progress through the cell cycle more slowly (Figure 4.9B).

To identify the mechanisms involved in remodeling of the cell cycle profile

described above, regulators of the cell cycle were analyzed. The miRNA cluster

miR-17-92 was found to be a target of c-myc in ESCs by ChIP-Chip analyses

(Figure 4.10A). This miRNA cluster is an established myc target in some

cancers, where it is a key regulator of cell cycle targets such as E2F1 and cyclin

D1, facilitating proliferation of the cancer cells 101, 145.

ChIP followed by q-PCR was carried out in independent samples to confirm the

miR-17-92 cluster is a target in ESCs (Figure 4.10B). Evidence of direct

regulation of the cluster by c-myc in pluripotent cells came from observations that

the expression of miR20a, one miRNA member of the cluster, is reduced in dKO

cells compared to Flox cells (Figure 4.11A). To determine if c-myc would

stimulate expression of miR20a, activation of the c-mycER fusion was carried out

in ESCs using 4-OHT. Upon stimulation of c-myc activity, there was an increase

in miR-20a levels (Figure 4.11B). These data confirm that the miR-17-92 cluster

is a direct target of c-myc in ESCs.

As the miR-17-92 cluster has been shown to regulate cell cycle targets,

expression of cell cycle regulators on modulation of myc/miR-17-92 levels was

examined. Rb2/p130, a cell cycle regulator implicated in restriction point control

is not typically expressed in pluripotent cells. Deletion of myc, with a concomitant decrease in miR20a levels led to increases in Rb2/p130 expression (Figure

4.12). These changes are temporally associated with remodeling of the cell cycle that is observed in dKO cells (Figure 4.9A).

c- and N-myc but not L-myc promotes ESC self-renewal

Differentiation occurs upon co-deletion of c-and N-myc in iPSCs. This indicates that although L-myc is expressed, it is not sufficient to maintain self-renewal. To determine if overexpression of L-myc could rescue self-renewal, an inducible system was used to compare stimulation of L-myc activity to c- and N-myc activity in iPSCs.

Addition of the drug, 4-OHT was used to stimulate activation of the c- N- or L- mycER constructs upon concurrent deletion of endogenous c- and N-myc.

Activation of c-mycER and N-mycER individually, confirmed the ability of c- or N- myc to promote self-renewal. There was a recovery of pluripotent stem cell morphology (Figure 4.13A), alkaline phosphatase staining (Figure 4.13B), as well as a reduction in the expression of endoderm markers FoxA2 and Sox17 (Figure

4.14). There were no significant changes in Brachyury expression upon stimulation by mycER as expected, since there was no change comparing dKO cells to Flox cells, (Figure 4.14).

On the contrary, the ability of L-myc to sustain self-renewal in the absence of the other family members is considerably reduced (Figure 4.13), and this confirms the observation of loss of self-renewal upon deletion of c- and N-myc in iPSCs even in the presence of L-myc.

N-or c-myc is required for proper multilineage differentiation

Although dKO cells spontaneously differentiate primarily to PrE, this may have

been because loss of c, N-myc precluded formation of other lineages. This was

tested by culturing embryoid bodies under conditions that normally give an outer

layer of PrE and an inner mass of cells comprising of derivatives of the primary

germ layers.

The first observation made from this analysis was that embryoid bodies

generated from dKO cells were markedly smaller that those generated from Flox

cells (Figure 4.15). This is partially due to a reduction in proliferation rates of

differentiated dKO cells (Figure 4.9), and possibly the absence of specific

lineages in generated embryoid bodies. To address this, qRT-PCR was carried

out to examine the levels of transcripts of lineage markers. There was

approximately a 40-fold increase in Gata6, >300-fold increase in FoxA2, and approximately a 200-fold increase in Sox17 transcript levels in dKO cells compared to much lower levels in Flox controls. Interestingly, there was a striking lack of expression of Brachyury and Fgf5 in dKO cells indicating a loss of capacity to generate lineages other than endoderm (Figure 4.16).

Another differentiation model was used to test this idea, as different cell types are

generated under different culture conditions. Differentiation was evaluated in

adherent culture following LIF withdrawal in the presence or absence of retinoic

acid. Retinoic acid stimulates neural differentiation, and LIF withdrawal in adherent culture also promotes mesoderm generation.

Similar to that observed during embryoid body differentiation, there were marked increases in the endoderm markers Gata6, Foxa2, and Sox17 (12 - 40 fold) for dKO cells compared to Flox cells. Although there were significant increases in

Brachyury, Fgf5 and Otx2 transcripts in Flox cells, these increases in expression levels were significantly reduced in dKO cells (Figure 4.17). Taken together, these data confirm that dKO cells are predisposed to form endoderm under conditions of LIF removal.

Myc represses endoderm differentiation by suppressing Gata6 activity

Since myc loss results in endoderm formation, the working hypothesis was that myc blocks differentiation of pluripotent cells to PrE. The mechanism by which myc represses endoderm was explored by examining the expression of c-myc in endoderm populations, and investigating the effects of stimulation of c-myc on the PrE specification factor, Gata6.

To determine the expression of c-myc in endoderm, an AFPGFP ESC line was utilized. AFP is expressed in visceral endoderm, and GFP is expressed in this cell line under control of the AFP promoter. Upon aggregation of ESCs in LIF, a layer of GFP positive PrE will form on the surface of the embryoid bodies 21.

FACS was carried out to separate GFP positive and negative populations. As anticipated, GFP positive cells expressed Gata6 and Foxa2, while GFP negative cells expressed higher levels of Nanog transcripts (Figure 4.18). As mentioned previously, Nanog and Gata6 expression is mutually exclusive, with Nanog specifying ESCs and Gata6 marking PrE. Interestingly, c-myc transcript was also reduced to levels similar to Nanog in the PrE population (Figure 4.18), consistent with the possibility that c-myc plays a role in PrE repression.

A search was carried out for c-myc target genes that had a potential role in PrE specification. After close examination of ChIP-Chip data, the promoter region of

Gata6, approximately 3kb upstream of the transcriptional start site was identified as a potential c-myc binding site (p<0.001) (Figure 4.19A). To confirm this finding, ChIP analysis was carried out on independent samples, and there was an approximately 7-fold increase in enrichment over the control c-myc null ESCs

(Figure 4.19B).

To directly evaluate the effects of c-myc on Gata6 expression, c-mycER cells were treated with 4-OHT. This decreased Gata6 transcript levels compared to the vector control in –LIF conditions, consistent with the possibility that myc represses Gata6 (Figure 4.20A). These results were confounded however by the presence of other lineages in the spontaneous differentiation that results under such differentiation conditions. To generate cells that are directed towards PrE, the protein tyrosine phosphatase inhibitor sodium orthovanadate, which induces

PrE formation through the Grb2/MEK dependent mechanism was used 21.

Indeed, upregulation of Gata6 was observed on addition of sodium orthovanadate in cell lines transfected with vector only, as well as the c-myc ER cell line (Figure 4.20B). The activation of c-mycER with 4-OHT blocked this increase in Gata6 transcripts. Moreover, the repression of Gata6 by c-myc is independent of Nanog, as Nanog transcript levels remained low upon stimulation of c-mycER (Figure 4.20B). Taken together, these data indicate that in pluripotent stem cells, myc inhibits PrE specification by repressing Gata6 in a

Nanog-independent manner.

Figure 4.1. iPSCs display characteristics of self-renewal and pluripotency. A. Phase contrast and alkaline phosphatase staining of N-MYCfl/fl;c-MYCfl/fl iPSCs showing typical pluripotent stem cell morphology. B. Immunostaining demonstrating the expression of pluripotency markers, Nanog, SSEA-1, c-myc, and N-myc in N-mycfl/fl;c-mycfl/fl iPSCs. Scale bar, 100 μm.

Figure 4.2. iPSCs differentiate in vitro and in vivo. A. qRT-PCR of Nanog, c- myc and markers for endoderm (Gata6), mesoderm (Brachyury), and primitive ectoderm/ectoderm (Fgf5, Otx2) demonstrate the ability of N-MYCfl/fl;c-MYCfl/fl iPSCs to differentiate into multiple germ lineages in vitro. Experiments were performed in triplicate, normalized to GAPDH and represented as mean ± s.d. B. Immunohistochemistry of neuron-specific enolase, glial fibrillary acidic protein, smooth muscle actin and H&E staining reveals similar multi-lineage differentiation potential in teratomas. Scale bar, 20 μm.

Figure 4.3. Experimental scheme depicting inactivation of c- and N-myc in iPSCs. A. Derivation of double knockout c-myc; N-myc iPSCs by transfection of CreGFP and isolation by FACS. B. Phase contrast and fluorescence images showing mixed populations of GFP positive and negative cells before separation by FACS. White arrows indicate Flox cells, and red arrows indicate dKO cells.

Figure 4.4.Cre excision facilitates deletion of c- and N-myc in iPSCs. A. Locus map of targeted alleles representing alleles before and after excision by Cre. Blue arrows indicate loxP sites. B. PCR using primers indicated in A to analyze genomic DNA isolated from c-MYCfl/fl;N-MYCfl/fl, CreGFP- (Flox) iPSCs, and CreGFP+ (dKO) iPSCs, confirming deletion of c- and N-MYC in dKO cells.

Figure 4.5. Deletion of c- and N-myc in iPSCs results in the loss of self- renewal. A. iPSCs transfected with CreGFP, FACS-isolated to separate dKO and Flox cells, and plated in LIF medium for 3 days; phase contrast images of dKO and Flox cells on gelatin (left panels), alkaline phosphatase images of dKO and Flox cells on gelatin (middle panels); alkaline phosphatase images of dKO and Flox cells on mEFs (right panels). Scale bar, 100μm. B. Quantitative analysis of alkaline phosphatase staining for iPSCs, Flox and dKO cells, n>150, for each condition.

Figure 4.6. Deletion of c- and N-myc in iPSCs results in an increase in endoderm marker transcript levels. qRT-PCR analysis of c-myc, N-myc, and marker expression of Flox and dKO cells. qRT-PCR of c-myc, N-myc, endoderm (Gata6 and Foxa2), mesoderm (Brachyury), and primitive ectoderm (Fgf5) markers indicated preferential endoderm differentiation in dKO cells, compared to Flox cells. Experiments were performed in triplicate, normalized to GAPDH and represented as mean ± s.d.

Figure 4.7. Deletion of c- and N-myc in iPSCs results in the upregulation of endoderm markers. Immunostaining for c-myc, N-myc; pluripotency markers, Nanog and SSEA-1; and endoderm markers, FoxA2 and Gata4 reveals the spontaneous differentiation to endoderm from loss of myc in iPSCs. Scale bar, 100 μm.

Figure 4.8. Co-deletion of c- and N-myc results in slight increases in apoptosis. TUNEL assay indicates that only small increases in apoptosis were observed for dKO cells compared to Flox cells. Scale bar, 100μm.

A. B.

Figure 4.9. Loss of c- and N-myc results in decreased proliferation and remodeling of the cell cycle. A. Cell cycle profile of Flox and dKO cells by flow cytometric analyses reveals a lengthening of G1 for dKO cells. B. Flow cytometric analyses showing a decrease in the percentage of S-phase cells and an increase in G1 for dKO cells. C. dKO cells exhibit reduced rates of proliferation compared to Flox cells, as observed with immunostaining after BrdU incorporation after addition for 2h or 24h. Scale bar, 100μm.

Figure 4.10. c-myc binds to the miR-17-92 cluster in ESCs. A. Plot showing enrichment of chromosomal regions in ChIP-Chip assays. Comparisons of immunoprecipitated chromatin and whole cell extracts indicate that c-myc binds to the upstream regulatory region of the miR-17-92 cluster; *p<0.001. B. Independent validation of ChIP-Chip analysis with ChIP-qPCR.

Figure 4.11. c-myc regulates the miR-17-92 cluster in ESCs. A. miR-20a transcript is downregulated in dKO cells compared to Flox cells. B. Activation of c-mycER with 4-OHT in ESCs increases miR-20a transcript over basal levels. Experiments were performed in triplicate, normalized to GAPDH and represented as mean ± s.d.

Figure 4.12. Rb2/p130 is upregulated upon loss of c- and N-myc. Immunostaining analysis reveals increased expression of the mir-17-92 target, Rb2/p130 in dKO cells compared to Flox cells. Scale bar, 100 μm.

Figure 4.13. Activation of c-myc and N-myc but not L-myc is able to rescue the self-renewal potential in dKO cells. A. iPSC morphology after co- transfection of CreGFP, along with 4-OHT-inducible myc family members, c- mycER, N-mycER and L-mycER in the presence or absence of 4-OHT. Scale bar, 100 μm. B. Quantitation of alkaline phosphatase staining showing % alkaline phosphatase positive versus % alkaline phosphatase negative, after co- transfection of CreGFP, along with 4-OHT-inducible myc family members, c- mycER, N-mycER and L-mycER in the presence or absence of 4-OHT; n>300 for each condition.

Figure 4.14. Stimulation of c- and N-myc activity blocks the increase in endoderm marker transcripts in dKO cells. Activation of 4-OHT-inducible myc inhibits the increase of Gata6 and Sox17 transcript as determined by qRT-PCR. Experiments were performed in triplicate, normalized to GAPDH and represented as mean ± s.d.

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Figure 4.15. dKO embryoid bodies are reduced in size compared to embryoid bodies derived from Flox iPSCs. Phase contrast images taken after embryoid body differentiation showing a reduction in size of dKO embryoid bodies compared to Flox embryoid bodies. GFP fluorescence was monitored to verify expression of the transgene. Scale bar, 100μm.

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Figure 4.16. dKO iPSCs are predisposed to form endoderm upon differentiation in suspension culture. qRT-PCR of Nanog; Gata6, Foxa2, Sox17 (endoderm); Brachyury (mesendoderm); and Fgf5 (primitive ectoderm) during embryoid body differentiation indicates that the loss of Myc predisposes iPSCs to PrE differentiation. Experiments were performed in triplicate, normalized to GAPDH and represented as mean ± s.d.

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Figure 4.17. dKO cells preferentially form endoderm in adherent differentiation conditions. qRT-PCR analysis of endoderm markers, Gata6, Foxa2, and Sox17; mesendoderm marker, Brachyury; primitive ectoderm marker, Fgf5; and ectoderm marker, Otx2 4 days after LIF removal with and without retinoic acid (RA). Triplicate experiments were performed, normalized to GAPDH and represented as mean ± s.d.

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Figure 4.18. c-myc expression is reduced in PrE. qRT-PCR analysis showing that similar to Nanog, c-myc transcript levels are reduced in PrE cells isolated from aggregated ESCs that express GFP under the AFP promoter.

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Figure 4.19. The Gata6 promoter is bound by c-myc in ESCs. A. Plot showing enrichment of chromosomal regions in ChIP-Chip assays. Comparisons of ratios of immunoprecipitated chromatin to whole cell extracts indicate that c-myc binds to the promoter region of the Gata6 in ChIP-Chip analysis, *p<0.001. B. Independent validation of Gata6 as a c-myc-bound target by ChIP-qPCR.

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Figure 4.20. c-myc represses Gata6 in ESCs. A.Activation of c-mycER with 4- OHT in ESCs represses Gata6 transcript levels upon LIF removal by qRT-PCR. B. qRT-PCR analysis showing that induction of Gata6 transcript by sodium orthovanadate is blocked by activation of c-mycER with 4-OHT in ESCs by qRT- PCR. Triplicate experiments were carried out and values normalized to GAPDH and represented as mean ± s.d.

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Discussion

Defining specific roles for myc in the establishment and sustenance of pluripotent

cells has remained elusive. In this study, my data define, for the first time, distinct roles for myc in the establishment and maintenance of self-renewing pluripotent cells. One major mechanism identified was the inhibition of PrE formation by a mechanism involving repression of Gata6.

Gata6 is an inducer of PrE differentiation in ESCs and parental cell types in the

embryo. In early embryogenesis, Rossant’s group demonstrated that as early as

the ICM stage, cells fated to become PrE and epiblast express Gata6 and Nanog

respectively in a mutually exclusive manner 4. Terada’s group subsequently

demonstrated that ESC populations are similarly heterogeneous, and

demonstrated that repression of Gata6 in ESCs is regulated by Nanog 41. These

studies complement the observation that Nanog null ESCs differentiate into PrE

40, and Gata6 overexpression is sufficient to drive PrE formation even in the

presence of LIF 42. Moreover, embryoid bodies generated from Gata6 null ESCs do not form the outer PrE derived layer, and fail to express other markers of PrE including Gata4 and AFP. Consistent with this, Gata6 null embryos fail to gastrulate due to defective visceral endoderm formation 146, 147. Altogether, these

data indicate the importance of Gata6 in the development of PrE.

107

The identification of Gata6 as a target regulated by myc represents a major

contribution to mechanisms by which myc maintains ESCs. Gata6 is a novel

target that was identified in my ChIP-Chip studies. Additionally, c-myc activation

results in a reduction of Gata6 transcripts, and conversely Gata6 transcript levels

increase upon loss of c- and N-myc. This indicates indirect regulation of PrE

formation through Gata6 by c-myc. This finding that c-myc binds to, and

represses Gata6 in a manner independent of Nanog, together with the striking

preferential PrE differentiation of c-, N-myc deleted iPSCs, indicates that c-myc imposes a differential blockade to PrE. This provides another central mechanism

for the regulation of self-renewal of pluripotent cells by myc.

My work has now addressed a major question relating to pluripotent stem cell

biology: 'is myc essential for maintenance of pluripotency?’ The simultaneous

inactivation of both c- and N-myc establishes, for the first time, the absolute

requirement for one of these two family members in the maintenance of self-

renewal.

Unlike c- and N-myc, L-myc could not promote self-renewal in my studies. This

is consistent with the spontaneous differentiation of dKO cells, which maintain L-

myc expression. The reasons for the inability of L-myc to maintain pluripotent cells may be related to its reduced transactivation and oncogenic potential, relative to c-myc 148. Additionally, while L-myc has the potential to activate

expression of similar target genes such as CAD, it positively regulates the target

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CCL6 to oppose the effect of c-myc 149, 150. L-myc could therefore have non-

overlapping or partially overlapping roles in ESC maintenance that have not been defined by these studies.

Pluripotent cells exhibit a characteristic cell cycle profile where cells spend

approximately 60% and 15% of time in S-phase and G1, respectively (see Figure

4.9). Although not formally proven yet, this is likely to be associated with

mechanisms underpinning pluripotency. A defining feature of early differentiation

is the restructuring of the cell cycle so that the gap phase becomes lengthened

and the proportion of S-phase cells reduced 28. Therefore, the replication

potential and cell cycle structure of dKO iPSCs was examined. Coinciding with

the expression of endoderm markers following myc deletion, a reduction in the

rate of proliferation was observed together with a remodeling of the cell cycle.

ChIP-Chip analyses indicated that c-myc occupied the upstream regulatory

region of the miR-17-92 cluster, and directly activates its expression (Figure

4.10). Regulation of the miR-17-92 cluster by myc 101, in turn controls the post-

translational synthesis of Rb2/p130, effecting changes in cell cycle control. This

miR-17-92/Rb2/p130 relationship is similar to one previously shown in lung

progenitor cells where miR-17-92 promotes proliferation by inhibiting Rb2/p130

107. My studies therefore provide direct evidence for the role of myc in

establishing the cell cycle structure of pluripotent cells, which is a key

characteristic of their self-renewal. It is also a possibility that the miR17-92/Rb2

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mechanism shown here is only one of several mechanisms by which myc

establishes the ESC cell cycle structure however, particularly because several

cell cycle regulators including cyclin D1 and Cdk4 were identified as myc targets

in these (Appendix A) and other studies 44.

Maintenance of ESC cycle structure, which is characterized by a short G1 with

relatively large proportions of cells in S-phase, is partially due to Rb

hyperphosphorylation, which results in E2F dependent transcription. Regulation

of the miR-17-92 cluster by myc, targets and inhibits one such cell cycle regulator

Rb2/p130, which is involved in establishment of the restriction point in G1-S

progression 24-26. Inhibition of Rb2/p130 by myc/miR-17-92 is therefore likely to

block the activation of such checkpoints in pluripotent cells, promoting continuous

entry into the S-phase and long-term proliferation capacity.

In addition to the regulation of self-renewal, co-deletion of c- and N-myc resulted

in marked defects in the capacity to form mesoderm and ectoderm. This was

seen in three differentiation systems including suspension embryoid body culture,

and adherent cultures with and without retinoic acid supplementation. Data

obtained in all differentiation systems indicated that in the absence of c- and N-

myc, pluripotent cells lose their multilineage differentiation capacity and are predisposed to form endoderm. The absence of mesendoderm and primitive ectoderm markers indicate that this is likely to be PrE. This therefore implicates

myc as having a role in proper lineage specification as early as the peri-

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implantation stage of embryonic development. This is a stage prior to that which has been shown by Cleveland’s laboratory, which found that c-myc null ESCs

were deficient in embryonic blood vessel formation and development, an

observation that is also consistent with the c-myc null phenotype 114, 124.

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CHAPTER 5

IDENTIFICATION OF MYC INTERACTING PROTEINS IN EMBRYONIC STEM CELLS

Background

The ability to interact and recruit other transcriptional and epigenetic regulators

by myc is well established, as it exists in numerous complexes in somatic cell

types 80. Recently, a study identified over 200 binding partners of myc in

HEK293 cells using total cellular extracts 110. Some myc-interacting proteins

include other transcription factors, as well as chromatin modification complexes.

For instance, myc interacts with TRRAP, which is a component of the GCN5 and

TIP60 HAT complexes 151, 152. Recruitment of such complexes, which facilitates

acetylation of target genes, may be a mechanism by which myc promotes transcriptional activation. In addition, myc also interacts with chromatin remodeling components such as members of the Swi/Snf family 110, 153. These

are of interest because such epigenetic regulators are also key regulators of self-

renewal of stem cell populations 70, 154.

The proteins that myc interact with in pluripotent stem cells provide a view into

possible complexes within which myc exists at regulatory regions of target genes.

Such complexes may reveal new roles for myc or offer insight into how myc is

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regulated in these cell populations. A proteomics approach was taken using

cellular extracts from a mESC line expressing epitope tagged c-myc.

Identification of myc-interacting proteins in ESCs should provide further insight

into the mechanisms behind myc action.

Results

To examine myc protein interaction networks in ESCs, affinity purification

coupled with mass spectrometry was used to identify c-myc interacting proteins

(Figure 5.1).

The c-mycMYC mESC line was used to isolate c-myc and associated protein

complexes. A c-myc null mESC line was used as a negative control to identify

non-specific interactions after immunoprecipitation (Figure 5.2A). Nuclear

extracts were used as a starting point for immunoprecipitations to reduce the contamination by some abundant cytoplasmic proteins. Proteins precipitated with the 9e10 (anti-myc tag) antibody were eluted from beads, subject to polyacrylamide gel electrophoresis, then analyzed by silver staining, immunoblot analysis and mass spectrometry.

Silver staining and immunoblot analysis indicated that myc was efficiently

enriched in these immunoprecipitates (Figure 5.2B, 5.3A).

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Trypsin digestion was carried out to obtain peptide fragments of myc-interacting

proteins, which were then subjected to mass spectrometry. Sequences of tryptic peptides obtained using mass spectrometry were screened against the non- redundant mouse database from the National Center for Biotechnology

Information using a SEQUEST search. These results were filtered with a standard of 99.9% confidence, 0.1% false discovery rate, and proteins identified

from negative control extracts manually excluded from the analysis.

This analysis identified a number of myc-interacting proteins with functions in

transcriptional regulation, cellular structure, DNA replication, nucleotide

processing or modification, metabolism or signaling, mRNA/protein transport,

protein degradation, protein synthesis, protein modification, and proteins with

miscellaneous function (Table 2).

There were several inherent controls that were present in this screen, in the form

of previously identified c-myc targets. Therefore, the identification of the c-myc

interacting protein, casein kinase II that phosphorylates myc 155 as well as the

obligate binding partner, Max, provided increased confidence that true binding

partners of c-myc were immunoprecipitated and identified by mass spectrometry.

Several other proteins of particular interest were identified by the mass spectrometry screen. These include Hmgb2 and Set, which co-exist in repressor complexes, and function to modulate chromatin accessibility 156, 157. Another

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protein, Cbx3, participates in transcriptional repression by binding to trimethylated lysine 9 of histone H3 158. Additionally, Smarca4/Brg1 is a Swi/Snf

family member that functions in chromatin remodeling and has been implicated in

regulation of self-renewal and pluripotency. The Smarca4 null mutation is lethal

at an early embryonic stage 159. Furthermore, Kidder and colleagues recently

implicated Smarca4 in the ESC core regulatory circuitry, as it occupies the

promoters of ESC regulators Oct4, Nanog, Sall4 and polycomb genes, as well as

some of their downstream target genes. Smarca4 is also a functional regulator

of the ESC state as knockdown of Smarca4 in ESCs resulted in downregulation

of ESC marker genes and subsequent differentiation 160. The interaction of c-

myc with these proteins could therefore regulate the transcriptional status of

target genes.

One possible mechanism by which c-myc may repress target genes is by

interaction with selected proteins of the co-REST corepressor complex including

Aof2/LSD1, a lysine specific histone demethylase, and known c-myc interactors

HDAC1 and HDAC2 110. LSD1 was identified as an interacting protein in this

screen, and therefore the interaction of c-myc with LSD1 and other proteins

(HDAC1, HDAC2) that are present in the co-REST complex was examined.

These interactions were successfully validated in co-

immunoprecipitation/western blot experiments (Figure 5.3B). This suggests that

c-myc possibly recruits the co-REST corepressor complex to some target genes.

Implications of these interactions will be discussed further in Chapter 6.

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Figure 5.1 Experimental setup for the identification of c-myc interacting proteins by co-immunoprecipitation/mass spectrometry analysis.

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Figure 5.2 Epitope tagged c-myc interacts with multiple proteins in ESCs. A. Expression of c-myc was analyzed by immunoblot in the c-mycmyc cell line and the c-myc-/- control cell line prior to immunoprecipitation. B. Silver stain of interacting proteins, also showing background protein present in eluate in the c- myc-/- cell line.

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Figure 5.3 c-myc interacts with components of the co-REST co-repressor complex in ESCs. A. Efficient immunoprecipitation of c-myc was detected by immunoprecipitation-immunoblot analysis. B. Validation of select proteins identified in mass spectrometry analysis by co-immunoprecipitation and western blot.

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Table 2. c-myc interacting proteins identified by mass spectrometry of ESC extracts. Gene Symbol Name DNA replication Hmgb2 high mobility group box 2 Mcm7 minichromosome maintenance deficient 7 Sumo-2 suppressor of mif two 3, homolog 1 DNA/ RNA processing/modification Acin1 apoptotic chromatin condensation inducer 1 Dhx9 deah (asp-glu-ala-his) box polypeptide 9 Raver1 ribonucleoprotein PTB-binding 1 Ruvbl1 ruvb-like protein 1 Sfrs11 splicing factor, arginine/serine-rich 11 Sfrs5 splicing factor, arginine/serine-rich 5 (srp40, hrs) Skiv2L2 superkiller viralicidic activity 2-like 2 (s. cerevisiae) Smarca4 swi/snf related, regulator of chromatin, subfamily a, member 4 Terf1 telomeric repeat binding factor 1 Transcription/chromatin Aof2 amine oxidase (flavin containing) domain 2 Cbx3 chromobox homolog 3 (drosophila hp1 gamma) Max max protein Phf5A phd finger protein 5a mRNA/protein transport Nup160 nucleoporin 160 Set set translocation (myeloid leukemia-associated) Protein Synthesis Igf2bp1 coding region determinant binding protein Eef2 eukaryotic translation elongation factor 2 Eif4a2 eukaryotic translation initiation factor 4a2 Protein transport/modification Cct3 chaperonin subunit 3 (gamma) Cct8 chaperonin subunit 8 (theta) Npm3 nucleoplasmin 3 Ppp1r10 protein phosphatase 1, regulatory subunit 10 Protein degradation Psme3 proteasome activator complex subunit 3 Metabolism/Signaling Aldoart1 aldolase 1, a isoform Eno1 alpha-enolase Csnk2B casein kinase ii, beta subunit Pgk1 phosphoglycerate kinase 1 Rangap1 ran GTPase-activating protein 1 Suclg2 succinate-coenzyme a ligase, gdp-forming, beta subunit Tkt transketolase Usp48 ubiquitin specific peptidase 48 Cellular structure Dynll2 dynein light chain lc8-type 2 Myo1C myosin ic Myl6 myosin, light polypeptide 6, alkali, smooth muscle and non-muscle Miscellaneous/Unknown Cdv3 carnitine deficiency-associated protein 3 Sod1 superoxide dismutase 1, soluble Ss18 synovial sarcoma-associated Ss18-alpha

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Discussion

A major objective of my work was to define the protein complexes into which myc

is assembled in ESCs. This objective was taken because identification of myc-

interacting proteins would further resolve the function of myc in ESCs by giving

insight into additional mechanisms of myc action.

ESC nuclear extracts were used in co-immunoprecipitation/mass spectrometry

experiments to identify myc binding partners. After subtractive analyses to

remove proteins identified in the c-myc null ESC line, 40 interacting proteins were

identified. Interacting partners were involved in transcription and chromatin accessibility and included histone modifying enzymes. Myc levels are strictly regulated at multiple levels including protein stability 18, and other proteins

identified were involved in regulation of myc levels particularly at the level of

translation. Also, as expected since myc has well-documented roles in

promoting progress through the cell cycle, some additional interacting proteins

are involved in include DNA binding proteins HMGB2 and MCM7.

One potentially important finding was that c-myc interacts with the Swi/Snf family

member Smarca4/Brg1. Smarca4 is the catalytic ATPase of a multi-component

chromatin remodeling complex that serve to disrupt histone-DNA interactions,

thus facilitating entry of transcription factors to promote transcription 161.

Smarca4 is essential for maintenance of ESCs 160, and is a requirement for early

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embryonic development 159. Inactivation of Smarca4 results in spontaneous

differentiation concomitant with upregulation of lineage markers such as Gata6.

Interestingly, Smarca4 binds to promoters of active and inactive genes in ESCs.

One such interesting target of Smarca4 is Sall4 160, another member of the ESC

core regulatory circuitry 137. Sall4 is also a target of c-myc that was identified in my studies (Appendix A). It is thus plausible that c-myc could recruit Smarca4 to targets such as Sall4 to facilitate its expression. Thus, myc may interact with epigenetic regulators such as Smarca4 at specific target genes to activate gene expression directly. Another possibility however, is that myc may act as a competency factor which acts to facilitate gene expression upon stimulation by other gene-specific transcription factors.

The identification of LSD1/Aof2 presents another interesting c-myc interacting protein. LSD1 can demethylate lysine 4 of histone H3, a histone modification that is normally associated with active gene transcription 162. Under such

circumstances, LSD1 is associated with gene silencing, and consistent with this

is the association of LSD1 with corepressor complexes such as co-REST 162, 163.

One role of complexes involving REST/co-REST is to suppress transcriptional activity of neural specification factors 164. One such neural gene identified in the

genome-wide location analysis of myc targets is Otx2. Transcript levels of Otx2

decrease when c-myc is activated. This suggests that c-myc represses Otx2.

The identification of LSD1 as a myc-interacting protein suggests that

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LSD1/REST/co-REST complexes could be mediating the repression of Otx2 by

c-myc.

It has been recently demonstrated that Nanog and Oct4 interact with similar

epigenetic modifiers including Smarca4 and LSD1, forming repressor complexes

154. Boyer and colleagues demonstrated that Nanog and Oct4 occupy the

promoters of repressed genes in ESCs 29, 30, and recruitment of repressor complexes such as those involving LSD1 is one mechanism by which they enact gene repression 154. Additionally however, as c-myc also interacts with these

epigenetic regulators, it is conceivable that c-myc could be present at the

promoters of some of the same target genes as Oct4 and Nanog. This could

have been missed in ChIP studies because of the large and complex nature of

these complexes, particularly if c-myc is bound at regions distant from the DNA.

On the contrary, c-myc could also be present in repressive complexes at different

promoters to suppress transcription of a different subset of target genes than

Oct4 and Nanog, thus acting in concert to maintain the ESC state.

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CHAPTER 6

FINAL DISCUSSION AND CONCLUSIONS

Although myc has been studied for almost 30 years, it remains somewhat of an

enigma even today. Its role as a regulator of transcription and its involvement in transformation and oncogenesis has been extensively studied 77, 96.

Nonetheless, views on its regulation of cellular processes are constantly

changing. For instance, until recently, myc was thought to be a gene-specific

transcription factor. However, substantial evidence has since been provided for

myc’s role as a more global regulator of transcription. Current work suggests c-

myc may influence expression of 10-15% of all cellular genes 97, 144, and recent studies indicate that, in addition to binding and regulating individual genes, myc may also regulate large domains of chromatin 70. Moreover, evidence of myc’s

ability to recruit a number of epigenetic modifiers 69, 108, suggests that myc

certainly has the potential for global regulation of the genome.

Studies carried out in our laboratory initially demonstrated the requirement for c-

myc in mESC maintenance by showing that expression of dominant negative c-

myc promotes differentiation 18. As both c- and N-myc bind to E-box target sites,

it is likely that overexpression of the dominant negative construct used by

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Cartwright and colleagues, displaced binding of both c- and N-myc from target

genes.

Spontaneous differentiation of dKO cells to endoderm unequivocally

demonstrates that myc is a requirement for self-renewal. This is a similar finding

to that observed for other central members of the ESC core transcriptional

regulatory circuitry including Oct4, Sox2, Sall4 and Nanog 29, 30, 44, 136, 137. Target

genes have been identified for these key ESC transcription factors, and for the

most part, these target genes segregate from myc targets 44. However, myc may participate in the regulation of their target genes through protein-protein interactions, particularly because Oct4 and Nanog were recently found to interact with Smarca4 and LSD1 in repressive complexes ESCs 154, two proteins

identified as binding partners in my proteomics analysis. Further investigations into whether myc, Oct4 and Nanog exist in same protein complexes are warranted. This would provide greater insight into mechanisms of repression of differentiation genes in pluripotent cells, and further the understanding of how myc is involved in target gene regulation in instances in which it exists in large macromolecular complexes.

The implications of myc-mediated endoderm repression may extend to the role of

myc in reprogramming of somatic cells. Sridharan and colleagues found that

significant gene expression changes occur upon introduction of exogenous c-

myc into fibroblasts 66. Hence, myc may be involved in the stepwise inhibition of

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expression of somatic genes along the dedifferentiation pathway. Gata6 may be

one such example where myc restricts the expression of lineage specific genes to allow reprogramming.

Another level of myc control of ESC self-renewal is that of the cell cycle.

Through regulation of the miR17-92 cluster, myc regulates the stability of

Rb2/p130 to impede establishment of the restriction checkpoint, thus facilitating

continuous re-entry into the cell cycle. A similar role for stimulating proliferation

has been observed in some cancer types where myc regulates expression of this

cluster to modulate activity of cyclin D1, p21, Rb2/p130 and E2F transcription

factors 101, 102, 105, 106, 145. This relationship highlights another similarity in the

relationship between pluripotent stem cells and tumor cells. Indeed, both cell types typically display high myc activity 18, 77, 79, 80, which facilitates infinite

proliferation potential and imposes differentiation blockades.

miRNAs have also been implicated in the regulatory circuitry that impedes

differentiation of pluripotent cells. Marson and colleagues determined that Oct4,

Sox, Nanog and Tcf3 occupy the promoters of the miR17-92 and miR290-295

cluster 49. Therefore, c-myc and other members of the transcriptional network

may cooperate to promote expression of the miR17-92 cluster.

In ESCs, the miR290-295 cluster also promotes rapid proliferation by inhibiting

the cyclinE/Cdk2 inhibitor, p21 27. More recently, this cluster has also been

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implicated as a substitute for c-myc in the reprogramming process. In this study,

Judson and colleagues found that expression of the miR290-295 cluster improves the efficiency of somatic cell reprogramming similar to myc through an unclear mechanism 50. This finding provides support for the deduction that one mechanism by which myc improves dedifferentiation is by increasing the capacity for cellular proliferation. Further investigations into whether the miR17-92 cluster would facilitate reprogramming in a similar manner in the absence of myc should provide insight into the effect of downregulation of other cell cycle controllers.

The identification of myc target genes provides insight into additional mechanisms through which myc may govern maintenance of the ESC state.

This is complemented by the identification of myc interacting proteins, which may influence the potential of myc to act as an activator or as an inhibitor of specific genes in pluripotent stem cells. That myc may have dual functions as an activator and an inhibitor is not a novel finding 77, 96, 151, 165, 166, however identification of mediators of this activity further defines how myc may act to regulate gene transcription in pluripotent stem cells.

Loss of myc likely has significant impact of the expression of target genes.

Downstream targets that require myc for direct stimulation would be silenced.

Targets for which myc acts as a licensing factor to recruit epigenetic modulators to modulate gene activity upon subsequent stimulation by gene-specific transcription factors would also be affected.

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In addition to being a requirement for pluripotent stem cells to self-renew, myc is

required for proper differentiation into germ layer derivatives. The precise means

by which myc facilitates generation of mesoderm, ectoderm and possibly

definitive endoderm is not well understood. One possibility is that c- or N-myc is required for cell cycle entry in epiblast derivatives. PrE could be the default cell type that remains because it is not highly proliferative, and cells therefore continue to replicate in the presence of L-myc only. Alternatively, c- and N-myc could be required for survival of differentiating cells. Slight increases were observed in apoptosis upon inactivation of myc in iPSCs, and myc may be required for survival of cellular derivatives of the embryonic germ layers.

My data therefore suggest that myc is an absolute requirement at earlier

developmental stages than the individual c- and N-myc null phenotypes suggest.

Furthermore, the data support the inference that c-myc and N-myc null embryos

survive until embryonic d10-12 because of compensation by the other family

member. It is expected that the phenotype for double knockout mouse would be

lethal at earlier developmental stages than previously observed 114, 116, and may

occur as early as implantation.

Prior to these studies, despite several reports identifying myc target genes in

pluripotent cells, significant gaps in the knowledge of myc’s role in self-renewal

and pluripotency still remained. This report identified myc target genes and novel

myc interacting proteins, and also defined key mechanisms of how myc regulates

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the ESC state (Figure 6.1). These included acting as a transcriptional repressor of the PrE specification factor, Gata6, as well as sustaining self-renewal through maintenance of the cell cycle structure that is characteristic of a pluripotent cell.

My studies thus implicate myc as a master regulator of the key characteristics of self-renewal and pluripotency in ESCs and iPSCs on multiple levels, including the regulation of target genes including transcription factors, signaling proteins and miRNAs (Figure 6.1).

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Self-renewal Myc

Transcription factors

Signaling proteins

miRNA

Gata6 Pluripotent Stem cells PrE

Pluripotency Myc

Ectoderm Mesoderm Endoderm

Figure 6.1.Model illustrating the central role of myc in maintaining self- renewal and pluripotency in ESCs and iPSCs. Myc sustains self-renewal by regulating the metabolic status and cell cycle of pluripotent stem cells. Through protein-protein interactions, and direct regulation of target genes such as Sall4, myc likely influences the transcriptional status of the core ESC regulatory circuitry. Myc is also important for proper differentiation into mesodermal and endodermal cell types. Although myc possibly enables differentiation into definitive endoderm, this was not addressed in these studies due to the overlapping nature of PrE and definitive endoderm marker genes.

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APPENDIX A. Myc target genes identified in ChIP-Chip

Targets identified in c-mycmyc and c-mycHA cell lines.

Gene c-mycmyc c-mycHA Total Gene c-mycmyc c-mycHA Total Abcc10 2 1 3 Atp5g2 3 2 5 Abce1 1 1 2 Atp5l 3 1 4 Adam19 1 1 2 Atp6v0d1 3 2 5 Adh5 1 2 3 Atp6v1a-Nat13 2 2 4 Adss 2 1 3 Atp6v1d-Eif2s1 2 1 3 Agpat5 3 3 6 Atpbd4 2 1 3 Ahctf1 2 1 3 Atpif1 1 1 2 Ahi1 1 1 2 Atrx 1 1 2 Ahsa1 1 1 2 Atxn3 1 1 2 AI316807 2 2 4 Aurkb 1 1 2 AI450540 3 3 6 Aven 1 1 2 AI847670 2 1 3 Avpi1 2 1 3 AK122525 2 2 4 B230219D22Rik 2 1 3 Akap1 3 2 5 B3gnt2 3 1 4 Alad 3 1 4 B3gntl1 1 1 2 Aldh9a1 2 1 3 Bat1a 3 1 4 Aldoa 3 1 4 BC024814 2 1 3 Anapc11 2 1 3 BC031781 1 1 2 Angptl4 2 1 3 BC031853 1 1 2 Ankrd28 2 1 3 BC038925 3 1 4 Ankrd49-Mre11a 2 1 3 BC048355 2 1 3 Apbb3 3 1 4 BC050210 2 2 4 Apex1 1 1 2 BC065397 3 3 6 Arhgdia 2 1 3 BC068281 1 1 2 Arid1a 3 3 6 Bcdin3d 2 1 3 Arid5b 2 1 3 Bckdha 2 1 3 Ars2 3 1 4 Bcl7b 2 2 4 Asah3-Clpp 2 1 3 Bet1l-Ric8 2 1 3 Ash2l 2 1 3 Birc2 2 2 4 Asxl2 3 3 6 Blm 2 1 3 Atad3a 2 1 3 Brca2 3 3 6 Atf2 2 2 4 Brd8-Kif20a 1 1 2 Atf7 2 1 3 Bre 1 1 2 Atf7ip 1 1 2 Btbd9 2 1 3 Atic 2 1 3 Btf3l4 2 1 3 Atox1-G3bp1 2 2 4 Bxdc1 1 1 2 Atp5b 2 1 3 Bxdc2 3 1 4 Atp5e 2 1 3 C130032J12Rik 2 3 5 Atp5g1 1 1 2 C330023M02Rik 2 1 3

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Targets identified in c-mycmyc and c-mycHA cell lines continued. c- c- c- c- Gene mycmyc mycHA Total Gene mycmyc mycHA Total Cacnb1-Rpl19 3 2 5 chr14:113924953-113925007 2 3 5 Cacybp 3 3 6 chr14:113925071-113925130 2 2 4 Canx 2 2 4 chr14:113925188-113925243 1 1 2 Caprin1 3 3 6 chr16:090033448-090033495 1 1 2 Casp2 2 1 3 chr16:090033607-090033656 1 1 2 Cbx1 1 1 2 chr17:032902608-032902652 1 1 2 Cbx3 2 1 3 chr2:128658145-128658204 1 1 2 Cbx5-Hnrpa1 2 3 5 chr2:128658315-128658373 2 1 3 Ccdc128 1 1 2 chr2:128659977-128660036 3 3 6 Ccdc38 1 1 2 chr2:128660133-128660189 1 2 3 Ccdc47-Ddx42 2 1 3 chr7:044115121-044115165 1 1 2 Ccdc95-Hirip3 1 1 2 chr7:044115238-044115282 1 1 2 Ccnd1 3 2 5 chr9:088318992-088319051 1 2 3 Ccnl1 2 1 3 chr9:088319884-088319943 1 2 3 Ccnt2 1 1 2 chr9:088320023-088320082 3 2 5 Cct4 2 1 3 chr9:088320128-088320184 2 1 3 Cct5 1 1 2 chr9:088320576-088320624 3 3 6 Cct6a 3 1 4 chr9:119170043-119170102 2 1 3 Cct7 3 2 5 chr9:119170218-119170276 1 1 2 Cdc20 3 1 4 chr9:119172093-119172141 1 1 2 Cdc26 2 1 3 chrX:048989873-048989932 1 1 2 Cdca2-Kctd9 3 3 6 chrX:048990245-048990293 1 1 2 Cdca7 3 1 4 Cidec-Jagn1 1 1 2 Cdca8 2 1 3 Ckb-6720458F09Rik 3 2 5 Cdk2ap1 2 1 3 Cks2 3 1 4 Cdk4 2 1 3 Clpp 2 1 3 Cdk5rap2 1 1 2 Clptm1 2 1 3 Cdkal1 1 1 2 Clta 2 1 3 Cdv3 2 2 4 Cnbp 3 3 6 Cenpp-Nol8 3 3 6 Cnot4 3 3 6 Chchd1 1 1 2 Cnot6l 1 1 2 Chchd2-2410018M08Rik 2 1 3 Commd5 1 1 2 Chchd4 2 2 4 Cops7a 2 1 3 chr10:122387965-122388024 1 1 2 Cox5a 1 1 2 chr14:077583871-077583917 1 1 2 Cox7b 1 1 2 chr14:113924274-113924318 3 3 6 Cox7c 1 2 3 chr14:113924469-113924523 3 3 6 Cpsf2 1 1 2 chr14:113924649-113924703 3 3 6 Crlz1 1 1 2 chr14:113924833-113924892 3 3 6 Csda 2 1 3

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Targets identified in c-mycmyc and c-mycHA cell lines continued. Gene c-mycmyc c-mycHA Total Gene c-mycmyc c-mycHA Total Cstf3 3 1 4 Eif3s1 1 1 2 Ctcf 2 1 3 Eif3s6 2 1 3 D19Bwg1357e 3 1 4 Eif3s7 2 1 3 D1Pas1 3 1 4 Eif4ebp1 3 1 4 D6Wsu163e 3 3 6 Eif5a 3 3 6 D8Ertd457e 3 1 4 Emp3-Ccdc114 3 1 4 Dak 1 1 2 Eno1 3 3 6 Dars2 2 1 3 Epb4.1l4a 2 1 3 Dars2-Cenpl 1 1 2 Epc1 1 1 2 Dbt 1 1 2 Ergic2 2 1 3 Dctd 2 1 3 Etf1 1 1 2 Ddah1 3 1 4 Etv5 2 1 3 Ddb1 2 1 3 Exosc1 1 1 2 Ddx21 3 3 6 Exosc2 1 1 2 Ddx3x 1 1 2 Exosc5 2 1 3 Dhodh 1 1 2 Exosc7 1 1 2 Dhps 3 3 6 Ezh2 1 1 2 Dkc1 2 3 5 Fancb 1 1 2 Dlg1 2 1 3 Fau 1 1 2 Dnajb4 2 2 4 Fbxl19 1 1 2 Dph1 2 1 3 Fbxl3 2 1 3 Dph5 2 1 3 Fbxo15 3 3 6 Dtl 2 1 3 Fbxw2 2 3 5 Dtymk 1 1 2 Fdps 1 2 3 Dus4l 2 1 3 Fem1a 1 1 2 E130012A19Rik 3 1 4 Fen1 1 1 2 E2f3 1 1 2 Fignl1 1 2 3 E2f8 1 1 2 Fiz1 3 2 5 Ebna1bp2 2 1 3 Fkbp11 1 1 2 Eed 3 2 5 Fnbp4 1 1 2 Eef1b2 2 1 3 Foxn2 3 1 4 Eef1d 1 1 2 Foxred2 1 1 2 Eef1e1 1 1 2 Fpgs 2 1 3 Eef2 3 1 4 Frat1 2 1 3 Eftud2 2 1 3 Ftsj3 2 1 3 EG623661 1 1 2 Fusip1 2 1 3 Ehmt2 3 1 4 Gars 1 1 2 Eif1a 3 3 6 Gart 3 3 6 Eif2s1 2 1 3 Gemin4 2 2 4

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Targets identified in c-mycmyc and c-mycHA cell lines continued. Gene c-mycmyc c-mycHA Total Gene c-mycmyc c-mycHA Total Gfer 1 1 2 Isg20l1 3 1 4 Gja1 2 1 3 Itpk1 2 1 3 Gjb3 1 1 2 Jagn1 1 1 2 Gnas 3 3 6 Jarid1b 3 1 4 Gnl3l 3 3 6 Jmjd5 3 1 4 Got1 2 1 3 Jph4 1 1 2 Gpatch4 2 1 3 Jtv1 1 1 2 Gpbp1 2 1 3 Jtv1-Pms2 1 1 2 Gpr19 1 1 2 Kars 1 1 2 Gpsn2 1 1 2 Kcmf1 3 3 6 Gtf2e1 2 1 3 Kif20a 2 1 3 Gtf3a 1 1 2 Kifc3 1 1 2 Gtrgeo22 1 1 2 Klf16 3 1 4 H2afy2 2 2 4 Klf7 2 1 3 Heatr1 3 1 4 Klf9 1 2 3 Hells 3 3 6 Kpna2 3 1 4 Hk2 3 1 4 Kpnb1 2 3 5 Hn1l 2 1 3 l7Rn6 2 1 3 Hnrpa1 3 3 6 Lactb2-Xkr9 1 1 2 Hnrph2 1 2 3 Laptm4a 1 1 2 Hnrpr 1 1 2 Lck 2 1 3 Hnrpu 2 1 3 Lig3 2 1 3 Hsp90aa1 2 1 3 Lin28 1 1 2 Hspa9 2 1 3 Lin28b 2 3 5 Hspbap1 3 2 5 Lmnb1 2 1 3 Hspd1 3 3 6 Lrmp 2 1 3 Hyou1 3 3 6 Lsm12-G6pc3 2 2 4 Ifrd2 2 1 3 Lsm3 3 1 4 Igf2bp3 1 2 3 Lsm4 2 1 3 Igfbp2 1 2 3 Luc7l 1 1 2 Ikbkap 1 1 2 Lyrm4 2 1 3 Il23a-Usp52 1 1 2 Magi1 1 1 2 Incenp 2 1 3 Manba 1 1 2 Inoc1 3 1 4 Map2k3 1 1 2 Ints6 2 1 3 Mars 3 1 4 Ipo13 3 3 6 Mat2a 3 3 6 Iqce-AA881470 3 1 4 Matr3 1 1 2 Iqcg-Rpl35a 3 2 5 Mbd3 2 2 4 Iqck 1 1 2 Mbtps2 3 3 6

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Targets identified in c-mycmyc and c-mycHA cell lines continued. Gene c-mycmyc c-mycHA Total Gene c-mycmyc c-mycHA Total Mcm3 1 1 2 Nfib 2 1 3 Mcm3ap 3 1 4 Nfx1 2 1 3 Mcm7 1 1 2 Nhp2l1 1 1 2 Mcm9 1 1 2 Nme1 2 1 3 Mdc1 2 2 4 Nme4 1 1 2 Med25-Fuz 1 1 2 Nme6 1 1 2 Mett10d 3 3 6 Noc3l 2 1 3 Mga 2 1 3 Nol1 3 1 4 Mid1ip1 1 1 2 Nol14 3 1 4 Minpp1 1 1 2 Nol5 3 3 6 Mki67ip 3 2 5 Nol6 1 1 2 Mlh1 1 1 2 Nol8 2 3 5 Mllt10 3 3 6 Nola2 3 1 4 Mmaa 2 1 3 Nola3 3 3 6 Mon2 3 3 6 Nolc1 2 1 3 Mrpl15 3 1 4 Nphp1 2 1 3 Mrpl17 1 1 2 Nrf1 1 1 2 Mrpl18 1 1 2 Nrp 2 3 5 Mrpl20 2 1 3 Nsun2 2 1 3 Mrpl50 3 3 6 Nudcd3 2 1 3 Mrps18b 3 3 6 Nudt9 1 2 3 Mrto4 2 2 4 Nup153 3 1 4 Msi2 2 1 3 Nup155 1 2 3 Msra 2 1 3 Nup50 1 2 3 Mt2 2 1 3 Nup85 3 2 5 Mtdh 1 1 2 Nupl1 3 2 5 Mtg1 1 1 2 Obfc2a 1 2 3 Mthfd1 2 1 3 Odc1 3 1 4 Mus81-Cfl1 2 1 3 Orc2l 1 1 2 Mybbp1a 2 2 4 Otx2 1 1 2 Mybl2 2 1 3 Pa2g4 2 1 3 Myef2 3 2 5 Pabpn1 1 1 2 Myo5c 2 1 3 Paics 2 2 4 Naca 3 1 4 Parl 1 1 2 Nasp 2 1 3 Parp1 2 1 3 Ncl 3 3 6 Parp11 2 1 3 Ndufa7 1 1 2 Parp2 3 3 6 Nedd4 2 1 3 Pbk 2 1 3 Nfe2l1 1 1 2 Pcgf2-Psmb3 1 1 2

145

Targets identified in c-mycmyc and c-mycHA cell lines continued. Gene c-mycmyc c-mycHA Total Gene c-mycmyc c-mycHA Total Pcmtd1 1 1 2 Ptrh2 1 1 2 Pdcd4 1 1 2 Pum2 1 1 2 Pdia4 2 2 4 Pus3 2 2 4 Pdk1 2 1 3 Pwp2 3 1 4 Pes1 3 1 4 Qtrtd1 2 2 4 Pex12 1 1 2 Rab30 1 2 3 Pfn1-Eno3 1 1 2 Rad1 1 1 2 Phf5a 2 1 3 Rad23b 2 1 3 Phf5a-Aco2 1 1 2 Rad50 2 3 5 Pif1 1 1 2 Rad51ap1-D6Wsu163e 3 3 6 Pim1 1 1 2 Rad51c 1 1 2 Pipox 1 2 3 Rad9 2 1 3 Pkm2 3 3 6 Rad9b 2 2 4 Pla2g6 2 1 3 Ran 3 3 6 Plagl2 1 1 2 Ranbp1 2 2 4 Plekhj1 1 1 2 Rangap1 1 1 2 Plekhq1-Pif1 1 1 2 Rbm13 3 1 4 Pno1-AI553587 3 1 4 Rbm26 1 1 2 Pola1 1 1 2 Rbm35b 1 1 2 Pold2 2 1 3 Rbm39 2 1 3 Ppid 1 1 2 Rbmx 1 1 2 Ppil3-Nif3l1 1 1 2 Rcc1 3 3 6 Ppil4 1 1 2 Rcc2 3 2 5 Ppm1a 1 1 2 Rcl1 2 1 3 Ppm1g 2 1 3 Rdh11 1 1 2 Ppp2r3c 2 1 3 Renbp 2 1 3 Prmt1 1 1 2 Rexo2 1 1 2 Prmt7 2 1 3 Rfc1 2 1 3 Prpf38b 2 1 3 Rfx2 1 1 2 Prps1 3 2 5 Rfx3 1 1 2 Psen1 2 2 4 Rg9mtd2 3 3 6 Psmb10 3 1 4 Rheb 1 1 2 Psme3 2 2 4 Rhof 1 1 2 Psrc1 3 1 4 Rlbp1-Fanci 2 3 5 Ptbp1 3 3 6 Rnaseh1 1 1 2 Ptcd3 3 3 6 Rnaseh2b 2 1 3 Ptcra-2310039H08Rik 3 3 6 Rnf12 1 2 3 Ptges3 1 1 2 Rnf13 2 1 3 Ptprk 1 1 2 Rnf145 3 3 6

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Targets identified in c-mycmyc and c-mycHA cell lines continued. Gene c-mycmyc c-mycHA Total Gene c-mycmyc c-mycHA Total Rnf151-Rps2 2 1 3 Rps14 3 1 4 Rnf207-Rpl22 1 1 2 Rps15a 3 3 6 Rnf4 2 1 3 Rps16 3 1 4 Rnps1 2 1 3 Rps17 1 1 2 Rpl10a 1 1 2 Rps18 3 2 5 Rpl11 3 2 5 Rps19 3 1 4 Rpl14 2 1 3 Rps20 2 1 3 Rpl15 3 1 4 Rps24 3 2 5 Rpl18 3 1 4 Rps25 2 1 3 Rpl18a-Mtap1s 1 1 2 Rps27a 3 3 6 Rpl22 2 1 3 Rps27l 2 1 3 Rpl23 3 3 6 Rps28-Ndufa7 2 1 3 Rpl23a 3 3 6 Rps29 1 3 4 Rpl24 2 1 3 Rps3 2 2 4 Rpl26 3 2 5 Rps4x 3 3 6 Rpl27 3 1 4 Rps5 2 1 3 Rpl27a 3 3 6 Rps6 2 1 3 Rpl29 3 1 4 Rps9 3 3 6 Rpl30 3 3 6 Rpusd4 1 1 2 Rpl30-BC030476 2 2 4 Rrp1b 2 1 3 Rpl31 3 1 4 Rrp9 1 1 2 Rpl32 2 1 3 Rsrc2 2 1 3 Rpl34 3 2 5 Sae2 3 1 4 Rpl35 1 1 2 Sall4 1 3 4 Rpl35a 3 2 5 Sdad1 1 1 2 Rpl36a 2 1 3 Seh1l 2 1 3 Rpl37 3 2 5 Sephs1 2 1 3 Rpl38 3 1 4 Serbp1 3 3 6 Rpl39 3 3 6 Set 3 1 4 Rpl3-Syngr1 3 3 6 Sf3b3-Cog4 2 1 3 Rpl5 3 1 4 Sfrs10 3 1 4 Rpl7 3 3 6 Sfrs3 2 1 3 Rpl7-Rdh10 3 1 4 Sh3pxd2a 1 1 2 Rpl8 3 1 4 Shmt1 3 2 5 Rplp1 3 2 5 Shmt2 2 1 3 Rplp2 2 1 3 Six6os1 2 1 3 Rpp14 2 1 3 Skiv2l2 2 1 3 Rpp38 3 1 4 Skp2 1 1 2 Rps10 2 1 3 Slc16a1 3 1 4

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Targets identified in c-mycmyc and c-mycHA cell lines continued. Gene c-mycmyc c-mycHA Total Gene c-mycmyc c-mycHA Total Slc16a3 2 1 3 Sugt1 1 2 3 Slc19a1 2 1 3 Sumo2 3 1 4 Slc1a5 1 1 2 Tada2l 2 1 3 Slc20a1 1 1 2 Taf1c 2 1 3 Slc25a39 3 3 6 Tardbp 1 2 3 Slc25a40 2 1 3 Tars2 3 3 6 Slc29a1 1 1 2 Tbc1d7 1 1 2 Slc35f2 1 1 2 Tbl1x 1 2 3 Slc38a4 3 2 5 Tbl1xr1 1 1 2 Slc39a10 1 1 2 Tbrg4 2 1 3 Slc3a2 3 3 6 Tcfap4 3 1 4 Slc44a1 1 1 2 Tcof1 2 1 3 Slc7a7-Mrpl52 2 2 4 Tcp1 2 1 3 Slc9a8 1 1 2 Tdh 1 1 2 Slmo2 1 1 2 Tdp1 1 1 2 Smarcad1 2 1 3 Tfdp1 3 3 6 Smc2 2 1 3 Tfpt-Prpf31 1 1 2 Smndc1 3 3 6 Thrap3 2 1 3 Smpdl3b 2 2 4 Thumpd1 2 1 3 Smyd5 2 1 3 Tial1 3 3 6 Snd1 2 1 3 Timm10 1 1 2 Snrpa 2 1 3 Timm23 3 1 4 Snrpa1 1 1 2 Tipin 1 1 2 Snrpd3 3 2 5 Tle1 3 1 4 Snrpg 1 1 2 Tmem103 1 1 2 Sntb2 2 1 3 Tmem109 1 1 2 Snx11 1 1 2 Tmem11 1 1 2 Snx5 3 2 5 Tmem16f 1 1 2 Sp1 1 1 2 Tmem24-Dpagt1 2 1 3 Sphk2 1 1 2 Tmem41b 1 1 2 Srfbp1 3 1 4 Tmem69 3 3 6 Srpr 1 1 2 Tmem79-Smg5 3 2 5 Ssrp1 2 1 3 Tmem97 2 1 3 St6galnac2 3 1 4 Tnfsf5ip1 1 2 3 Stag3 2 1 3 Tnpo1 1 3 4 Stam2 2 1 3 Tnpo2 2 2 4 Stat3 2 1 3 Tomm20 2 1 3 Stt3b 2 1 3 Top2a 2 2 4 Suclg1 3 1 4 Topors 1 2 3

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Targets identified in c-mycmyc and c-mycHA cell lines continued. Gene c-mycmyc c-mycHA Total Gene c-mycmyc c-mycHA Total Trap1 1 1 2 Zcchc10 2 1 3 Trap1a 1 2 3 Zdhhc5 1 1 2 Trappc4-Rps25 1 1 2 Zfp106 2 1 3 Trib1 1 1 2 Zfp110 3 1 4 Trim28 3 1 4 Zfp146 2 1 3 Trpm7 1 1 2 Zfp207 3 2 5 Tsc22d1 1 1 2 Zfp27 2 2 4 Tsr1 1 1 2 Zfp286 2 1 3 Tubb5 2 1 3 Zfp451 1 1 2 Txnl4 3 2 5 Zfp566 2 1 3 U2af1 2 1 3 Zfp57 1 2 3 U2af2 2 1 3 Zfp706 1 2 3 Uba52 2 2 4 Zgpat 1 2 3 Ubap2 1 1 2 Znrf2 1 1 2 Ube2b-Cdkl3 3 3 6 Zwilch 1 1 2 Ube2c 1 1 2 0610009O03Rik 1 1 2 Ube3a 2 2 4 1110005A03Rik 2 1 3 Ubxd6 2 1 3 1110005A23Rik 3 2 5 Uhrf1 1 1 2 1190005P17Rik 1 1 2 Usp1 3 1 4 1700021C14Rik-Cct3 2 1 3 Usp10 2 1 3 1700022C21Rik 1 1 2 Usp14 2 2 4 1700029J07Rik 1 1 2 Usp28 1 1 2 1700065D16Rik 2 1 3 Usp53 1 1 2 1810055G02Rik 1 2 3 Utp14a 3 3 6 2010305C02Rik 3 1 4 Wdr12 3 3 6 2010315L10Rik 1 1 2 Wdr36 1 1 2 2210411K11Rik 1 1 2 Wdr4 2 1 3 2310003F16Rik 1 1 2 Wdr4-1500032D16Rik 1 1 2 2310005N03Rik 1 2 3 Wdr5 1 1 2 2310044G17Rik 1 1 2 Wdr74 2 1 3 2310073E15Rik 2 1 3 Wipf2 2 1 3 2410002O22Rik 1 1 2 Xbp1 2 1 3 2410015N17Rik 2 1 3 Xpnpep3 1 1 2 2610318N02Rik 2 1 3 Xpo4 3 3 6 2610528E23Rik 3 3 6 Xpo7-Dok2 1 1 2 2610529C04Rik- 1 2 3 Cox7b Xrcc5 3 2 5 2700094K13Rik 2 2 4 Ybx1 3 1 4 2810004N23Rik 2 1 3 Ywhaq 2 1 3 2810030E01Rik-Mllt10 1 1 2

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Targets identified in c-mycmyc and c-mycHA cell lines continued. Gene c-mycmyc c-mycHA Total 2810403A07Rik 1 1 2 2810410M20Rik 2 1 3 2810432D09Rik 2 1 3 3110009E18Rik 3 2 5 3110023B02Rik 1 1 2 5430437P03Rik 1 1 2 5730590G19Rik 1 1 2 5730596K20Rik 2 3 5 5830457O10Rik 1 2 3 5830457O10Rik-Cirh1a 2 1 3 5830483C08Rik 3 1 4 6030429G01Rik 2 1 3 6030458C11Rik-Rnasen 1 1 2 6330548G22Rik 2 1 3 6720458F09Rik 2 1 3 8030462N17Rik 2 3 5 8430406I07Rik 2 1 3 9430010O03Rik 1 1 2 A130010J15Rik 2 1 3 A730055C05Rik 1 1 2 A830007P12Rik 1 1 2 A830093I24Rik 2 1 3

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Targets identified in the c-mycmyc cell line. Gene c-mycmyc Gene c-mycmyc Gene c-mycmyc AA408296 1 Cyct 2 Lhx1 1 Acin1 1 Cyp1b1 1 Lyar 1 Actr3b 2 Dbi 1 Map4k4 1 Adam17 1 Dclre1c 1 Mapk11 1 Adam32 1 Ddx5 1 Marcks 2 Arhgap11a 2 Dmrt3 1 Mcm5 1 Asb1 1 Dnajc12 1 Mkrn3 1 Atf1 1 Dpysl2 1 Mkx 1 AU020772 2 Drg1 2 Moap1 2 AW456874 1 Dusp4 1 Myoz3 1 B4galnt2 1 Dut 1 Nf2 1 Barx1 1 Ebf2 1 Nkx2-5 1 Bat2 2 Ebf3 1 Npas3 1 BC049762-Clk4 1 Eda2r 1 Nr2f1 1 Bckdha-Exosc5 1 Eif2b4 1 Nsbp1 1 Bmf 2 Emx1 2 Ntrk3 1 Cad 2 Epha7 1 Nucks1 1 Cadm1 1 Fancl 1 Nutf2 1 Cart1 1 Fbxo42 1 Osr2 1 Ccdc90b 1 Fgf8 1 Otx1 1 Cct5-A930016P21Rik 1 Fkbp3 1 Pank2 2 Cd3eap-Ppp1r13l 1 Foxg1 1 Pcdh10 1 Cdca4 1 Foxl2 1 Pcdhga9 1 Cenpk 1 Fzd10 1 Pfkfb2-Yod1 1 Cenpl 1 Gata6 1 Plekhg3 2 chr10:122387854-122387913 1 Gdi2 1 Pml 1 chr11:102634140-102634199 1 Glrx5 2 Pnpla6 1 chr17:039452885-039452929 1 Grwd1 1 Pole 1 chr17:039453294-039453338 1 Gse1 3 Polr3g 1 chr2:129391647-129391706 1 Hoxb6 1 Ppp1r14b 1 chr3:041660511-041660570 1 Hsf4 1 Prdm4 1 chr7:044114733-044114777 1 Hspa2 1 Prox1 1 chr9:119169719-119169778 1 Insm2 1 Ptp4a1 1 chrX:048989601-048989658 1 Irf4 1 Pus1 1 chrX:048989745-048989800 1 Irx5 1 Rbm38 1 chrX:048990018-048990077 1 Isl2 2 Rnf32 1 Cops3-Nt5m 1 Junb 1 Rpl39l 1 Crebzf 1 Kbtbd3 1 Rtn4rl2 2 Cyb5d2 1 Kif2c 1 Sall3 1

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Targets identified in the c-mycmyc cell line continued. Gene c-mycmyc Gene c-mycmyc Scamp1 1 2410016O06Rik 1 Scye1 2 2610029G23Rik 1 Sema6a 1 2700049P18Rik 1 Sfi1 1 5630401D24Rik 1 Sim1 1 5730410I19Rik 1 Six2 1 6030446N20Rik-Riok3 1 Slc16a6 1 8030451K01Rik 1 Slc2a1 1 A930008G19Rik 1 Smad4 1 Smap1 1 Smtnl2 1 Ssbp4-Lrrc25 1 St13-Xpnpep3 1 Sycp3 1 Tceal8 1 Tdrkh 1 Thrap2 1 Thtpa 1 Tm7sf3 1 Tmem166 1 Tmem39a 1 Tmem4 1 Tmpo 1 Traf4 1 Trappc5 2 Trp73-Wdr8 1 Uchl1 2 Wbp11-BC049715 1 Wdr23 3 Xab2 1 Xpo5 1 Yap1 1 Yrdc 1 Zfhx2 1 Zic1 1 Zmiz1 1 1110054O05Rik 1 1700012A16Rik 1

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Targets identified in the c-mycHA cell line. Gene c-mycHA Gene c-mycHA Aasdhppt 1 Atp6v0a1 1 Abhd2 1 Atpbd3 1 Acp1 1 Aurka 1 Acp6 3 Aurka-Cstf1 1 Actn4 1 AW049765-Mpnd 1 Adipor2 1 AW549877 1 Adprh 1 B3gnt5 1 Adprh-Cd80 1 B4galt3 1 Afmid 1 Bag2 1 Agbl5 1 Bax 1 Agrp 1 Baz1b 1 AK129128 1 BC022593 1 Akap12 3 BC024479 1 Akap8 1 BC027231 1 Akp2 1 BC050210-Cox5b 1 Akt1s1 1 BC055111 1 Alg5 1 BC057627 1 Alg8 1 Bcar1 1 Ankrd10 1 Bcat2 1 Ankrd13b-Git1 1 Bcl7a 1 Ankrd37 1 Bdp1 1 Ankrd37-Lrp2bp 1 Blcap 1 Anln 1 Bnip1 1 Ap1g2 1 Bop1 1 Aph1c 1 Brd2 1 Apoc1 1 Brd8 1 Aqp3 1 Btf3l4-Txndc12 1 Arf6 1 Bysl 1 Arhgef12 2 C230021P08Rik 1 Arih2 1 C230052I12Rik 1 Arl10 1 C230081A13Rik-Hmg20a 1 Armetl1 1 C530044N13Rik 1 Arpm1-Mynn 1 C78339 1 Arv1 1 Calcoco1 1 Asb15 1 Calm2 1 Ash2l-Kcnu1 1 Calr3 1 Atad2-2410187C16Rik 1 Calr3-1700030K09Rik 1 Atf4 1 Cbll1 1 Atp5a1 2 Ccdc111 1

153

Targets identified in the c-mycHA cell line continued. Gene c-mycHA Gene c-mycHA Ccdc117 3 Cox6b1 1 Ccdc124 1 Cpsf3 1 Ccdc5-Atp5a1 1 Cradd 1 Ccdc95 1 Crtap-Glb1 1 Ccne1 1 Crtc1 1 Cct3 1 Csnk1a1 1 Cct6b 1 Cspp1 1 Cd37 1 Csrp2bp 1 Cd97 1 Cstf1 1 Cenpc1-AI586015 1 Cwf19l2 1 Cenpq 1 Cyb5r3 1 Cep63 1 D10Wsu102e 1 Chchd5 1 D13Wsu177e 1 Chek1 1 D14Ertd500e 1 Cherp 1 D3Ertd300e 1 Chmp4b 1 D4Ertd22e 1 chr11:095520141-095520194 1 D4Ertd22e-Fbxo42 1 chr11:116306802-116306847 1 D730040F13Rik 1 chr14:077583982-077584041 1 Dag1 1 chr14:077584093-077584152 1 Dars 1 chr16:090033320-090033375 1 Daxx 1 chr18:061774479-061774527 1 Dcps 1 chr18:061774748-061774807 1 Dcun1d1 1 chr2:128658509-128658554 1 Ddx11 1 chr2:128659855-128659914 2 Ddx25-Pus3 1 chr2:128660298-128660357 1 Ddx31 1 chr3:041660765-041660812 1 Ddx39 1 chr5:125682731-125682788 1 Ddx56 1 chr7:044114868-044114912 1 Dhcr24 1 chr9:119171949-119172008 1 Dhx15 1 chr9:119172447-119172502 1 Dhx33 1 chrX:048990348-048990395 1 Dirc2-Hspbap1 1 chrX:048990458-048990517 1 Dnajc1 1 Cks1b 1 Dnmt3b 1 Clcn3-Nek1 1 Dpagt1 1 Cln3-Apob48r 1 Dph1-Rtn4rl1 1 Cmas 1 Dpm2 1 Cnot6 1 Dpp4 1 Cnot8 1 Dppa5 3 Coq2 1 Dr1 1

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Targets identified in the c-mycHA cell line continued. Gene c-mycHA Gene c-mycHA Dscr3 1 Foxh1 1 Dtymk-Ing5 1 Foxh1-Ppp1r16a 1 Dus2l 1 Foxo3a 1 Dus3l 1 Ftsj3-Psmc5 1 Dusp16 1 G3bp2 1 Dusp6 1 Gcnt2 1 Dyrk3 1 Gemin6 1 Ece2 1 Git2-Ankrd13a 1 Ect2 1 Glb1 1 Edg5 1 Gls2 1 Eif2a 1 Gm1019 1 Eif3s10 1 Gm603 1 Eif3s9 1 Gmfb 1 Eif4a1 1 Gmnn 1 Elp2 1 Gnl3 1 Enoph1 1 Gnpat 1 Epb4.9 1 Got2 1 Ercc5 1 Gp5 1 Erp29 1 Gpa33 1 Esco2-Ccdc25 2 Gpd1l 1 Etv1 1 Gpx1 1 Etv2 1 Grpel1 1 Etv4 1 Grsf1 1 Exosc10 1 Gsg1 1 F3 1 Gsk3b 1 Fancc 1 Gsr 1 Fanci 1 Gsta4 1 Farsa 1 Gstp2 2 Fastkd2 2 Gstt2 1 Fbxo22 1 Gtf2e1-Rabl3 1 Fdft1 1 Gtf2f2 1 Fem1c 1 Gtf2h1 1 Fhod3 1 Gtf2i 1 Fkbp2 1 Gtpbp3 1 Fkbp5 1 Gtpbp5 1 Fkbp8 1 Guf1 1 Flcn 2 Gzf1 1 Fntb 1 H2afj 1 Folr1 1 Hat1 1 Hdhd2 2

155

Targets identified in the c-mycHA cell line continued. Gene c-mycHA Gene c-mycHA Helb 1 Ktelc1 1 Herc4 1 L3mbtl2 1 Hes1 1 Lars 1 Hiat1 1 Ldlr 1 Hisppd1-Zh2c2 1 Lemd1 1 Hist1h2ag-Hist1h2bj 1 Liph 2 Hist1h3c-Hist1h2bb 1 Lmbr1 1 Hist1h3f 1 Lpl 1 Hist1h4h 2 Lrdd-Rplp2 1 Hist2h2ab 1 Lsg1 1 Hist2h4-Hist2h3c1 1 Lyplal1 1 Hist4h4 1 Lyrm7-Hint1 2 Hmg20b-F630110N24Rik 1 Map2k5 1 Hmga1 1 Mat2b 2 Hmga2 1 Max 1 Hmgb2l1 1 Mcph1 1 Hmgcr 1 Mett11d1 1 Hnrpm 1 Mettl3 1 Iars 1 Mettl4 1 Ifitm2-Ifitm1 1 Mettl5 1 Ifrd1 1 MGC67181 1 Igsf8 1 Mgrn1 1 Impa2 2 Mif 1 Impact 1 Mink1 1 Ing4 1 Mip 2 Ints2 1 Mlh3 3 Ints3 1 Mllt11 1 Irak3-Tmbim4 1 Mmachc 1 Isy1 1 Mobkl1a 1 Itgb3bp 1 Mog 1 Itgb3bp-BC020077 1 Morc1 1 Itih1-Nek4 2 Morc3 2 Ivns1abp 1 Mpdu1 1 Jam2 1 Mphosph10 1 Jmjd1a 1 Mrm1 1 Jund1 1 Mrpl1 1 Kctd6 1 Mrpl13-Mtbp 2 Kif11 1 Mrpl24 1 Klf15 1 Mrpl36 1 Klf6 1 Mrpl39 1 Klhdc4 1 Mrpl43-Peo1 1

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Targets identified in the c-mycHA cell line continued. Gene c-mycHA Gene c-mycHA Mrps12 1 Nudt21 1 Mrps18a 1 Nufip1 1 Mrps18b-Ppp1r10 2 Nvl 1 Mrps33 1 Nxt1 1 Msh6 3 Oaz2 1 Mt1 1 ORF34 1 Mtap7 1 Osgep 1 Mtbp 1 Otub1 1 Mtf2 1 Oxnad1 2 Mthfd2 1 P2rx3-Ssrp1 1 Mtif2 1 P4hb 1 Mtnr1a 1 Pabpc1 1 Mxd3 1 Park7-Tnfrsf9 1 Myo10 2 Parn-Bfar 1 Myst2 3 Patz1 1 Nadk 1 Pcbp4 1 Narg1l 1 Pcna 1 Nbn 1 Pcolce2 1 Ncaph2 1 Pcyox1l 1 Ncor1-Pigl 1 Pdcd11 1 Ndufa5-Asb15 1 Pde8a 1 Ndufs8 1 Pdss1 1 Neil1 1 Pdzd4 1 Nek6 1 Pebp1 1 Nek8 1 Pelp1 1 Nfe2l2 1 Peo1 2 Ngdn 1 Pet112l 1 Ngrn 1 Pfdn2 1 Nhedc1 1 Pfkl 1 Niban 1 Pfkp 1 Nmb 1 Pgam1 1 Nmd3 1 Pgd 1 Nol10 1 Pgk1 1 Nosip 1 Pias4-Eef2 1 Notch4 2 Picalm 1 Nrip1 1 Pigl 1 Nsf 2 Pih1d1 1 Nt5dc2 1 Pim3 1 Nub1 1 Plaa 1 Nubp2 1 Plk3 1 Nudcd2 1 Plod2 1

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Targets identified in the c-mycHA cell line continued. Gene c-mycHA Gene c-mycHA Pmm2 1 Rbm8a 1 Pno1 1 Rbpms 1 Pnpo 1 Rccd1 1 Pnrc1 2 Rdm1 1 Pola2 1 Rmi1 1 Polh 1 Rmnd1 1 Polr2e 1 Rnf168 1 Polr2i 1 Rnf38 1 Pols 1 Rnmt 1 Pomt1 1 Rnuxa 1 Pou2f1 1 Rpa2 1 Ppan 1 Rpap1 1 Ppard 1 Rpl22l1 1 Ppp1cc 1 Rpl36al 1 Ppp1r9a 1 Rpo1-3 1 Ppp2r1a 1 Rpo1-4 1 Pprc1 1 Rpp25 1 Pps 1 Rps21 1 Prdm5 1 Rps27 1 Prdx1 1 Rpsa 1 Prdx6 1 Rragc 1 Prei3 1 Rrm1 1 Prkcbp1 1 Rrp15 1 Prpf3 1 Rsl1d1 1 Psmb1-Tbp 1 Rsph1 1 Psmc1 1 Rtel1 1 Psmd7 1 Rttn 1 Pspc1 1 Ruvbl2 1 Ptcd3-Rpo1-4 2 Sae1 1 Ptpn21 1 Sart1 1 Ptprv 1 Sc4mol 1 Qrich1 1 Scgb3a1 1 Rab9 1 Sdccag10-3110031B13Rik 1 Rabif 1 Sec23a-Sip1 2 Rac1 1 Sephs2 1 Rad21 1 Sertad2 1 Rai14 1 Setd4 1 Rassf3 1 Setd4-Cbr1 1 Rbbp4-Zbtb8os 1 Setd8 1 Rbbp7 1 Sf3a1 1 Rbm4 1 Sf3b4 1

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Targets identified in the c-mycHA cell line continued. Gene c-mycHA Gene c-mycHA Sfi1-Eif4enif1 1 Sulf2 1 Sfrs11 1 Supt16h 1 Sgol1 1 Supt4h1 1 Shkbp1 1 Suz12 2 Sirt6 1 Taf13 1 Siva1 1 Taf5l 1 Slc12a8 1 Taf6 1 Slc19a2 1 Tagap1 1 Slc25a32 1 Tbc1d17-Akt1s1 1 Slc25a36 3 Tbn-Ccnd3 2 Slc2a5 1 Tbrg1 1 Slc30a6 1 Tcl1 1 Slc35e1 1 Tcp11 1 Slc35f5 1 Tfrc 1 Slc6a15 1 Thex1 1 Slc7a1 1 Thg1l 1 Slc7a3 1 Timm44 1 Slc7a7 1 Tmc6-Tmc8 1 Sltm 1 Tmed1 1 Smad7 1 Tmem107 1 Smarcc1 1 Tmem142c 1 Smyd1-Krcc1 1 Tmem161b 1 Smyd2 1 Tmem50a 2 Snag1 1 Tmem50a-D4Wsu53e 1 Snrp70 1 Tmem69-Gpbp1l1 3 Snx11-Cbx1 1 Tmem85-2410042D21Rik 1 Sod1 1 Tnfrsf19 1 Sos1 1 Tnfsf13 1 Spnb2 1 Tomm7 1 Spp1 1 Tor1aip1 1 Spred1 2 Tpk1 1 Src 1 Tpr 1 Srd5a1-Nsun2 1 Trappc4 1 Srebf2 1 Trappc6a 1 Srp14 1 Trh 1 Srrm1 1 Trim13 1 Ss18 1 Trim25 1 Ssbp1 1 Trim59 1 Ssbp3 1 Tsr2 1 St13 1 Tubb2b 1 Stmn1 1 Tulp2 1

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Targets identified in the c-mycHA cell line continued. Gene c-mycHA Gene c-mycHA Txn1 1 Zc3h3 1 Txndc12 1 Zc3h8 1 Ube1c-Arl6ip5 1 Zc3hc1 1 Ube1x 1 Zcchc7 1 Ube2s 1 Zcchc8 1 Ube2t 1 Zdhhc24 1 Ucp2 1 Zfand2a 1 Umps 1 Zfp13 1 Unk 1 Zfp13-Zscan10 1 Urod-Hectd3 1 Zfp192 1 Usp15 1 Zfp219 1 Usp45 1 Zfp341 3 Usp5 1 Zfp444 1 Uspl1 1 Zfp593 1 Utp15 1 Zfp704 1 Vamp4 1 Zfp740 1 Vars 1 Zfp91-Lpxn 1 Vegfc 1 Zic3 1 Vil2 1 Zic5-Zic2 1 Wasf3 1 Zwint 1 Wdr13 2 1-Sep 1 Wdr18 1 2-Sep 1 Wdr42a 1 15-Sep 2 Wdr51b 1 0610006I08Rik 1 Wdr55 1 0610009B22Rik 1 Wipi2 1 0610009O20Rik 1 Wnk1 2 1110002N22Rik 1 Wtap 1 1110004F10Rik 1 Wwp2 1 1110038F14Rik 1 Xkr5 1 1110067D22Rik 1 Xrcc4 1 1600012F09Rik 1 Xrn2 1 1700030K09Rik 1 Yars 1 1700061G19Rik 1 Yars2 1 1810035L17Rik 1 Ywhae 1 2010111I01Rik 1 Ywhaz 1 2010309E21Rik 1 Yy1 1 2310004I24Rik 1 Zbed3 1 2310037I24Rik 1 Zbtb38 1 2410008K03Rik 1 Zbtb41 1 2410015M20Rik-Rpl36 1 Zbtb8os 1 2410017P07Rik 1

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Targets identified in the c-mycHA cell line continued. Gene c-mycHA 2410018C20Rik 1 2410042D21Rik 1 2410127L17Rik 1 2410166I05Rik 1 2610101N10Rik 1 2610110G12Rik 1 2810417H13Rik 1 2810422O20Rik 1 3110031B13Rik 1 3200002M19Rik 1 4632434I11Rik-Prcp 1 4833401D15Rik 1 4833418A01Rik 1 4930452B06Rik 1 4930535B03Rik 2 4930550C14Rik 1 4932417I16Rik-Gabarapl2 1 4933405K07Rik 1 4933422H20Rik 1 4933426M11Rik 1 4933435A13Rik 1 5730536A07Rik 1 5830482F20Rik 1 5930416I19Rik 1 6230410P16Rik 1 6330408A02Rik 1 6330416G13Rik 1 A930016P21Rik 1

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