A Dissertation Entitled SWI/SNF Chromatin Remodeling Enzymes

A Dissertation

Entitled

SWI/SNF Chromatin Remodeling Enzymes as Regulators of Neural-crest Derived Cell Differentiation by Himangi Marathe

Submitted to the Graduate Faculty in partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Science

Dr. Ivana de la Serna, (Committee Chair)

Dr. William Maltese, (Committee Member)

Dr. Kam Yeung, (Committee Member)

Dr. David Giovannucci, (Committee Member)

Dr. Robert Trumbly, (Committee Member)

Dr. Patricia R. Komuniecki, Dean, College of Graduate Studies

The University of Toledo August 2013

This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of

SWI/SNF Chromatin Remodeling Enzymes as Regulators of Neural-crest Derived Cell

Differentiation

Himangi Marathe

Submitted to the Graduate Faculty as partial fulfillment of the requirement for the

Doctor of Philosophy degree in Biomedical Sciences

University of Toledo,

May 2013

Neural crest cells are multi-potent cells that migrate along predefined paths as progenitor cells and differentiate into a plethora of cell types upon reaching their specified locations in the embryo. Melanocytes and Schwann cells are two cell types derived from a bipotent precursor of neural crest origin, which perform the important function of melanin synthesis and myelination respectively. The SWI/SNF complex has been implicated in the regulation of differentiation of multiple cell lineages as well as in the maintenance of embryonic pluripotency. In the current study we demonstrate that the SWI/SNF complex, which was previously shown to interact with and required by MITF to regulate melanocyte gene expression, also plays a critical role in SOX10 mediated regulation of Schwann cell as well as melanocyte specific gene expression. SOX10 is a Sry-related high mobility (HMG)-box transcriptional factor that promotes differentiation of neural crest precursors into Schwann cells and melanocytes.

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Our studies indicate that SOX10 can reprogram cells derived from NIH3T3 fibroblasts to express, in a SWI/SNF dependent manner the most abundant protein component of peripheral myelin, Myelin Protein Zero (MPZ) and Myelin Basic Protein (MBP), another abundant component of myelin in both Schwann cells and oligodendrocytes. Transient transfections of dominant negative BRG1 and BRM, the ATPases of the SWI/SNF complex in an immortalized Schwann cell line significantly abrogated SOX10 mediated activation of MPZ and MBP. We provide evidence that SOX10 physically interacts with and promotes recruitment of BRG1, an ATPase of the SWI/SNF complex, to regulatory regions of MPZ. siRNA mediated downregulation of SOX10 compromised expression of MPZ and also led to significantly lower enrichment of BRG1 at the MPZ regulatory regions. Thus SWI/SNF enzymes are essential for, and co-operate with SOX10 to directly activate genes encoding essential components of myelin, which is a key function of Schwann cells.

In the second study we have shown that SOX10 mediated regulation of melanocyte specific gene expression is also dependent on the chromatin remodeling activity of the SWI/SNF complex. We have further probed if SWI/SNF enzymes are downstream modulators of a physiologically important inducer of melanin synthesis namely Alpha-MSH. Our studies indicate that SOX10 both individually and in synergy with MITF, promotes expression of two key melanin synthesizing enzymes in a SWI/SNF dependent manner in reprogrammed melanocytes obtained from NIH3T3 fibroblasts. Moreover our studies indicate that SOX10 and MITF play an independent role in the recruitment of BRG1 to the enhancer and promoter regions of the Tyrosinase related protein-1 (TRP-1) gene respectively.

Chromatin immunoprecipitation experiments in cells that are depleted of MITF show reduced BRG1 enrichment at the Trp1 promoter, whereas the BRG1 enrichment at the enhancer is unaffected, further strengthening the observation that MITF and SOX10

iv individually recruit BRG1 to the Trp1 promoter and enhancer respectively. Moreover knockdown experiments of SOX10 indicate reduced chromatin accessibility suggesting that SOX10 mediated SWI/SNF recruitment may remodel the chromatin structure at the enhancer leading to a more accessible conformation. These observations collectively place the SWI/SNF complexes as downstream modulators of alpha-MSH mediated melanin synthesis and as co-effectors of the two critical regulatory factors namely MITF and SOX10.

The concluding section of the study points out an exciting phenomenon wherein MITF may play a role in preferentially directing the bi-potent precursor towards melanocyte specific gene expression by modulating the SWI/SNF complex availability for SOX10 mediated expression of Schwann cell specific genes. Knockdown studies of MITF in melanoblasts strongly up-regulates expression of Myelin Protein Zero (MPZ). Moreover Chromatin immunoprecipitation studies indicate increased enrichment of BRG1 and transcriptionally permissive histone modifications in MITF knockdown cells, whereas SOX10 enrichment was unaffected. These observations suggest that MITF may play a role in modulating SWI/SNF recruitment and other chromatin remodelers to Schwann cell specific targets. These observations combined with the recent observations that both melanocytes and Schwann cells can de-differentiate and revert to a neural crest like progenitor has significant implications in their utility for regenerative medicine. Our studies which implicate SWI/SNF complex as a downstream modulator of lineage specific transcription factors may open new avenues for developing therapies for patients suffering from disorders that affect neural crest cell derived cells.

Acknowledgements

I would like to first and foremost acknowledge my advisor Dr. Ivana de la Serna for being one of the most accessible, considerate and supportive person I have met professionally. Working with her has been an incredible experience and has taught me to be thorough and professional in my attitude towards research. She has given me a great deal of exposure to learning new methods and techniques and has been very patient in doing so. Her support in sending me to multiple conferences has given me the opportunity to interact with diverse group of academicians thus enriching my graduate school experience. I admire the sincerity and passion in her approach towards science and hope to imbibe that from her in my career henceforth.

I owe a great deal of knowledge to my interactions with Dr. Kam Yeung. He always encouraged me to go into the minute details of experiments and data and thus hone my critical thinking abilities. Working with him gave me my earliest first author publication. I am also indebted to my committee members Dr. William Maltese, Dr. Manohar Ratnam and Dr. David Giovannucci for taking out time and providing me with valuable inputs regarding my project. Dr. Maltese’s sense of commitment to his appointments truly touched me and inspired me to imbibe the same in my future career. Dr. Ratnam always urged me to look at the “BIG Picture” thus making me conscious of how important it is to be aware of the clinical relevance and significance of the work being carried out. It helped me to gain a holistic view point of the involved intricacies. Dr. Giovannucci has always been a very friendly and approachable committee member, and challenged me with intellectual questions during my committee meetings. I would like to thank Dr. Randall Ruch for being a friendly mentor during my initial days of rotations and helping

vi me become comfortable in my course. I also would like to extend a warm note of thanks to Dr. Trumbly for agreeing to be my substitute committee member at short notice.

Sincere appreciation is also due to all my past and current lab members. Dr. Bridget Keenen and Dr. Srinivas Saladi were instrumental in helping me learn and understand experiments and techniques during my rotation days. Thanks are also due to my senior Dr. Mithun Khattar for helping me gain the necessary information and knowledge regarding the formal norms of my thesis and the entire graduation process. Moreover new additions to the lab with respect to my current colleagues Aanchal, Shweta, Ila, Archit, Gaurav and Phil kept the atmosphere enthusiastic and conducive to further collaborative research, discussions and a very productive lab. I also owe them a vote of thanks for all their help during my qualifying exam and thesis preparation and various oral presentations.

Handling the orders of the lab would have been short of a nightmare if not for the constant support extended towards me by the lovely ladies of our Department of Cancer Biology, Jenifer Zak and Anna Chlebowski.

Lastly and most importantly I am indebted to my mother Mrs. Manjiri Marathe for being incredibly brave and selfless in letting me study so far from her and for being so understanding and supportive throughout my graduate school journey. One of the most significant person in my life who made my journey so far worthwhile and provided me with the necessary criticism, encouragement, support and love is my husband Dr. Souma Chowdhury. Every achievement becomes more significant when you have loved ones who revel in your success and I am incredibly lucky to have a very loving and a supportive family of in-laws, who make me feel so accomplished at the smallest of achievements. Lastly I would like to remember my Dad Late. Dr. Girish Marathe for giving me a truly blessed childhood and molding me into the person I am today.

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Contents

Abstract iii

Acknowledgements vi

Contents viii

List of Abbreviations x

Chapter 1: Introduction and Literature Review 1-17

 Neural Crest cells and derivatives  Chromatin Remodeling Enzymes  MITF and SOX10: The major transcriptional regulators

Chapter 2: Materials and Methods 18-22

Chapter 3: SWI/SNF enzymes promote SOX10 mediated activation of myelin gene expression 23-59

 Abstract  Introduction  Results  Discussion  Figure Legends  Figures  References

Chapter 4: SWI/SNF chromatin remodeling enzymes are downstream effectors of Alpha-MSH mediated melanocyte differentiation and act as transcriptional co factors for both MITF and SOX10. 60-91

 Abstract  Introduction  Results  Discussion  Figure Legends  Figures

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Chapter 5: Mitf preferentially directs melanocyte specific gene expression in presence of SOX10 and Brg1 92-104

 Introduction  Results  Discussion  Figure Legends  Figures

Chapter 6: Conclusion 105

References 106-114

List of abbreviations

Alpha-MSH: Alpha- melanocortin stimulating hormone

BRG1: Brahma related gene 1

BRM: Brahma

CHD: Chromodomain

CNS: Central Nervous System

ISWI: Imitation Switch

MBP: Myelin Basic Protein

MC1R : Melanocortin -1 receptor

MITF: Microphthalmia transcription factor

MPZ: Myelin Protein Zero

NC: Neural crest

NF1: Neurofibromatosis-1

PNS: Peripheral Nervous System

SCP: Schwann cell precursor

SOX10: Sry-related HMG box domain containing factor 10

SWI/SNF: Mating type switching/sucrose non fermenting

Trp1: Tyrosinase related protein 1

Trp2/Dct: Tyrosinase related protein 2/ Dopachrome tautomerase

Tyr: Tyrosinase

UVR: Ultra violet radiation

CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW

Neural Crest Cells and their Derivatives:

The Neural crest cells are a primitive mass of multipotent cells that migrate along predefined paths as progenitor cells and then undergo differentiation into a vast plethora of cell types depending upon the specific location in the embryo. These cells were first identified in the developing chick embryo by Wilhelm His in 1868 [1]. The cells derived their name based on their location at the “Crest” of the closing neural tube. [1] The neural crest cells are a unique characteristic of vertebrates and though derived from the ectoderm are often also referred to as the “Fourth Germ layer” [2] since they give rise to cells that are of both ectodermal and mesodermal nature [3].

The neural crest cells apart from being multi-potent also exhibit self-renewal properties thus making them akin to stem cells. The multi-potency of neural crest cells was demonstrated by cell-labeling experiments using cell autonomous dye lysinated rhodamine dextran (LRD) that was injected into an individual neural tube cell of the dorsal region in the chick embryo. The labeled individual cell was then found to give rise to multiple derivatives including sensory neurons, presumptive pigment cells, and supportive ganglionic cells [3] [4]. Furthermore, serial dilutions and clonal density culturing of neural crest cells isolated from the rat neural tube demonstrated that self- renewal ability could be maintained up to 10 days in culture [3]. The neural crest cells arising at the dorsal region and which migrate along the trunk give rise to the pigment cells and the sensory neurons and glia [5].

Analysis of factors regulating proper development of neural crest derivatives is critical, since perturbed NC development leads to diverse disorders which are collectively known as Neurocristopathies [6]. The notable disorders affecting the neural crest derivatives of interest include Hirschsprung disease, Neurofibromatosis type-1, Peripheral demyelinating neuropathy, Waardenburg syndrome, Vitiligo Vulgaris, melanotic nevi and melanoma to mention a few. Most of these disorders are associated with mutations in the major transcription factors and/or the signaling pathways regulating the development and differentiation of the melanocytes and the Schwann cells. Thus understanding the mechanism of their development downstream of these regulators is essential to obtain potential therapeutic targets for the above disorders.

Figure 1: Neural crest Derivatives [5]

Neuro-Glial Cells: The Schwann cells

An interesting and important fact about the higher vertebrate brain is that about 90% of the brain is comprised by glial cells [7]. The term “Neuro-Glia” (Nerve-cement) was coined by the pathologist Rudolph Virchow [7]. Glial ensheathment of axons and subsequent formation of the Myelin sheath is critical for normal neuronal function.

Myelination of axons in the Central Nervous System (CNS) is carried out by oligodendroglia whereas the Schwann cells perform this function in the Peripheral

Nervous System (PNS).

Schwann cells were so named after the scientist who first discovered them Theodor

Schwann, who described these cells as “a cell of the peripheral nervous system that wraps around a nerve fiber, jelly-roll fashion, forming the myelin sheath” in 1839 [8] [9].

Additional evidence that Schwann cells in addition to other cell types, were indeed derived from Neural Crest cells was provided by LeDouarin in 1973 using heterochromatin staining in quail-chick chimeras [10].

The two types of Schwann cells, namely the myelinating and the non-myelinating cells are the two main types of peripheral glial cells. The peripheral glia is also comprised of satellite cells and enteric glia. Multiple evidences in literature indicate that these neural crest derived glial cells which includes the Schwann cells belonging to the PNS retain plasticity throughout life [11-14]. Schwann cell differentiation occurs in three stages, the first stage is the formation of the Schwann cell precursor (SCP) from the neural crest cells, followed by the formation of an immature Schwann cell from the SCP. The last stage consists of the differentiation of the immature Schwann cell into myelinating and non-myelinating Schwann cells. These stages have been shown to be reversible [13].

Moreover dedifferentiation of adult Schwann cells to an immature Schwann cell phenotype has been observed in vivo and is essential for regeneration of the axon [15-17].

Thus Schwann cell plasticity in vivo is controlled by both positive and negative regulators[15]. The SCP has been shown to be reprogrammed in vitro into other neural crest derivatives. Recent publications utilizing lineage tracing methods have established the SCPs as the in vivo cellular source of melanocytes, thus highlighting their bi-potent and plastic nature [18, 19]. Further analysis in mice revealed that myelinating Schwann cells could also form melanocytes if challenged with a new microenvironment which included loss of contact with peripheral nerves [18, 19]. It remains to be determined if non-myelinating Schwann cells could also act as cells that could give rise to melanocytes.

Further reports indicating the de-differentiation potential and phenotypic plasticity of

Schwann cells showed that these cells could revert back to a bi-potent glial melanocyte progenitor and could give rise to myofibroblasts in-vitro [20-24]. Similar observations are also observed in-vivo where in sciatic nerve injury leads to pigment formation around the damaged nerves [11, 22] These observations imply that growing peripheral nerves can be regarded as stem/progenitor cell niches which can act as a source of diverse cell types [18]. Adult Schwann cells apart from being involved in myelination of the PNS are also responsible for regeneration of multiple types of neurons in the adult spinal cord as well as for axonal regrowth into peripheral nerve grafts [25-27]. Schwann cells isolated from rat adult nerve were shown to significantly promote neurite growth in retinal ganglion cells of the human eye [25, 28]. Transplantation of adult Schwann cells isolated and cultured from peripheral nerve into the CNS have also proved useful in repair of demyelinated lesions of the CNS that have been observed in diseases such as multiple

4 sclerosis and other neural injuries [25]. In very recent developments the FDA has approved the first clinical trial involving 8 patients led by the University of Miami-Miller

School of Medicine, to evaluate the safety of transplanting Schwann cells isolated from each patient’s leg to cure them of paralysis [29]. Thus Schwann cells have tremendous practical potential for therapy related to the nervous system and this underscores the importance of understanding the mechanisms regulating Schwann cell development.

Apart from being a valuable therapeutic source, the Schwann cell and its precursors have been implicated in neurofibromatosis-1 (NF1) [18]. NF1 is a cancer of Schwann cells, melanocytes, axons, and fibroblasts. It is characterized by abnormal pigmentation and simultaneous occurrence of malignant melanoma [30]. Genetic analysis of NF1 patients identified Schwann cells as the tumorigenic source [31]. Further inactivation analysis of

NF1 in an early Schwann cell stage lead to induction of neurofibromas which were associated with hyperpigmentation [18]. These studies suggested that SCPs formed the origin of melanocytes in the NF1 tumors. Melanotic schwannoma is another disorder characterized by co-occurrence of pigmented melanocytes and Schwann cells in the same tumor which are found deep inside the nerve sheaths [18]. This brings into question the origin of the tumorigenic melanocytes at these unusual locations further strengthening the need to understand how the SCPs can give rise to melanocytes and if there is any mechanism that dictates the direction of differentiation the SCPs take towards either

Schwann cells or melanocytes.

Pigment producing cells: Melanocytes

Melanocytes are the only cell type in the skin that contributes to skin pigmentation by means of melanin synthesis [32]. Melanin synthesis is a key function of a mature melanocyte and the pigment is responsible for photo-protection and thermoregulation

[33]. Apart from populating the skin, melanocytes are also present in the inner eye, hair follicle, heart and the Harderian gland [34]. Melanocytes were previously thought to originate directly only from the dorso-laterally migrating neural crest cells. Recent experimental evidence indicates that melanoblasts, the precursors of melanocytes, are also obtained from the SCPs which migrate ventrally [18, 19, 32]. Cell tracing experiments in mice have shown that that majority of the cutaneous pigmented melanocytes present in post natal skin are derived from SCPs [18, 19, 32]. Recently it was reported that melanoblasts are multipotent and can be induced to differentiate to glial as well as neural cells [35]. A melanoblast is said to have differentiated to a melanocyte when it exhibits melanin synthesis and shows structural characteristics such as formation of dendrites and small granular structures containing melanin, known as melanosomes

[33, 36]. Melanin is an excellent photoprotectant since it absorbs UV radiation and transforms it to harmless heat, with about 99% efficiency [37]. Moreover, melanin also plays an important role as an antioxidant and in neutralizing the effects of harmful free radicals, and this property seems to be lost during progression of melanoma [33].

Melanin synthesis in melanocytes is regulated by three major enzymes, namely

Tyrosinase (Tyr), Tyrosinase related protein-1(Trp1) and Dopachrome tautomerase (Dct) or Tyrosinase related protein-2 (Trp2). Tyrosinase is the rate-limiting enzyme of this process [33, 38]. The expression of these enzymes is highly specific to melanocytes and

6 hence their expression is often utilized as indicators of melanocyte differentiation.

Literature states that failure of differentiation is necessary for dysplasia, and dysplastic nevi on further accumulation of mutations can lead to melanoma [39, 40]. Apart from its implications in melanoma, disrupted melanocyte differentiation or absence of melanocytes is observed in congenital pigmentary disorders such as Waardenburg syndrome and Tietz syndrome. These two disorders are characterized by either patches of white pigmentation or albinism and a few patients also have neurological disorders and facial abnormalities. There are four subtypes of Waardenburg syndrome with characteristic abnormalities that are associated with mutations in key factors implicated in regulating melanocyte development and differentiation [41-44]. Vitiligo is another rather frustrating disorder that involves loss of melanocytes and leads to de-pigmented skin lesions, that can spread to other body areas, but the cause of which has not yet been deciphered. Similar to their Schwann cell counterparts, specified and pigmented melanocytes also display phenotypic instability, and were found to differentiate into other

NC derivatives [20, 45]. This ability of mature melanocytes to de-differentiate and revert to an uncommitted and pluripotent NC precursor is enhanced by endothelin-3 [20, 46] .

Similarly, de-differentiation of melanocytes and re-activation/ over-expression of early developmental genes have been observed in melanoma along with reports of transdifferentiation of melanocytes to activate myelin gene expression [46-48]. Thus understanding the mechanisms regulating development of melanoblast from the SCP and its further differentiation into melanocytes is essential to analyze how perturbation of these mechanisms may lead to the associated disorders including melanoma.

CHROMATIN REMODELING ENZYMES

Chromatin can be considered as one of the most efficient forms of a packaging process, where by a string of about 6 billion base pairs that constitute the human DNA in a diploid cell is neatly organized and stored into the microscopic nucleus of a cell. This compaction is reversible and is mediated by unique proteins termed histones.

Approximately 146bp of DNA is wrapped around an octamer of core histones H2A,

H2B, H3 and H4 each of which is present as a dimer. This arrangement of DNA around the histone octamer constitutes the fundamental unit of chromatin known as the nucleosome. Histone H1 functions as the linker histone by binding to the DNA between two adjacent nucleosomes and mediating further compaction and stabilization of higher order structures [49, 50]. This compaction of DNA is obstructive to a majority of cellular processes such as cell growth, development, differentiation all of which require access to the genetic information contained in the DNA. Moreover, higher order chromatin structure by means of its spatial distribution can be classified into transcriptionally

“active” euchromatin or into transcriptionally “repressive” or “inactive” heterochromatin conformations, thus suggesting that specific alteration of chromatin structure is required for gene expression [51]. Two most frequently observed mechanisms of altering chromatin structure include disruption of histone-DNA contacts by ATP-dependent chromatin remodeling enzymes and by histone modifying enzymes which covalently modify histone tails [51, 52].

ATP- DEPENDENT CHROMATIN REMODELING ENZYMES

The ATP-dependent chromatin remodeling enzymes have been grouped into four major families based on ATPase homology and presence of other characteristic catalytic domains. These families are the SWI/SNF family, ISWI family, CHD family, and the

INO80 family. The SWI/SNF family was the first chromatin remodeling complex to be identified. These were identified in yeast screens for mutants affecting “mating-type switching” (SWI) and “Sucrose Non-Fermenting” (SNF). These mutants displayed altered nuclease sensitivity at the suc2 gene and suppressors of these mutants were genes coding for histones and other chromatin associated proteins, thus indicating their role in alteration of chromatin to mediate gene expression [51, 53-56].

Figure 2: Families of ATP-dependent chromatin remodeling enzymes [57]

The Imitation Switch (ISWI) family chromatin remodelers were first identified in

Drosophila embryo extracts in assays for nucleosome remodeling activities [53]. The

ATPases of this family are characterized by the presence of two unique domains in addition to the conserved ATPase domains namely SANT and SLIDE. These domains collectively constitute a nucleosome recognition unit which allows it to bind to DNA and unmodified H4 tails [53, 57]. Acetylation of H4K16 has been shown to reduce ISWI activity and is targeted away from transcriptionally active chromatin[53]. There are two mammalian homologues of the ISWI family snf2H and snf2L which can function as monomers/dimers or by associating with multiple subunits to form larger complexes known as CHRAC (chromatin accessibility complex), WICH (WSTF- ISWI chromatin remodeling complex) and the NURF (nucleosome remodeling factor) complex. These complexes have been implicated in regulation of several functions such as heterochromatin formation, DNA replication, transcriptional repression as well as in embryonic stem (ES) pluripotency [53, 57, 58].

The Chromodomain-Helicase-DNA binding (CHD) family contains two chromodomains at the N-terminal. The family consists of 9 members that have been further classified into

3 sub-families CHD1, Mi-2 and CHD7 [53, 57]. The functions of the CHD family members that have been characterized so far seem to be very diverse. The CHD members can function as monomers or associate with other factors to form complexes, of which the best characterized is the Nucleosome remodeling and deacetylase (NURD) complex.

This complex is implicated in transcriptional repression by mediating removal of active histone marks and also as regulator of genomic stability [53, 57].

The Inositol requiring 80 (INO80) family has a unique ATPase domain that is split in the middle due to the presence of a spacer region and hence is referred to as the split ATPase domain [59]. This characteristic domain retains ATPase activity and also acts as a scaffold protein allowing association of a bacterial helicase RuvB thus implicating them in binding of Holliday junctions and replication forks [53, 57, 59]. Another unique aspect of this complex is that it exhibits DNA helicase activity and has an affinity for H2A variants, H2AZ and H2AX. The complex also plays an important role in DNA repair where it mediates nucleosome eviction at double strand breaks and also promotes homologous recombination repair. Finally the complex has been shown to be essential for regulating telomere length and stability [53, 57, 59].

SWI/SNF family of chromatin remodeling enzymes

The SWI/SNF family is an evolutionarily conserved multisubunit family of chromatin remodeling enzymes. The central catalytic ATPase of the complex is either Brahma

(BRM) or Brahma related gene-1 (BRG1). BRG1 was the first chromatin remodeling protein to be identified and was characterized in yeast [54, 60]. Brm was first identified in Drosophila as a suppressor of body segment defects observed in Polycomb mutants.

This antagonism with a known chromatin regulator coupled with identification of several co-associated subunits implicated in transcription established the SWI/SNF complex as proteins that modified chromatin structure to activate transcription [56, 61-66]. These co- associated subunits were termed as BRG1/BRM associated factors (BAFs). The

SWI/SNF complex does not possess any sequence specificity and is recruited to target

11 regions primarily via interactions between BAFs and key transcriptional modulators [67,

68]. The yeast SWI/SNF complex was found to be a 1.14MDa complex with about 8-11 subunits and multiple research groups contributed to the observation that the mammalian

SWI/SNF complexes are more diverse than the yeast complex with the incorporation of multiple unique subunits in addition to retaining about five ySWI/SNF orthologues [69-

71]. The resulting mammalian complex is about 2MDa which is larger than the total sum of the known subunits, thereby suggesting that multiple subunits of this complex are yet to be identified [53]. SWI/SNF activity has been shown to be required for an extensive list of physiological processes including maintenance of embryonic pluripotency [72-75], development of neural progenitors [76-79], cardiac development [80, 81], liver development [82], erythropoiesis [83-85] and differentiation of a plethora of cell types including muscle differentiation[86-88], hematopoietic[89, 90], neural differentiation[78,

79, 91], in adipogenesis [92] and very recently in melanocyte differentiation [68, 93].

SWI/SNF activity in each of the above processes was mediated by interactions between characteristic BAFs with key regulatory factors. Further evidence supporting the requirement for BAFs is derived from mouse knockouts of these factors which results in a myriad of phenotypes such as liver failure, abnormal cardiac development, placental defects and defects in neural development [94]. Moreover many of the BAFs have been established as bonafide tumor suppressors which get inactivated in multiples malignancies as well as in cancer cell lines [95-99]. The two SWI/SNF ATPases, BRG1 and BRM, are very similar and share about 74% sequence identity and have been shown to exert similar biochemical activities in vitro [100]. The function of

12 these two ATPases in vivo is very diverse as is indicated by the disruption/ inactivation studies in mice, where in BRG1 inactivation is embryonic lethal in homozygotes, and heterozygotes show accelerated development of tumors, erythropoietic defects along with other abnormalities. BRM inactivated mice were about 15% larger than control mice thus exhibiting mild proliferation defects and showed upregulation of BRG1 in certain tissues, indicating that BRG1 can compensate for BRM loss in several cases [83, 101, 102].

BRG1 and BRM have also been shown to exhibit selectivity in interactions with transcription factors. BRG1, unlike BRM was found to interact with zinc finger proteins whereas BRM was found to preferentially interact with ankyrin repeat proteins [103].

Histone modifying enzymes

The second class of chromatin modifying enzymes comprises of those that post- translationally modify histones. Examples of histone modifying enzymes are Histone acetyltransferases (HATs), Histone deacetylases (HDACs), kinases, histone methyltransferases (HMT) and demethylases. These enzymes post translationally modify histone tails by means of addition or removal of methyl, acetyl or phosphate groups (43).

Two or more different types of modifications may coexist on genes and usually constitute a histone code that is indicative of the transcriptional status of a gene. Lysine residues of

Histone 3 and 4 are most commonly modified. Histone 3 lysine 4 di or tri methylation

(H3K4me2/3) and Histone 4 acetylation (H4Ac) when present at target promoters indicate active transcription, whereas Histone 3 lysine 9 methylation(H3K9me) or

Histone 3 Lysine 27 trimethylation (H3K27me3) are marks associated with silent genes and in certain situations is also present along with H3K4 marks forming bivalent domains, often observed in embryonic stem cells. Thus the histone code along with chromatin remodeling functions to mark and regulate the transcriptional status of genes.

Sry- like HMG box 10 (SOX10)

SOX10 belongs to the high mobility group (HMG) domain containing family of transcription factors. The Sox family of proteins has been termed as “omnifunctional” due to the diverse role played in the regulation of stem cell plasticity, lineage restriction, and differentiation [104]. A mouse model for Waardenburg Shah syndrome was genetically linked to a truncating SOX10 mutation affecting its transactivation domain and thus was responsible for discovering the role of SOX10 in melanocyte differentiation

[42]. Since then numerous SOX10 mutations, spanning almost the entire length of the

SOX10 coding region have been identified and linked to a variety of disorders such as

Yemenite deaf-blind hypopigmentation syndrome, Waardenburg syndrome, peripheral demyelinating neuropathy, central dysmyelination, as well as primary melanoma [41,

104, 105]. All members of the Sox family share a highly conserved HMG domain, and can regulate transcription by a variety of methods including, direct or indirect interaction with the general transcription machinery, they can affect availability of transcription factors by sequestering them into complexes or they also lead to bending of DNA and mediate promoter-enhancer interactions [43, 104, 106]. The SOX10 protein is characterized by the presence of an HMG box, a DNA dependent dimerization domain

14 and two transactivation domains, one at the carboxyterminus and one located centrally

[43, 104].

SOX10 is critical for survival of undifferentiated neural crest cells as well as for differentiation of glial committed progenitors to mature Schwann cells [43]. SOX10 expression is initiated in early migrating neural crest cells and continues throughout

Schwann cell development. SOX10 mutant mice show severe and early defects specifically leading to inability of neural crest cells to be specified as melanocytes and glial cells [42, 104, 107]. SOX10 directly regulates expression of glial differentiation markers such as Myelin Protein Zero (MPZ); a highly abundant protein of peripheral myelin sheath having restricted expression, Myelin Basic Protein (MBP) and Connexin32

(Cx32); a gap junction protein are also directly regulated by SOX10 [41, 108, 109].

SOX10 physically interacts and shows synergy with the SP1 transcription factor for MBP expression and shows synergistic activation of Cx32 along with KROX20.

Moreover SOX10 in addition to MITF has also been shown to transcriptionally regulate all the three major enzymes of pigmentation namely Dct, Tyr and Trp1 thus playing an important function in the melanocyte lineage [6, 107, 110-112]. SOX10 was shown to directly bind to the enhancer elements in all the three genes and showed synergistic activation. Thus SOX10 plays an important role in both the glial and the melanocyte lineage. Despite this information, the exact mechanism by which SOX10 co-regulates

MITF target genes in pigmentation is yet to be well characterized. Involvement of

SWI/SNF enzymes for SOX10 was not tested in any of these studies, and this adds a novel aspect to my project.

Microphthalmia transcription factor (MITF)

MITF is so called since it was discovered as an inheritable mutation in mice leading to small eyes. Pigment cell defects and deafness were also observed in mice as a result of the gene name microphthalmia (mi) [42, 113]. MITF is one of the earliest markers of the melanocyte lineage [114, 115]. MITF is a basic helix loop helix transcription factor, in which the basic domain is responsible for DNA binding and the helix-loop-helix- leucine zipper (HLH-LZ) mediates homo/heterodimerization with TFE3, TFEB or TFEC which belong to the same family. The bHLH-LZ family of transcription factors, including

MITF, recognizes and binds to E-box elements with the canonical consensus sequence

CATGTG. Another sequence that was identified at multiple MITF target promoters was the E-box sequence flanked by a thymidine or adenosine (TCATGTG, CATGTGA or

TCATGTGA). These sequences were then collectively known as M-Box sequences [47,

115, 116]. There were three key experiments in the literature that led to MITF being known as the Master regulator of melanocyte differentiation [36, 117-119]. Additional observations supporting the importance of MITF in pigmentation arose when mouse models with pigmentary disturbances including the Waardenburg syndrome and Tietz syndrome, also involved mutations in the DNA binding domain or the transcriptional activation domains of MITF. Moreover mouse homozygous for null mutations in MITF completely lack melanocytes [36, 116]. Apart from regulating genes involved in melanocyte differentiation, MITF also regulates genes required for melanocyte

16 proliferation and survival. In later stages of melanoma, MITF functions as a lineage survival oncogene [120].

In spite of being designated as master regulator of melanocyte differentiation, there were also several reports indicating that MITF alone was insufficient to activate melanin production [104, 121, 122]. With respect to activating pigmentation gene expression, our lab was the first to establish a novel requirement of SWI/SNF enzymes for MITF activity

[68, 93]. This report showed that MITF physically interacts with the core ATPase of the

SWI/SNF complex and also with other subunits of the complex. It was also demonstrated that MITF recruitment to the TRP1 promoter does not depend on SWI/SNF remodeling activity and that MITF mediates SWI/SNF recruitment to target promoter.

CHAPTER-2: MATERIALS AND METHODS

Cell Culture and expression Plasmids: SOX10 cDNA was provided by Dr. William

Pavan (NIH) and the KROX20 cDNA was provided by John Svaren (University of

Wisconsin). Each cDNA was subcloned into the pBabe retroviral vector [36]. Dominant negative BRM (H17) and dominant negative BRG1 (B22) cell lines inducibly express

ATPase deficient, dominant negative alleles of BRM or BRG1 in a tetracycline dependent manner [37]. Cells were cultured in the presence (dominant negative expression OFF) or absence (dominant negative expression ON) of tetracycline for 3 days and were infected with pBabe-SOX10, pBabe-KROX20 retrovirus, or with retrovirus generated from the empty pBabe vector as previously described [26] for 30 hours. A low serum medium containing 2% horse serum was then added, and cells were harvested 64 hours later. Immortalized rat Schwann cells (S16) were purchased from ATCC and maintained in media with 10% fetal calf serum. Mouse melanoblasts (Melba) were obtained from Welcome Trust (UK). The cells were cultured in RPMI media with 10% fetal bovine serum. 40 pm Fibroblast growth factor (FGF) and 0.08 ng Stem cell factor

(SCF) were added to the growth medium. Differentiation was induced by adding 2 uM

NDP-MSH and 200 nM PMA to the medium and replenished every alternate day.

RNA isolation and Quantitative Real Time PCR: Total RNA was isolated using Trizol

(Invitrogen) and c-DNA was prepared using the Qiagen Quantitect Reverse Transcription kit. Quantitative real time PCR was performed in SYBR Green master mix (Qiagen)

18 with an Applied Biosystems 7500 PCR and analyzed with the SDS software as described

[38]. Primers for the various gene used were as follows; mouse and rat MPZ [39]:

5’-GCCCTGCTCTTCTCTTCTTT-3’ 5’-CCAACACCACCCCATACCTA-3’ mouse MBP:

5’-TACCCTGGCTAAAGCAGAGC-3’ 5’-GAGGTGGTGTTCGAGGTGTC-3’,

Mouse KROX20:

5’-TTGACCAGATGAACGGAGTG-3’ 5’-ACCAGGGTACTGTGGGTCAA-3’. mRNA levels were normalized to mouse RPL7:

5’-GGAGGAAGCTCATCTATGAGAAGG-3’ 5’-AAGATCTGTGGAAGAGGAAGGAGC-3’

mouse MITF:

5’-CAGACCCACCTGGAAAACC-3’ 5’-ATGCTGAGCTCAGGACTTGG-3’

mouse Tyrosinase:

5’-CGGCCCAAATTGTACAGAGAAGC-3’ 5’-CTGCCAGGAGAAGAAGGATTG-3’

mouse TRP1:

5’-GCCCCAACTCTGTCTTTTCTCAAT-3’ 5’-GATCGGCGTTATACCTCCTTAGC-3’

19 mouse TRP2:

5’-GGACCGGCCCCGACTGTAATC-3’ 5’-GGGCAACGCAAAGGACTCAT-3’

Rat KROX20:

5’-CCTGGGTGTGTGTACCATGT-3’ 5’-GAGAGGAGGTGGAAGTGGTG-3’,

Rat MBP:

5’-GGCACGCTTTCCAAAATCT-3’ 5’-CGGGATTAAGAGAGGGTCTG-3’

Rat 18SrRNA:

5’-AGTCCCTGCCCTTTGTACACA-3’ 5’-GATCCGAGGGCCTCACTAAAC-3’

siRNA Knockdown: siRNA targeting rat SOX10 (5’-

CUGCUGUUCCUUCUUGACCUUGC-3’) as reported in [40] and a non-targeting siRNA (5′-UUCUCCGAACGUGUCACGU-3′) were obtained from Dharmacon

(Lafayette, CO) and transfected according to manufacturer’s instructions. Cells were harvested 96 hours post transfection. Acell SMART Pool siRNAs targeting MITF (E-

008674-00-0050) and red non-targeting siRNAs D-001960-0150) were purchased from

Dharmacon Inc. (Chicago, Il.) as reported in [93]. Control and shRNA constructs targeting BRG1/BRM were provided by Stephen Smale (Howard Hughes Medical

Institute, UCLA).

Cell extracts and immunoblot analysis: Western blots were performed as described [41].

Antiserum to BRG1 was previously described [41]. The SOX10 (N-20) antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The KROX20 antibody was from Covance (Princeton, New Jersey, USA). The FLAG antibody and the MITF antibody were from Sigma (St. Louis, MO). Total-ERK and tubulin antibodies were from Cell Signaling Technology (Boston, MA, USA).

Chromatin immunoprecipitations (ChIPs): ChIPs were performed as described [38].

Primers that amplify the MPZ promoter and intron in mouse or rat were previously described in [42]. ChIP signal at the MPZ locus was normalized to control IgG and to a control region with primers that amplify the mouse SCN2A1 promoter or rat Ig2a enhancer [42, 43] ChIP signal for histone modifications at the TRP1 locus was normalized to input and to a control region with primers that amplify the mouse IgH enhancer. The primers used were as below; mouse TRP1 Promoter 5’-GCAAAATCTCTTCAGCGTCTC-3’ 5’-AGCCAGATTCCTCACACTGG-3’ mouse TRP1 Enhancer 5’-GGGAGTGGGTGTGGTATGAG-3’ 5’-CCTCACCCTCTTCTTGGTCA-3’ mouse IgH Enhancer

5’-GCCGATCAGAACCAGAACACC-3’ 5’-TGGTGGGGCTGGACAGAGTGTTTC-3’

Fluorescence- activated cell sorting (FACS): Roughly 1x106 were fixed with 100% ethanol for 1 hour and stained with PI-RNAse solution for 30mins and loaded on a

FACS-Calibur (BD Biosciences, San Jose, CA, USA at the University of Toledo Flow

Cytometry Core Facility). Data was analyzed using Cell Quest Pro (BD Biosciences).

Chromatin Accessibility assay: Roughly 10 million cells were trypsinized and washed once with 1X PBS. The nuclei were extracted using dounce homogenization in chilled

NP-40 lysis buffer. Nuclei were pelleted and washed with MNase digestion buffer containing 1mM calcium chloride. 100ul of nuclei were digested with 1unit of

Micrococcal Nuclease (MNase) for series of time periods. Digestion was stopped by addition of STOP buffer, 10%SDS and MNase digestion buffer. 25ug/ul Proteinase K was added and incubated at 37degrees overnight. Samples were extracted to phenol- chloroform and precipitated overnight with 1/10th volume of 3M sodium acetate and 2.5 volumes of 100% ethanol. Precipitated material was suspended in 100ul TE. 2ul sample was used for each well in qRT-PCR analysis with primers specific to region of interest.

Accessibility was calculated as difference in signal intensity between control region and test region. Primers utilized for TRP1 and MPZ have already been described in the ChIP section.

Statistical Analysis: Statistical significance was calculated by the Student’s t test when comparing two sets of data and a one way ANOVA followed by the Tukey-Kramer multiple comparison tests for comparing more than two sets of data.

CHAPTER-3 (Accepted Manuscript)

SWI/SNF enzymes promote SOX10- mediated activation of

myelin gene expression

1Marathe, Himangi, G., 1Mehta, Gaurav, 1Zhang, Xiaolu, 1Datar, Ila, 1Mehrotra, Aanchal,

1Yeung, Kam, C., and 1*de la Serna, Ivana, L.

1University of Toledo College of Medicine, Department of Biochemistry and Cancer

Biology, 3035 Arlington Ave, Toledo, OH 43614

Toledo, Ohio 43614

* Corresponding Author. Mailing address: University of Toledo College of Medicine,

Department of Biochemistry and Cancer Biology, 3035 Arlington Ave, Toledo, OH

43614. Phone: (419) 383-4111. FAX: (419) 383-6228. E-mail:

[email protected]

ABSTRACT

SOX10 is a Sry-related high mobility (HMG)-box transcriptional regulator that promotes differentiation of neural crest precursors into Schwann cells, oligodendrocytes, and melanocytes. Myelin, formed by Schwann cells in the peripheral nervous system, is essential for propagation of nerve impulses. SWI/SNF complexes are ATP dependent chromatin remodeling enzymes that are critical for cellular differentiation. It was recently demonstrated that the BRG1 subunit of SWI/SNF complexes activates SOX10 expression and also interacts with SOX10 to activate expression of OCT6 and KROX20, two transcriptional regulators of Schwann cell differentiation. To determine the requirement for SWI/SNF enzymes in the regulation of genes that encode components of myelin, which are downstream of these transcriptional regulators, we introduced SOX10 into fibroblasts that inducibly express dominant negative versions of the SWI/SNF ATPases,

BRM or BRG1. Dominant negative BRM and BRG1 have mutations in the ATP binding site and inhibit gene activation events that normally require SWI/SNF function. SOX10 activated expression of the endogenous Schwann cell specific gene, myelin protein zero

(MPZ) and the gene that encodes myelin basic protein (MBP) in cells derived from

NIH3T3 fibroblasts, thus reprogramming these cells into myelin gene expressing cells.

Ectopic expression of KROX20 was not sufficient for activation of these myelin genes.

However, KROX20 together with SOX10 synergistically activated MPZ and MBP expression. Dominant negative BRM and BRG1 abrogated SOX10 mediated activation of MPZ and MBP and synergistic activation of these genes by SOX10 and KROX20. Our data suggest that SOX10 is required to recruit BRG1 to the MPZ locus. Similarly, in

24 immortalized Schwann cells, BRG1 recruitment to SOX10 binding sites at the MPZ locus was dependent on SOX10 and ectopic expression of dominant negative BRG1 inhibited expression of MPZ and MBP in these cells. Thus, SWI/SNF enzymes cooperate with

SOX10 to directly activate genes that encode components of peripheral myelin.

INTRODUCTION

Glial cells insulate axons by forming a lipid rich structure called the myelin sheath [1].

Two types of myelinating cells, oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS) are essential for nervous system development and for proper conduction of nerve impulses. De-myelinating diseases, such as multiple sclerosis of the CNS [2], and neuropathies such as Charcot-

Marie-Tooth Disease of the PNS cause severe sensory and motor defects [3]. Inherited neuropathies of the PNS are characterized by mutations in genes that encode essential components of myelin and transcriptional regulators of Schwann cell development.

SOX10 is a Sry-related high mobility (HMG)-box transcriptional regulator that promotes differentiation of neural crest precursors into the glial lineage and is also involved in melanocyte differentiation [4]. The critical function of SOX10 in Schwann cell development and function is underscored by the occurrence of demyelinating neuropathies that result from SOX10 mutations [3]. SOX10 not only has a role in the commitment and early differentiation of neural crest cells into Schwann cell precursors, it is also required for their maturation into myelinating Schwann cells [4]. During early stages of differentiation, SOX10 promotes expression of low levels of myelin protein

25 zero (MPZ), a major component of myelin that is specifically expressed in Schwann cells

[5]. At later stages, SOX10 drives the myelination process through a stepwise feed forward mechanism. SOX10 first activates the POU homeo-domain transcription factor,

OCT6 [6] which then cooperates with SOX10 to activate expression of the zinc finger transcriptional regulator, KROX20 [7]. In the next step, pro-myelinating Schwann cells transition to myelinating cells as SOX10 and KROX20 synergistically activate high levels of MPZ and the expression of genes encoding other components of myelin [8, 9].

As a transcriptional activator, SOX10 and other SOX proteins bind to AT rich sequences in the minor groove and promote DNA bending [10]. The ability of SOX proteins to bend DNA and potentially change the architecture of target loci may promote transcription by facilitating interactions between target promoters and distal regulatory elements. However, the exact mechanisms by which SOX proteins promote transcription are poorly understood. A recent study suggests that SOX10 mediated transcriptional activation involves recruitment of SWI/SNF chromatin remodeling enzymes [11].

Mammalian SWI/SNF enzymes are evolutionarily conserved, multi-protein complexes that contain one of two ATPases, BRM or BRG1, and utilize the energy of ATP to disrupt chromatin structure and render chromatin permissive to the transcriptional machinery [12, 13]. In vitro, chromatin remodeling is achieved by a core complex containing BRG1 or BRM, the INI1 subunit, BAF 170, and BAF 155, while in vivo, additional BRG1/BRM-associated factors (BAFs) are required for interactions with

26 transcriptional activators and repressors which help recruit the SWI/SNF complex to specific genomic loci [14,15,16,17,18,19].

BRG1 and a number of other SWI/SNF components promote embryonic stem cell pluripotency and self-renewal and are essential for mouse development [20, 21, 22, 23,

24]. SWI/SNF enzymes also function in cell cycle regulation, genome organization, and cellular differentiation [25]. Interactions between SWI/SNF components and lineage specific factors have been shown to drive muscle, neuron, adipocyte, melanocyte, myeloid, and more recently Schwann cell and oligodendrocyte differentiation [11, 26, 27,

28, 29, 30, 31]. During differentiation, SWI/SNF mediated chromatin remodeling promotes transcription of previously silent genes by facilitating stable pre-initiation complex formation and/or binding of gene specific activators or alternatively by promoting later stages of transcription [32,33,34]. Thus, the mechanisms by which

SWI/SNF enzymes promote transcription depend on the context of the promoter.

Two recent studies indicate that conditional deletion of the BRG1 component of the

SWI/SNF complex in mice results in loss of pro-myelinating transcription factors and severely inhibits Schwann cell differentiation [11, 35]. In one study, BRG1 was found to activate SOX10 expression via interactions with NF-Kappa B [35]. A different study found that BRG1 interacts with SOX10 to activate OCT6 and KROX20 expression [11].

However, neither study probed the direct requirement for SWI/SNF enzymes in the transcriptional regulation of myelin specific genes downstream of these transcriptional regulators.

In the current study, we tested the hypothesis that SWI/SNF enzymes are directly required to activate genes that encode components of peripheral myelin. In order to bypass the requirement for SWISNF enzymes in the activation of transcription factor expression, we utilized an in vitro model of differentiation in which SOX10, KROX20, or

SOX10 together with KROX 20 were ectopically expressed in NIH 3T3-derived cells that inducibly express dominant negative versions of the BRM or BRG1 ATPase of the

SWI/SNF complex [35]. Dominant negative BRM and BRG1 have mutations in the ATP binding site, thus are deficient for ATPase activity and cannot remodel chromatin. They have been shown to inhibit gene activation events that normally require SWI/SNF function [35]. We found that SOX10 can activate expression of two myelin genes, myelin protein zero (MPZ) and myelin basic protein (MBP) in these cells and that induction of dominant negative BRM or BRG1 inhibits expression of both myelin genes. We then focused on the requirement for the BRG1 ATPase because previous studies indicated that disruption of BRG1 in mice severely inhibits myelination while disruption of BRM has no obvious myelination defect [11,30,36].

In order to investigate the requirement for BRG1 in the transcriptional regulation of myelin genes, we ectopically expressed SOX10, KROX20, and SOX10 together with

KROX20 in a cell line that inducibly expresses dominant negative BRG1. Expression of only KROX20 was not sufficient to activate expression of MPZ and MBP, but KROX20 together with SOX10 resulted in synergistic activation of these genes. Synergistic activation of MPZ and MBP was inhibited by dominant negative BRG1. We found that

SOX10 promotes recruitment of BRG1 to the MPZ locus. SOX10 has previously been

28 shown to activate an MBP reporter in NIH3T3 cells [37], however, to our knowledge, this is the first report that indicates SOX10 can reprogram NIH3T3 derived cells into cells that express endogenous myelin genes. We also demonstrate a direct requirement for SWI/SNF enzymes in the SOX10-mediated activation of myelin gene expression. In a complementary approach, we found that depletion of SOX10 in immortalized Schwann cells significantly decreased BRG1 occupancy at the MPZ locus. Furthermore, transient transfection of immortalized Schwann cells with dominant negative BRG1 decreased myelin gene expression. In combination, our data indicate that the chromatin remodeling domain of BRG1 is required to directly activate myelin gene expression in Schwann cells through SWI/SNF interactions with SOX10.

RESULTS

Induction of myelin gene expression by SOX10 is inhibited by dominant negative BRM and BRG1.

We previously described NIH3T3 derived cell lines, H17 and B22,that inducibly express dominant negative versions of BRM or BRG1 respectively under the control of the tetVP16 activator [35]. These cell lines have previously been used to test the requirement for SWI/SNF enzymes in tissue culture models of muscle, adipocyte, and melanocyte differentiation promoted by ectopic expression of the appropriate lineage specific factors

[25,28,32]. In order to determine whether SOX10 can convert these cells into myelin gene expressing cells and to test the requirement for SWI/SNF enzymes in the activation of these genes, we introduced SOX10 by retroviral infection into a dominant negative

BRM cell line (H17), and a dominant negative BRG1 cell line (B22) that had been grown

29 in the presence or absence of tetracycline and then cultured in low serum media to promote differentiation. Fig. 1A shows ectopic expression of the SOX10 protein and

FLAG-tagged dominant negative BRM and BRG1 expression in H17 and B22 cells respectively, when the cells were cultured in the presence or absence of tetracycline.

Interestingly, we found that SOX10 modestly but significantly increased expression of

KROX20 at the mRNA level by approximately two fold in H17 (left) and B22 (right) cells that were differentiated in the presence of tetracycline (dominant negative BRM and

BRG1 off (Fig.1B). Activation of KROX20 was partially inhibited by dominant negative

BRM and BRG1 when cells were differentiated in the absence of tetracycline (Fig. 1B).

SOX10 robustly induced the expression of two myelin genes, myelin protein zero (MPZ)

(Fig. 1C) and myelin basic protein (MBP) (Fig. 1D) in H17 and B22 cells that were differentiated in the presence of tetracycline (dominant negative BRM and BRG1 off).

However, expression of these genes was dramatically inhibited by the presence of dominant negative BRM and BRG1 when cells were differentiated in the absence of tetracycline. These results indicate that SOX10 can reprogram NIH 3T3 cells to express myelin genes by a mechanism that is dependent on SWI/SNF enzymes.

Synergistic activation of myelin genes by SOX10 and KROX20 requires BRG1

Neural crest precursors, and pro-myelinating Schwann cells express low levels of some myelin genes and the expression of these genes is highly up-regulated during myelination

[44]. Myelination involves a transcriptional cascade by which SOX10 first activates

KROX20 expression and then cooperates with KROX20 to synergistically activate the

30 promoters of a subset of myelin genes. We focused on the BRG1 ATPase of the

SWI/SNF complex, because BRG1 has been found to be required for the formation of myelin by Schwann cells in mice [11,30]. To determine if the observed requirement for

BRG1 in the activation of MPZ and MBP expression (Fig. 1 C and D) is solely due to the requirement for BRG1 in the activation of KROX20 (Fig. 1B) or due to a requirement for

BRG1 in the direct activation of these genes, we expressed SOX10 alone, KROX20 alone, and SOX10 together with KROX20 in B22 cells that were differentiated in the presence or absence of tetracycline (Fig. 2A). Our data indicate that expression of

KROX20 alone is not sufficient to activate MPZ and MBP expression in NIH3T3 derived cells (Fig. 2B). Interestingly, when cells were differentiated in the presence of tetracycline (dominant negative BRG1 off), MPZ mRNA levels were approximately eight-fold higher and MBP mRNA levels were approximately three-fold higher in cells expressing both KROX20 and SOX10 compared to cells expressing only SOX10.

Expression of dominant negative BRG1 caused a significant inhibition of the synergistic activation of MPZ and MBP expression by SOX10 and KROX20. Thus, the inhibitory effect of dominant negative BRG1 on MPZ and MBP expression was not rescued by

KROX20 expression. Furthermore, these data suggest that SWI/SNF enzymes are not only required downstream of KROX20 for activation of low levels of MPZ and MBP expression by SOX10 but are also required for the synergistic activation of MPZ and

MBP expression by SOX10 and KROX20 .

Cell cycle arrest occurs independently of SWI/SNF enzymes

Cessation of DNA synthesis and withdrawal from the cell cycle is coordinated with myelination during Schwann cell differentiation [45]. In order to determine whether

SWI/SNF enzymes are required for cell cycle withdrawal in this tissue culture model of differentiation, we cultured cells expressing either SOX10, KROX20, or SOX10 with

KROX20 in low serum media in the presence or absence of dominant negative BRG1. At the end of differentiation, cells were stained with propidium iodide to determine the number of cells in the different phases of the cell cycle. Fig. 3 indicates that expression of dominant negative BRG1 did not affect the ability of SOX10 and KROX20 expressing cells to arrest in the G1 phase of the cell cycle. This suggests that the catalytic domain of

BRG1 is required for activation of myelin gene expression by these transcriptional regulators but is not required to promote cell cycle withdrawal under these conditions.

Ectopic expression of SOX10 promotes recruitment of SWI/SNF enzymes to the MPZ regulatory region in NIH 3T3 cells.

MPZ is a Schwann cell specific component of myelin that is expressed at basal levels early in neural crest development and is highly activated during Schwann cell myelination [44]. A 1.1 kb region of the MPZ promoter that contains high affinity

SOX10 binding sites is sufficient for basal expression of MPZ while additional sequences including a region in the first intron that contains low affinity SOX10 binding sites as well as a KROX20 binding site are important for high levels of MPZ expression [8,41].

In order to elucidate the mechanisms by which components of the SWI/SNF complex are

32 recruited to the MPZ promoter, we performed chromatin immunoprecipitations (ChIPs) in control cells expressing empty vector (EV), and in cells expressing KROX20 or

SOX10. We found that ectopic expression of KROX20 alone did not significantly increase BRG1 association with either the promoter or intronic region of MPZ when compared to the empty vector control (Fig. 4A). In contrast, ectopic expression of SOX10 resulted in a significant increase in the recruitment of BRG1 to the MPZ promoter and to a lesser but significant extent to the MPZ intron (Fig. 4A). BRG1 enrichment on these sites was not significantly affected by the expression of dominant negative BRG1 in cells that were cultured without tetracycline. This is consistent with previous findings that dominant negative BRG1 associates with other components of the SWI/SNF complex and is recruited to its target loci, but is not functional at these loci [28,33].

SOX10 enrichment on the MPZ promoter was substantially higher than on the MPZ intron in B22 cells that ectopically express SOX10 (Fig. 4B). Thus, ectopic expression of

SOX10 in NIH3T3 derived cells results in SOX10 association with the same regions of the MPZ locus as was previously observed in myelinating rat sciatic nerve [43].

Furthermore, we found that SOX10 occupancy at these sites was not significantly affected by the expression of dominant negative BRG1. In combination, these data suggest that the mechanism by which SOX10 reprograms NIH3T3 cells to express this myelin gene involves binding to the MPZ regulatory regions and recruiting BRG1.

SOX10 promotes BRG1 recruitment to MPZ regulatory regions in immortalized

Schwann cells

A recent report demonstrated a requirement for BRG1 in Schwann cell differentiation by virtue of SOX10 dependent recruitment of BRG1 to the regulatory regions of genes encoding the transcription factors, OCT6 and KROX20, in myelinating sciatic nerve and in immortalized Schwann cells [11]. However, this report did not investigate whether

SOX10 recruits BRG1 to the regulatory regions of genes that encode components of myelin, such as MPZ. We performed ChIPs to assess if SOX10 also recruits BRG1 to the

MPZ locus in immortalized Schwann cells (S16) that were transfected with control siRNA or with siSOX10. Western blotting indicated that siSOX10 efficiently depleted

SOX10. Depletion of SOX10 abrogated KROX20 expression at the protein level (Fig. 5A and B) but did not affect the expression of BRG1 (Fig.5A). SOX10 depletion also abrogated KROX20 mRNA levels (Fig. 5C) and to a greater extent, MPZ and MBP mRNA levels (Fig. 5D and E).

Consistent with a previous study in myelinating sciatic nerve [43] as well as our data in

B22 cells (Fig. 4B), we detected strong enrichment of SOX10 on elements in the MPZ promoter and weaker enrichment on elements in the first intron of MPZ in these cells

(Fig. 5F). Transfection with siSOX10 significantly reduced SOX10 enrichment on both these regulatory regions. In contrast to SOX10, KROX20 was strongly associated with the intron and only weakly associated with the promoter (Fig. 5G). Mutation of SOX10 binding sites in the MPZ intron was shown not to interfere with KROX20 binding on

34 naked DNA templates [46], however it is not known whether SOX10 is required for

KROX20 to bind its recognition sites at the endogenous MPZ locus. We found that depletion of SOX10 resulted in a small but significant decrease in KROX 20 association with both the MPZ promoter and intron (Fig. 5G). However, due to the effects of SOX10 depletion on KROX20 protein levels (Fig. 5A and 5B), it is unclear whether SOX10 is directly required for KROX20 to associate with these regions or the effects are a reflection of reduced KROX20 levels. Interestingly, as we observed in B22 cells (Fig.

4A), BRG1 was highly enriched on the MPZ promoter in control cells and to a lesser extent on the intron (Fig. 5H). Occupancy of BRG1 was dependent on SOX10, as demonstrated by the significant decrease in enrichment of BRG1 on both the promoter and intron in cells that were depleted of SOX10. Thus, the enrichment profile of BRG1 closely parallels that of SOX10 on these MPZ regulatory regions and is dependent on

SOX10 expression.

Dominant negative BRG1 abrogates myelin gene expression in immortalized Schwann cells

Mice with a Schwann cell specific deletion of BRG1 display a dramatic reduction in the number of myelinating KROX20 positive cells and a decrease in the expression of myelin genes [11]. However, it was not determined from this study if the catalytic domain of

BRG1 is required for expression of these genes. In order to determine if the catalytic domain of BRG1 is required for maintenance of Schwann cell specific gene expression, we ectopically expressed a dominant negative version of BRG1 into the S16 immortalized Schwann cell line. We found that transient transfection of S16 cells with

35 dominant negative BRG1 did not decrease SOX10 or KROX20 at the protein level

(Fig.6A). Dominant negative BRG1 also did not significantly inhibit SOX10 expression at the mRNA level (Fig. 6B) and had a small but significant inhibitory effect on KROX20 mRNA levels (Fig. 6C). Expression of MPZ, and MBP was significantly inhibited by dominant negative BRG1 (Fig. 6D, E). Thus, the chromatin remodeling activity of BRG1 is likely to be required for maintenance of myelin gene expression in differentiated

Schwann cells.

DISCUSSION

It is well established that cellular differentiation is highly dependent on SWI/SNF chromatin remodeling activity to render previously silent lineage specific loci permissive for transcription [24]. Multiple studies have found that an important mechanism by which

SWI/SNF enzymes regulate lineage specific gene expression is through interactions with transcriptional regulators that likely recruit SWI/SNF enzymes to the relevant genomic sites [26,28,32,47]. Studies on muscle differentiation suggest that SWI/SNF recruitment is precisely orchestrated to establish the temporal patterning of gene expression that ultimately transforms undifferentiated cells into terminally differentiated cells [33,48].

By somewhat different mechanisms, SWI/SNF activity is also modulated during neurogenesis to regulate the timing of terminal differentiation [49]. Thus, there are likely to be lineage specific mechanisms that operate through chromatin remodeling to precisely regulate the timing of cellular differentiation. However, very little is known about these mechanisms in other lineages.

Early studies implicated SWI/SNF enzymes in neural crest differentiation and gliogenesis

[50,51]. The requirement for SWI/SNF enzymes in the specification of neural crest precursors into oligodendrocytes of the central nervous system and Schwann cells of the peripheral nervous system as well as the transcriptional interactions that regulate

SWI/SNF recruitment have only recently been elucidated [11,30,31]. During Schwann cell differentiation, the BRG1 component of the SWI/SNF complex was found to interact with NFKappaB to activate SOX10 expression and in turn to interact with SOX10 to activate the expression of the transcriptional regulators, OCT6 and KROX20 [11,30]. Our study uncovers a requirement for BRG1 in the direct regulation of genes encoding components of myelin through BRG1 interactions with SOX10. Thus, SWI/SNF enzymes are extensively required for the activation of multiple classes of genes that ultimately result in myelin gene expression in terminally differentiated Schwann cells (Fig. 7).

Master regulators of differentiation have been identified by their ability to convert heterologous cells into cells that express lineage specific genes. We found that SOX10 can activate two myelin genes, one of which is a Schwann cell specific marker.

Interestingly dominant negative BRM and BRG1 severely inhibited activation of these genes. We focused on BRG1 for the remainder of our studies because mice with conditional disruption of BRG1 exhibit myelination defects while BRM disrupted mice do not [11,30,36]. Although our data indicate that BRM may also promote myelin gene expression, the requirement for BRM is likely to be at least partially compensated by

BRG1 in vivo.

Our study in transdifferentiated B22 cells indicates that SOX10 promotes recruitment of

BRG1 to two regulatory regions of the MPZ locus. Importantly, we found that KROX20 in the absence of SOX10 cannot activate myelin gene expression, nor can KROX20 promote the recruitment of BRG1 to these two MPZ regulatory sites. This is consistent with a previous study in which KROX20-mediated activation of MPZ transcription depended on SOX10 binding sites [43]. Although KROX20 cannot promote BRG1 recruitment in the absence of SOX10, our data do not rule out that KROX20 either directly or indirectly contributes to BRG1 recruitment in conjunction with SOX10.

Alternatively, SWI/SNF mediated chromatin remodeling may be required for KROX20 to bind to cognate sites in the MPZ regulatory region if they are embedded in repressive chromatin structure. Ongoing studies are currently dedicated toward elucidating the dependency of KROX20 binding on BRG1 and the dependency of BRG1 recruitment on

KROX20 and on characterizing the chromatin structure of the MPZ regulatory region.

BRG1 recruitment to the MPZ promoter and intron was also dependent on SOX10 in immortalized Schwann cells. Interestingly, although the enrichment profile of SOX10 and BRG1is similar in B22 cells that express SOX10 and in immortalized Schwann cells that express SOX10, there appears to be greater enrichment of these factors on the MPZ promoter in Schwann cells (compare Fig. 4A and 4B and Fig. 5F and 4H). Dominant negative BRG1 dramatically inhibited SOX10 mediated activation of MPZ as well as synergistic activation of MPZ by SOX10 and KROX20 in B22 cells (Fig. 1C) but had a modest effect on MPZ expression in Schwann cells (Fig. 6D). The less dramatic effect of dominant negative BRG1 on MPZ expression in Schwann cells may reflect the transient

38 nature of dominant negative BRG1 expression and possibly a need to compete with higher levels of wild type BRG1 on the MPZ promoter. However, it is important to note that induction of MPZ expression was assayed in B22 cells, whereas maintenance of

MPZ expression was assayed in Schwann cells. Studies on the PHO and GAL genes in yeast suggest that SWI/SNF enzymes are required to increase the initial rates of transcription induction [52,53]. Thus, although BRG1 contributes to the maintenance of myelin gene expression, there may be a greater requirement for SWI/SNF mediated chromatin remodeling in promoting the initial rate of MPZ activation. In combination, these data demonstrate a direct role for BRG1 in the regulation of myelin gene expression that is dependent on SOX10.

SOX10 may be classified as a master regulator or lineage determination factor of

Schwann cell differentiation in part through its ability to promote the recruitment of

SWI/SNF enzymes and thereby activate myelin gene expression in heterologous cells.

However, since SOX10 also promotes differentiation of neural crest precursors into other lineages, including oligodendrocyte and melanocyte, the role of SOX10 as a master regulator or determination factor of Schwann differentiation is likely to be modulated by additional levels of regulation.

The transition from an immature promyelinating cell to a myelinating cell is regulated by the concerted activity of SOX10 and several other transcription factors. During myelination, SOX10 activates expression of the transcriptional regulator, KROX20 and then synergizes with KROX20 to activate genes encoding myelin components [54]. We

39 found that although KROX20 alone cannot activate MPZ or MBP expression in NIH3T3 derived cells, ectopic expression of KROX20 together with SOX10 leads to synergistic activation of MPZ and MBP in NIH3T3 derived cells, thus mimicking the myelination step during Schwann cell differentiation. Furthermore, synergistic activation of MPZ and

MBP by SOX10 and KROX20 was inhibited by dominant negative BRG1, suggesting that SWI/SNF complexes are directly required for promoting basal expression of MPZ and MBP as occurs in neural crest precursors and promyelinating Schwann cells and for activated levels of transcription as occurs in myelinating Schwann cells [44]. Thus,

BRG1 regulates myelin gene expression at multiple phases of Schwann cell differentiation.

SOX10 is utilized at multiple steps during neural crest differentiation and is required for differentiation into pro-myelinating as well as myelinating cells, in addition to neural crest differentiation into oligodendrocyte and melanocyte lineages [55]. The requirement for this lineage determination factor for late stages of Schwann cell differentiation is in contrast to the requirement for the lineage determination, factor, MYOD during late stages of muscle differentiation. MYOD is a master regulator of muscle differentiation that can activate the muscle specific gene expression program in heterologous cells

[56,57]. MYOD commits precursors to the muscle lineage and is critical for early stages of muscle differentiation, including the activation of two transcriptional regulators, myogenin and MEF2 [58]. This cascade of transcription factor activity is then critical for controlling the timing of late muscle specific gene expression in part by regulating BRG1 recruitment to the regulatory regions of late muscle specific genes [48,59]. Elucidation of

40 the role of other transcriptional regulators for the recruitment of SWI/SNF components to

Schwann cell specific loci is likely to provide insight into the mechanisms that regulate temporal patterning of gene expression during early neural crest differentiation into several different lineages as well as for myelination of Schwann cells.

By utilizing a dominant negative approach in both a reprogramming model of differentiation as well as in immortalized Schwann cells, our work strongly suggests that

SWI/SNF chromatin remodeling activity is required for SOX10 mediated activation of genes that encode myelin constituents. Future studies will investigate the types of chromatin modifications that SWI/SNF enzymes catalyze for the activation of these genes and how they impinge upon the DNA bending properties of this multifunctional

HMG domain transcriptional regulator.

Acknowledgements

ILD was supported by National Institute of Health R01(ARO59379). Benjamin

Chojnacki (University of Toledo Flow Cytometry Core Facility) for help with FACS analysis.

FIGURE LEGENDS

Figure 1: Dominant negative BRM and BRG1 inhibit SOX10-mediated activation of myelin genes.

Cell lines that express dominant negative BRM or BRG1 were either infected with a pBabe control vector or pBabe-SOX10 in the presence or absence of tetracycline and then cultured in low serum media to promote differentiation. (A) Western Blot showing expression of SOX10 in cells that were cultured in the presence and absence of tetracycline and the expression of FLAG-tagged dominant negative BRM (left) in the

H17 cell line and BRG1 (right) in the B22 cell line, when cells were cultured in the absence of tetracycline. Protein expression was detected from cell extracts and ERK1/2 was used as a loading control (B) Quantitative RT-PCR (qRT-PCR) of SOX10 target genes from pBabe or pBabe-SOX10 infected H17 (left) and B22 (right) cells. KROX20,

MPZ, and MBP expression was normalized to expression of RPL7. The data are representative of at least four experiments and are the average of two independent experiments performed in triplicate. Standard error bars and statistical significance are shown (**p<0.01).

Figure 2: Dominant negative BRG1 inhibits synergistic activation of myelin genes by

SOX10 and KROX20.

Cell lines that express dominant negative BRG1 were infected with an empty vector, pBabe-SOX10, pBabe-KROX20, or pBabe-SOX10 together with pBabe-KROX20 in the presence or absence of tetracycline and then cultured in low serum media to promote

differentiation. (A) Western Blot showing expression of SOX10 and KROX20 in the presence and absence of tetracycline and the expression of FLAG-tagged dominant negative BRG1 in the B22 cell line when cells were cultured in the absence of tetracycline. Protein expression was detected from nuclear extracts and a non-specific band was used as a loading control. (B) Quantitative RT-PCR (qRT-PCR) of SOX10 target genes from pBabe or pBabe-SOX10, pBabe-SOX10, pBabe-KROX20, or pBabe-

SOX10 together with pBabe-Krox20 infected cells.

Expression of MPZ and MBP was normalized to expression of RPL7. The data are representative of greater than three experiments and are the average of two independent experiments performed in triplicate. Standard error bars and statistical significance are shown (**p<0.01).

Figure 3: FACS analysis of differentiated B22 cells.

B22 cells were infected with an empty vector, pBabe-SOX10, pBabe-KROX20, or pBabe-SOX10 together with pBabe-KROX20 in the presence or absence of tetracycline and differentiated in low serum media. Propidium iodine stained samples were FACS sorted and analyzed using Cell Quest Pro (BD Biosciences). The data are the average of three independent experiments. Standard error bars are shown.

Figure 4: BRG1 is recruited to the MPZ promoter as a result of SOX10 mediated differentiation

Chromatin immunoprecipitations (ChIPs) were performed with control IgG, antisera to

BRG1, or an antibody to SOX10 on chromatin from B22 cells that were infected with

empty vector (EV), KROX20, or SOX10 retrovirus and cultured as described in Fig. 1.

Enrichment at the MPZ promoter and intron is relative to control IgG and normalized to a control region, SCN2A1. There was minimal variation in the ChIP signal at the SCN2A1 region. (A) Detection of BRG1 interactions with the MPZ promoter and intron. (B)

Detection of SOX10 interactions with the MPZ promoter and intron. This data are representative of at least three experiments and are the average of two independent experiments that were assayed four or more times, each in triplicate. Standard errors bars and statistical significance are shown (**p<0.01, * p<0.05).

Figure 5: SOX10 is required to recruit BRG1 to the MPZ promoter in S16 Schwann cells.

(A) Cell extracts were prepared from S16 cells that were transfected with siControl or siSOX10 and subjected to Western analysis with antibodies to SOX10, KROX20, and antisera to BRG1 (The starred top band (*) in the KROX20 Western may be a non- specific band. It was not used in the quantitation of KROX20 expression shown in Fig.

5B. ERK1/2 is shown as a loading control. B. Quantitation of KROX20 protein levels relative to ERK1/2 protein levels by Image J software. The data are the average of three independent experiments. Standard error bars and statistical significance are shown

(**p<0.01). C-E. Quantitative RT-PCR (qRT-PCR) of KROX20, MPZ, and MBP mRNA levels. F-H.ChIPs were performed with control IgG, antibodies to SOX10 or KROX20, or antisera to BRG1 on chromatin from S16 cells that were transfected with siControl or siSOX10. Enrichment on the MPZ promoter and intron is relative to control IgG and normalized to a control region, Ig2a enhancer. There was minimal variation in the ChIP

signal at the Ig2A enhancer. (F) Detection of SOX10 interactions with the MPZ promoter. (G) Detection of KROX20 interactions with the MPZ promoter. (H) Detection of BRG1 interactions with the MPZ promoter. The data are representative of three independent experiments that were assayed three times. Standard errors bars and statistical significance are shown (**p<0.01, *p<0.05)

Figure 6: Dominant negative BRG1 inhibits expression of SOX10 target genes in S16

Schwann cells.

S16 cells were transfected with GFP (not shown), empty vector (EV), or pBabe-dominant negative BRG1 (dnBRG1). Transfection efficiency was monitored by GFP and was determined to be approximately 30%. (A) Cell extracts were prepared and subjected to

Western analysis with antibodies to the Flag epitope, SOX10, or KROX20. Tubulin is shown as a loading control. Quantitative RT-PCR (qRT-PCR)of SOX10 (B), KROX20

(C), MPZ (D), and MBP (E) from empty vector (EV) or pBabe-dnBRG1 transfected cells. Expression of each gene was normalized to that of 18S rRNA. The data are the average of three independent experiments performed in triplicate. Standard error bars are shown (**p<0.01).

Figure 7: The requirement for BRG1 during Schwann cell differentiation and myelination.

A model illustrating the stepwise requirement for BRG1 during Schwann cell differentiation and myelination based on the current study as well as two recent studies

[11, 30].

FIGURES

Fig.7

Manuscript

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CHAPTER- 4

SWI/SNF enzymes are downstream modulators of Alpha-MSH

mediated melanocyte differentiation and act as transcriptional co-

factors for both MITF and SOX10 in regulating melanocyte

specific gene expression

ABSTRACT

Microphthalmia -Associated Transcription Factor (MITF) is the master regulator of melanocyte differentiation and activates the expression of genes important for melanin synthesis and melanocyte function [123, 124]. MITF also regulates aspects of melanoma tumorigenicity [47, 114, 120, 124, 125]. The alpha melanocyte stimulating hormone

(Alpha-MSH) promotes melanocyte differentiation and protects cells from UVR induced damage [125-128]. Importantly, Alpha-MSH activated gene expression is mediated through a transcriptional mechanism involving MITF. However, transcriptional activation during cellular differentiation and in response to many environmental cues requires epigenetic changes that convert repressive chromatin structure to a transcriptionally permissive conformation. We previously determined that MITF interacts with SWI/SNF chromatin remodeling enzymes to activate melanocyte specific gene expression [68, 93].

SWI/SNF enzymes are multi-subunit complexes that alter chromatin structure in an ATP dependent manner and play important roles in regulating gene expression. Components of the complex are required for stem cell plasticity as well as for differentiation into multiple cell lineages [72, 75, 82, 85, 87]. We tested the hypothesis that SWI/SNF enzymes are downstream modulators of Alpha-MSH signaling and cooperate with MITF and SOX10 to epigenetically regulate the expression of pigmentation specific genes.

Our data indicate that Alpha-MSH promotes SWI/SNF recruitment and histone post- translational modifications on MITF target genes. Moreover siRNA mediated depletion of BRG1/BRM reduced the observed transcriptionally permissive histone modifications and compromised the pigmentation as compared to control cells. This suggests that

SWI/SNF enzymes are downstream modulators of Alpha-MSH mediated melanocyte differentiation. We have also found that MITF requires SOX10 to activate high levels of melanocyte specific gene expression. SOX10 also exhibits SWI/SNF dependent activation of melanocyte specific target genes. Thus SWI/SNF enzymes act as transcriptional partners for both MITF as well as SOX10.

INTRODUCTION

The ability of skin to turn dark when exposed to sunlight comprising of UV radiations is called tanning. Tanning response is the first line of defense against the harmful effects of

UV radiation and subsequent skin cancer. One of the major mechanisms of initiation of the tanning response is through the activation of melanocortin receptor 1 (MC1R) by the

Alpha-MSH [128, 129]. The MC1R is a seven transmembrane domain G-protein coupled

receptor. Binding of this receptor by Alpha-MSH activates the G-protein receptor associated adenylyl cyclase that leads to an increase in cyclic-AMP which acts a second messenger, finally leading to transcriptional activation of MITF [130]. MITF then leads to transcriptional activation of the pigmentation genes, ultimately leading to increased melanin content visible as tanning. Certain genetic variants of the MC1R locus and

Alpha-MSH gene lead to a tan resistant red hair phenotype that is associated with significantly higher risk of skin cancer[125, 129, 131, 132]. Also, synthetic Alpha-MSH analogs are being clinically evaluated as a means of inducing pigmentation mediated protection against UV in light skin tan resistant individuals [133, 134]. Tanning occurs in response to an external stimulus. Physiological responses to external stimuli, leading to either gene activation or repression usually requires changes in chromatin. Yet the effect of Alpha-MSH induced pigmentation gene expression on chromatin structure has not been explored. Thus, analyzing the mechanism of pigmentation mediated by Alpha-

MSH, through MITF might help in design of additional therapeutic targets that would reduce risk of UV induced damage.

MITF also known as the master regulator of melanocyte differentiation, directly regulates most of the key enzymes required for activating melanin synthesis and its distribution

[113, 124]. MITF activation of pigmentation genes is dependent on SWI/SNF chromatin remodeling enzymes [68, 93]. MITF physically interacts with BRG1, the core ATPase and multiple subunits of the SWI/SNF complex, and dominant negative forms of the

BRG1/BRM inhibit MITF mediated pigmentation [68].

Another factor that emerged in the literature as an important regulator of pigmentation is known as the SRY-like HMG box10 (SOX10) transcription factor [42]. Humans and mice models having characteristic SOX10 mutations with normal MITF expression show visible pigmentary abnormalities and hearing loss. SOX10 in addition to MITF has been shown to transcriptionally regulate all the three major enzymes of pigmentation namely

Dct, Tyr and Trp1 thus playing an important function in melanocyte lineage [110-112,

121, 135]. SOX10 was shown to synergistically activate genes by directly binding to the enhancer elements in all the three melanocyte differentiation genes. Despite this information, the exact mechanism by which SOX10 co-regulates MITF target genes in pigmentation is yet to be well characterized. Involvement of SWI/SNF enzymes for

SOX10 was not tested in any of these studies, and this adds a novel aspect to my project.

RESULTS

Alpha-MSH induces robust pigmentation gene expression in melanoblasts.

The melanoblasts are the immature unpigmented precursors of melanocytes and have been described in the previous chapter. These cells were incubated in differentiation inducing medium and harvested for gene expression analysis at specific time points post treatment. Upon treatment with Alpha-MSH containing differentiation medium, it is observed that there is robust induction of expression of all three pigmentation enzymes required in melanin synthesis (Fig 1A-C). Moreover a visible effect on the cell pellets was also observed, where in the melanoblasts treated with differentiation inducing medium were significantly darker than the control cells (Fig. 1D). Thus Alpha-MSH can induce expression of the pigmentation enzymes as well as lead to visibly pigmented cells.

Alpha-MSH mediated melanocyte differentiation involves increased recruitment of

BRG1 to target promoters.

MITF is an established regulator of the melanocyte differentiation enzymes. Moreover previous reports from the lab have shown that MITF utilizes the SWI/SNF complex in order to activate melanocyte specific gene expression. In order to analyze role of BRG1 downstream of Alpha-MSH, we performed chromatin immunoprecipitations for BRG1 in melanoblasts incubated in differentiation inducing medium. It was observed that there was increased recruitment of BRG1 at target promoters post Alpha-MSH treatment

(Fig.2A). Normal human melanocytes are pigmented cells having base level expression of all the pigmentation enzymes. Upon performing ChIP analysis for BRG1, it was observed that BRG1 was also physically associated with the promoters of Tyr and Trp1

(Fig. 2B). Thus BRG1 is physically associated with target promoters in normal human melanocytes as well as is recruited to target promoters during Alpha-MSH mediated activation of melanocyte gene expression.

Alpha-MSH mediated melanocyte differentiation involves structural changes in chromatin leading to a more accessible chromatin conformation.

The eukaryotic genome is highly compacted and this compaction is often repressive to many cellular processes which rely on information obtained from the DNA.

Transcriptional activation is a multi-step process and requires the necessary gene regulatory elements to be accessible to specific transcription factors which may then recruit the basal machinery and thereby activate transcription. Structural changes in

chromatin are often necessary for the required regulatory elements to be accessible.

Using the ability of micrococcal nuclease (MNase) which cleaves the DNA between nucleosomes, one can look for any changes in accessibility at a particular region of interest. Melanoblasts incubated in differentiation medium were harvested at specific time points, the chromatin was isolated and subjected to MNase digestion and the Trp1 promoter region was analyzed for changes in accessibility. A progressive increase in accessibility at the Trp1 promoter as the melanoblasts undergo differentiation was observed (Fig. 3). This indicates that Alpha-MSH mediated differentiation involves changes in chromatin landscape making it more accessible to transcription.

Alpha-MSH leads to increased enrichment of transcriptionally permissive histone modifications.

Activation of gene expression is often also associated with changes in histone modifications. We then performed chromatin immunoprecipitations for transcriptionally permissive histone modification, H3K4-trimethylation (H3K4me3) and transcriptionally repressive modification, H3K9 trimethylation (H3K9me3) in melanoblasts which were either control or treated with Alpha-MSH containing differentiation medium. It was observed that Alpha-MSH led to increased enrichment of H3K4me3 at both Tyr and Trp1 promoters (Fig. 4A). Turning on expression also requires removal of previously established repressive modifications, and Alpha-MSH mediated gene activation included a significant reduction of the repressive mark on both Tyr and Trp1 promoters (Fig. 4B).

Thus Alpha-MSH mediated melanocyte specific gene expression involves characteristic changes in chromatin modifications to make it transcriptionally permissive.

Increased enrichment of transcriptionally permissive modifications is observed in an amelanotic melanoma cell line after ectopic expression of BRG1.

Sk-MEL5 is a BRG1 deficient melanoma cell line that is unpigmented even while retaining MITF expression and activity, suggesting that pigmentation was inhibited downstream of MITF. Stable Sk-MEL5+BRG1 cells were generated by retroviral transfection of epitope tagged BRG1 [93]. Ectopic expression of BRG1 in these cells enhanced pigmentation and increased expression of Tyr and Trp1, along with increased recruitment of BRG1 to these promoters [93]. Also, increased H3K4me2 levels were observed in the Sk-MEL5 BRG1 cells upon ectopic BRG1 expression (Fig. 5A). A similar reduction in transcriptionally repressive mark H3K9me3 was also observed (Fig.

5B). Thus ectopic expression of BRG1 in a melanoma cell line produces a similar effect on gene expression and histone modifications as that observed by Alpha-MSH mediated melanocyte differentiation.

Brg1 and/or Brm are required for establishment of the transcriptionally permissive histone modification H3K4me3.

BRG1 and BRM were depleted in Melba cells by means of shRNA targeting a common region of both BRG1 and BRM. Control transfected and anti BRG1/BRM transfected cells were incubated in differentiation containing medium and chromatin was isolated

24hours post incubation in differentiation medium. Chromatin immunoprecipitations were performed with antisera to BRG1 and H3K4me3. Decreased enrichment of BRG1 was observed at Trp1 promoter in cells transfected with shRNA to BRG1 and BRM

(Fig.6A). Depletion of BRG1/BRM severely compromised enrichment of the transcriptionally permissive histone modification H3K4me3 (Fig. 6B). This indicates that

BRG1 plays an essential role in the establishing the H3K4me3 modification downstream of Alpha-MSH.

SOX10 mediated activation of Tyrosinase and Trp1 is inhibited by dominant negative

BRG1.

SOX10 is known to directly bind to the enhancers of Tyrosinase and Trp1 and also synergizes with MITF to activate expression of these genes [110, 112, 122]. MITF was previously shown to require and recruit BRG1 containing complexes to the above gene promoters. To test the requirement for BRG1 in SOX10 mediated activation of Trp1 and

Tyrosinase, we utilized the B22 cells which have been described in the previous sections.

SOX10 and MITF were ectopically expressed as previously described. SOX10 shows

BRG1 dependent activation of Trp1 (Fig. 7A) and its synergy with MITF to activate Trp1 and Tyrosinase is also dependent on BRG1 (Fig. 7A and 7B). Thus BRG1 acts as a transcriptional partner of both MITF and SOX10 in order to activate expression of key melanocyte differentiation genes.

Time-course analysis indicates that SOX10 binding at TRP1 enhancer precedes binding of MITF at the promoter during Alpha-MSH mediated melanocyte differentiation.

MITF, SOX10 and SWI/SNF co-regulate expression of Trp11in response to Alpha-MSH.

MITF has binding sites in the promoter region of Trp1 and SOX10 has binding sites in the enhancer region of Trp1 (Fig. 8A). The promoter and the enhancer regions were

included in analysis of binding by MITF, SOX10 and BRG1. In order to understand the dynamics of recruitment and binding of these factors in response to Alpha-MSH, we performed chromatin immunoprecipitations with antisera to BRG1, MITF and SOX10 over a time period post treatment with Alpha-MSH. MITF recruitment at the Trp1 promoter occurs at 12 hours post treatment with Alpha-MSH and increases modestly at

24 hours post treatment (Fig. 8B). MITF is known to recruit BRG1 to the Trp1 promoter and BRG1 recruitment at the Trp1 promoter occurs at 12hours post treatment similar to

MITF (Fig. 8C). SOX10 ChIP indicates that it’s the earliest factor to come and associate with the Trp1 enhancer, since it is already present at the Trp1 enhancer and is further enriched at 6 hours post treatment (Fig. 8D). This indicates that SOX10 binding is independent of MITF and MITF mediated recruitment of BRG1 at the promoter. BRG1 enrichment was also observed at the enhancer at a later time period of 12hours. Since there was no MITF enrichment observed at the promoter, and BRG1 does not have any sequence specificity, it is possible that SOX10 might be recruiting BRG1 at the enhancer albeit with a slight delay.

SOX10 depletion leads to reduced accessibility of chromatin at the Trp1 enhancer.

SOX10 enrichment at 12 hours is lower than that observed at 6 hours post Alpha-MSH treatment. Increased enrichment of BRG1 occurs at 12hours post treatment. In order analyze the function of SOX10 with respect to BRG1 and in the context of chromatin, we performed chromatin accessibility analysis in melanoblasts depleted of SOX10 by siRNA transfection. Western blotting indicates that there was efficient depletion of SOX10 (Fig.

9A). Chromatin accessibility assay was performed in melanoblasts transfected either with

control siRNA or siSOX10 and treated with Alpha-MSH for 12 hours. Accessibility at the Trp1 promoter was found to be significantly less than that observed at the enhancer.

Furthermore siSOX10 containing cells exhibit significantly lower enrichment at the enhancer (Fig. 9B). This suggests that SOX10 can probably bind to the enhancer without the help of BRG1 but may require it for bringing about structural changes to chromatin in order to make it more accessible. In order to recruit the BRG1 containing complex,

SOX10 would require mediating physical interactions with BRG1 directly or through the

BAFs. Upon performing co-immunoprecipitation analysis SOX10 was found to physically interact with BRG1 (Fig. 9C). This suggests that SOX10 interacts with the

SWI/SNF ATPase BRG1 and may play a role in recruiting the complex to the Trp1 enhancer to bring about changes in chromatin accessibility.

MITF is required for recruitment of BRG1 specifically to the Trp1 promoter but not the enhancer.

MITF mediated recruitment of BRG1 to pigmentation genes was previously demonstrated in fibroblasts transformed to melanocytes and in melanoma cells. In order to characterize this phenomenon in a more physiologically significant model, we analyzed recruitment of BRG1 by MITF in melanoblasts treated with Alpha-MSH. We performed siRNA mediated depletion of MITF and performed ChIP analysis using antisera to MITF, BRG1 and H3K4me3 to determine the role of MITF in recruitment.

Depletion of MITF leads to a significant reduction of MITF interaction with the Trp1 promoter (Fig. 10A). Furthermore depletion of MITF significantly compromised BRG1 recruitment at the Trp1 promoter (Fig. 10B). Also similarly compromised enrichment

occurred with respect to a transcriptionally permissive histone modification, H3K4me3

(Fig. 10C). BRG1 and BRM depletion was previously shown to compromise this specific modification, thus indicating that reduced BRG1 recruitment due to depletion of MITF leads to the observed reduction in H3K4me3 modification. Also depletion of MITF and its effect on BRG1 recruitment was found to be specific to the TRP1 promoter, since it did not significantly affect BRG1 recruitment to the Trp1 enhancer (Fig. 10D) thus further suggesting that SOX10 independently mediates recruitment of BRG1 to the enhancer.

DISCUSSION

Tanning that occurs in response to UV exposure is one of the most conspicuous examples of human adaptation to an external stimulus. Synthesizing melanin is one of the central functions of a mature differentiatied melanocyte. Physiologically a critical inducer of melanocyte specific pigmentation is Alpha-MSH. Perturbed Alpha-MSH signaling is associated with a significantly increased risk of developing melanoma. With this effect multiple clinical trials are exploring synthetic analogs of Alpha-MSH in order to provide increased pigmentation in fair skinned individuals as well as patients suffering from photosensitizing disorders[133, 136, 137].

MITF is known to be a downstream effector of Alpha-MSH mediated melanocyte differentiation. Also MITF was shown to require the SWI/SNF chromatin remodeling complex to activate target genes in transformed fibroblasts as well as melanoma cell lines

[68, 93]. Furthermore BRG1 was shown to be required for a subset of MITF target gene

expression in melanoma cell lines. Transformed fibroblasts and melanoma cell lines may not perfectly recapitulate the mechanisms of gene expression as that of normal melanocytes. Thus there is a need to reaffirm the observations made in previous studies in a more physiologically relevant model and also link these disparate observations into a holistic physiological cascade thus making it clinically relevant.

Our current studies demonstrate application of Melba cells which are unpigmented precursors of melanocytes that differentiate to pigmented melanocytes upon treatment with Alpha-MSH. Robust activation of pigmentation enzymes was observed with visible changes in pigmentation of cells treated with differentiation inducing medium.

Furthermore we characterized the effects of Alpha-MSH signaling on chromatin of target genes, where in increased accessibility was observed in Alpha-MSH treated cells.

Increased enrichment of transcriptionally permissive modifications was also observed as a result of Alpha-MSH mediated differentiation. Similar pattern of histone modification changes were observed upon ectopic expression of BRG1 in an amelanotic melanoma cell line. The characteristic increase in transcriptionally permissive histone modifications was abrogated upon depletion of BRG1 and BRM, the two ATPases of the SWI/SNF complex, thus suggesting that SWI/SNF complex plays a role in mediating downstream epigenetic modifications in response to Alpha-MSH.

MITF was previously shown to interact with and recruit SWI/SNF complexes to target promoters. ChIP analysis in MITF depleted melanoblasts treated with differentiation inducing medium show significantly compromised recruitment of BRG1 to a target

promoter. BRG1 interactions with the Trp1 enhancer were not significantly affected by

MITF depletion. SOX10 is another transcription factor that is required in addition to

MITF to achieve optimum gene expression of all three pigmentation enzymes. SOX10 binding elements are all present in the upstream enhancer region of the three genes.

SOX10 synergizes with MITF to activate Trp1 expression.

Further analysis of SOX10 mediated activation by means of dominant negative cell lines indicated that SOX10 activity is SWI/SNF dependent. Thus the SWI/SNF complex functions as a transcriptional cofactor for both MITF and SOX10. Time course ChIP analyses indicate that SOX10 is associated with the Trp1 enhancer prior to treatment with differentiation inducing medium, and is further enriched upon treatment. MITF recruitment occurs at the promoter at later time points and coincides with enrichment of

BRG1 at the promoter. BRG1 enrichment at the enhancer occurs at a later time point as compared to SOX10 enrichment. This pattern of recruitment indicates that SOX10 binding at the Trp1 enhancer is independent of MITF and/or SWI/SNF binding.

SOX10 depletion analyses leads to reduced chromatin accessibility at the enhancer, thus suggesting that SOX10 probably requires the SWI/SNF chromatin remodeling activity to mediate structural changes in the chromatin thus making it more accessible. Further strengthening this possibility is the observed physical interaction of SOX10 with BRG1 in control and differentiation induced melanoblasts. The observation of SOX10 and

MITF synergy is known since long, an exact mechanism of the same remains elusive.

The SOX10 binding site in the enhancer is approximately 15kb away from the promoter.

More often than not communication between enhancer and promoters so distant from each other involves chromatin looping and is considered as a fairly prevalent mode of enhancer function [138, 139]. Furthermore SOX10 has been known to possess DNA bending abilities and also the SWI/SNF complex has been implicated in meeting the energy requirements to mediate chromatin looping [140, 141]. Also, both SOX10 and the

SWI/SNF complex are known to interact with the basal transcriptional machinery [85, 92,

142]. Further analysis including ChIPs over a time course for RNA pol II, and chromosome capture (3C) experiments would help in dissecting if chromatin looping is indeed at play.

There are multiple mechanisms of synergy between two factors, one of which is to mediate co-operative binding. In case of MITF and SOX10 this particular mechanism seems to be unlikely since SOX10 is present early on at the Trp1 enhancer, and moreover depletion of SOX10 did not significantly affect chromatin accessibility at the promoter.

In order to disprove of this mechanism, further analysis would be required with respect to

MITF binding at the promoter in SOX10 depleted cells. Also till date there are no reports of any physical contacts between MITF and SOX10. The mechanism of synergy could also include individual effects on multiple stages of transcription, such as formation and stability of the PIC, and rate of elongation. Further analysis by means of nuclear run-on assays and ChIPs for PIC complex factors would help shed light on the exact mechanism of synergy between MITF and SOX10.

FIGURE LEGENDS

Figure 1: Alpha-MSH induces robust pigmentation in (Melba) melanoblasts

Unpigmented immature melanoblast cell line Melba was incubated in differentiation inducing medium containing NDP-MSH which gets processed as the active form Alpha-

MSH. The cells were harvested for qRT-PCR analysis at fixed time intervals post treatment. qRT-PCR of Tyrosinase, Trp1 and Trp2, the three major enzymes of melanin synthesis (A-C). Expression was normalized to expression of RPL7. The data are average of two independent experiments performed in triplicate. Standard Error bars and statistical significance are shown (*<0.05). Visible changes in pigmentation are seen in cells pelleted from incubation in differentiation inducing medium as opposed to incubation in normal growth medium (D).

Figure 2: Increased recruitment of BRG1 occurs at target promoters post Alpha-MSH treatment.

Melba cells were incubated in differentiation inducing medium for 48 hours and harvested for chromatin immunoprecipitation (ChIP). Enrichment of BRG1 at Tyrosinase and TRP1 promoters in control cells and Alpha-MSH treated cells (A). Enrichment of

BRG1 at Tyrosinase and Trp1 promoters in normal human melanocytes (B).

Figure 3: Alpha-MSH treatment promotes a more accessible chromatin conformation.

Melanoblasts were incubated in differentiation inducing medium for fixed time interval, chromatin was isolated and subjected to Micrococcal nuclease (MNase) digestion for

specific time periods, digested material was precipitated, purified and subjected to qRT-

PCR analysis. Trp1 promoter exhibits increasingly accessible chromatin over course of

Alpha-MSH induced melanocyte differentiation. Trp1 promoter signal was normalized to mouse IgH enhancer control region. The data are average of two independent experiments performed in triplicate. Standard error bars and statistical significance are shown (*<0.05).

Figure 4: Alpha-MSH leads to an increase in transcriptionally permissive chromatin modifications.

Chromatin immunoprecipitations (ChIPs) were performed with control IgG, antisera to

H3K4me3 and H3K9me3 on chromatin from melanoblasts treated with differentiation inducing medium for 48 hours. Enrichment of H3K4me3 and H3K9me3 at Tyrosinase and Trp1 promoter is normalized control antibody and to mouse IgH enhancer. Detection of enrichment of histone modification H3K4me3 at Tyrosinase and Trp1 promoters (A).

Detection of H3K9me3 enrichment at Tyrosinase and Trp1 promoters (B). The data are representative of four independent experiments and are average of two independent experiments done in triplicates. Standard Error bars and statistical significance are shown

(*<0.05).

Figure 5: Ectopic expression of BRG1 promotes similar transcriptionally permissive histone modifications at target promoters in an amelanotic melanoma cell line.

Sk-Mel5 cell line is a human unpigmented melanoma cell line, lacking BRG1 and is described in [93]. ChIP analysis was performed in Control transfected and BRG1

transfected cells with antisera to H3K4me2 and H3K9me. Enrichment at target promoters was normalized to enrichment observed at the CD25 control region. Increased enrichment of H3K4me2 is observed at Tyrosinase and Dopachrome tautomerase (DCT/ TRP2) in cells containing BRG1 (A). Similarly decreased enrichment of transcriptionally repressive H3K9me3 mark is observed at target promoters in cells expressing BRG1 (B).

The data are average of two independent experiments performed in triplicate. Standard error bars are shown.

Figure 6: BRG1 and/or BRM are required for establishing the transcriptionally permissive H3K4me3 modification at the Trp1 promoter

BRG1 and BRM were depleted by means of shRNA as described previously [93]. Cells transfected with control and shBRG1/BRM were incubated in differentiation inducing medium at 48 hours post transfection. Cells were harvested for chromatin immunoprecipitation at 24 hours post incubation in differentiation inducing medium.

ChIPs were performed with antisera to BRG1 and H3K4me3. Decreased enrichment of

BRG1 is observed in cells transfected with shRNA to BRG1/BRM (A). Cells depleted of

BRG1/BRM show significantly reduced H3K4me3 enrichment at Trp1 promoter (B). The data are average of two independent experiments performed in triplicate. Standard error bars and statistical significance are shown (*<0.05).

Figure 7: Dominant negative BRG1 inhibits SOX10 alone and SOX10-MITF mediated synergistic activation of melanocyte specific genes.

Cell lines that express dominant negative BRG1 were either infected with empty vector control, pBabe-MITF, pBabe-SOX10 or pBabe-SOX10 together with pBabe-MITF in the presence or absence of tetracycline and then cultured in low serum media to promote differentiation. Tyrosinase expression and Trp1 expression was normalized to expression of Rpl7. SOX10 exhibits BRG1 dependent activation of Trp1, and its synergistic activity

(log scale) with MITF in mediating Trp1 expression is also dependent on wild type

BRG1 (A). Similarly SOX10- MITF synergy in mediating Tyrosinase expression also shows requirement of wild type BRG1 (B). The data are average of two independent experiments performed in triplicate. Standard error bars and statistical significance are shown (*<0.05).

Figure 8: Time course analysis of MITF, BRG1 and SOX10 binding at the Trp1 promoter and enhancer

Melba cells are incubated in differentiation inducing medium and harvested at specified time intervals for ChIPs. ChIPs were performed with antisera to MITF, BRG1 and

SOX10. Schematic of the Trp1 gene shows the binding sites of MITF in the promoter and that of SOX10 in the distal enhancer (A). Detection of MITF interactions with the Trp1 promoter and enhancer (B). Detection of BRG1 recruitment to the Trp1 promoter and enhancer (C) and detection of SOX10 interaction with the Trp1 enhancer and promoter

(D). The data are representative of at least four independent experiments and are an average of two independent experiments performed in triplicates. Standard error bars are shown.

Figure 9: Depletion of SOX10 compromises chromatin accessibility at Trp1 enhancer and physically interacts with BRG1.

SOX10 was depleted in Melba cells using siRNA approach. Control transfected and cells transfected with siSOX10 were incubated in differentiation inducing medium for 12 hours. Chromatin was isolated and subjected to micrococcal nuclease digestion (MNase) and region of interest was probed with specific primers. Trp1 signal was normalized to mouse IgH enhancer control region. Western blotting analysis indicates efficient depletion of SOX10 (A). Changes in chromatin accessibility at the Trp1 promoter and enhancer, in control and siSOX10 cells (B). The data are average of two independent experiments performed in triplicate. Standard error bars and statistical significance are shown (*<0.05). Co-immunoprecipitation analysis indicates physical interaction between

SOX10 and BRG1 (C).

Figure 10: MITF is essential for BRG1 recruitment and increased enrichment of

H3K4me3 on the Trp1 promoter.

MITF was depleted in Melba cells using siRNA transfections. Control transfected and siMITF transfected cells were incubated in differentiation inducing medium for 24 hours.

Chromatin was isolated and subjected to ChIP analysis. ChIPs were performed with antisera to BRG1, MITF and H3K4me3. MITF depleted cells show decreased enrichment of MITF at the Trp1 promoter (A). MITF depleted cells show significantly compromised enrichment of BRG1 on the Trp1 promoter (B). Similarly reduced enrichment of

H3K4me3 modification is observed in MITF depleted cells (C). MITF depletion does not significantly affect BRG1 recruitment to the Trp1 enhancer (D). Data are average of two experiments performed in triplicate. Standard error bars and statistical significance are shown (*<0.05).

FIGURES

A Fig. 1

C Fig. 1

D Alpha-MSH

0hr 48hr

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Fig. 3

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A Fig. 5

Performed by Dr. de la Serna

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SOX10 BRG1

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CHAPTER- 5

MITF preferentially directs the bi-potent precursor towards

melanocyte specific gene expression

Introduction

A major population of post-natal melanocytes in the skin is derived from the SCP. The

SCP is a bi-potent precursor that has the potential in-vivo to differentiate into either the

Schwann cell lineage or the melanocyte lineage [18, 19, 32]. The SWI/SNF complex is essential to obtain expression of key target genes of both lineages [68, 93, 143, 144].

Moreover the transcription factor SOX10 is also known to be an important regulator of the target genes of both Schwann cells and melanocytes. The SCP precursor expresses

SOX10 and its expression is maintained throughout Schwann cell differentiation as well as melanocyte differentiation [42, 145]. MITF is the earliest marker of the melanocyte precursor, the melanoblast, and is considered as the master regulator of melanocytes

[124]. Most of the factors known to induce MITF expression are present in the neural crest cell precursors as well as the SCP, yet the SCP does not express MITF, but instead has high expression of FoxD3 [146]. FoxD3 is a winged helix transcription factor expressed in early migrating neural crest cells as well as in glial and neural precursors

[146].

FoxD3 expression is downregulated in melanoblasts but is retained in Schwann cells

[147]. Ectopically introduced FoxD3 was found to repress melanogenesis and down regulate MITF expression, leading to trans-differentiation of the cells to a glial phenotype

[146]. Thus MITF levels are turned on during the formation of the melanoblast, whereas

MITF expression is inhibited in the Schwann cell lineage. Yet the functional significance of MITF inhibition has not been analyzed till date.

RESULTS

Melanocyte differentiation program inhibits Schwann cell gene expression.

Melanoblasts are the immature precursors of melanocytes. The Melba cell line is a clonal cell line of mouse melanoblasts isolated from new born mouse epidermis. This cell line is well characterized and is used as a model of melanocyte differentiation [148-150]. The

Melba cells are responsive to treatment with the pro-melanocyte differentiation hormone

(Alpha-MSH) which induces differentiation of melanoblasts to pigmented mature melanocytes. Upon incubating the cells in a differentiation inducing medium, increased expression of MITF is observed (Fig. 1A), with a concomitant reduction in expression of the bonafide Schwann cell marker gene MPZ (Fig. 1B). Thus melanocyte differentiation involves increased expression of MITF and repression of Schwann cell specific gene expression.

MITF depletion in melanoblasts leads to increased expression of Myelin Protein Zero.

Reports in literature suggest that during glial differentiation MITF expression is inhibited.

Our results indicate that during melanocyte differentiation MITF expression increases in

the melanoblasts and concomitantly expression of glial marker gene MPZ is reduced. In order to determine if changes in MITF expression are functionally significant to glial differentiation, we performed siRNA mediated knockdown of MITF in melanoblasts treated with differentiation inducing media. Western blotting shows that siMITF efficiently depleted MITF in the melanoblasts (Fig. 2A). Melanoblasts with depleted

MITF exhibited significantly up-regulated expression of MPZ (Fig. 2B). This data combined with evidence from the literature suggests that MITF plays an inhibitory role in glial differentiation, and hence MITF expression is downregulated during Schwann cell differentiation so as to promote formation of differentiated Schwann cells.

MITF inhibits the ability of SOX10 to promote expression of the glial maker MPZ.

SOX10 is a critical regulator of multiple stages of glial development and Schwann cell differentiation. It directly regulates MPZ and several other key target genes required for

Schwann cell differentiation. Downregulation of MITF was co-related with significant upregulation of MPZ. MPZ is directly regulated by SOX10 and hence to further analyze the effect of MITF downregulation on MPZ expression, we utilized the B22 cell model which has been previously described in Chapter 2. Ectopic introduction of SOX10 in presence of wild type BRG1 lead to robust activation of MPZ expression. Upon ectopic expression of MITF in addition to SOX10 and wild type BRG1, the previously observed upregulation of MPZ was severely compromised (Fig. 3). This suggests that MITF inhibits the ability of SOX10 to up-regulate MPZ expression.

MITF reduces the recruitment of BRG1 and enrichment of H3K4me at the MPZ promoter.

It was shown previously that MITF interacts with BRG1 and multiple BAFs of the

SWI/SNF complex [68, 93]. MITF was also shown to recruit BRG1 to specific targets required for melanocyte differentiation. Similarly experiments done in the previous chapter indicate that SOX10 also requires the SWI/SNF complex to activate gene expression of MPZ, a key target gene in Schwann cell differentiation. Hence we wanted to analyze if MITF was playing a role in mediating availability of BRG1 at the MPZ promoter. We performed siRNA mediated depletion of MITF in melanoblasts both untreated and treated with differentiation inducing medium. Chromatin immunoprecipitations show a modest increase in enrichment of MITF at the MPZ promoter in melanoblasts treated with differentiation inducing medium as opposed to untreated cells. MITF depleted cells show a modest reduction in MITF at the promoter

(Fig. 4A). MITF depletion leads to a significant increase in BRG1 recruitment at the

MPZ promoter (Fig. 4B) as well as increased enrichment of the transcriptionally permissive histone mark H3K4me (Fig. 4C). MITF depletion did not have any significant effect on SOX10 enrichment (Fig. 4 D). This suggests that MITF may play a role in

BRG1 availability at the MPZ promoter and further affect the transcriptionally permissive modifications leading to repression of MPZ.

DISCUSSION

The bipotency of SCPs to give rise to Schwann cells and melanocytes, coupled with their high plasticity and their occurrence in adult tissues has tremendous implications in therapy for neurodegenerative and pigmentary disorders. The SWI/SNF complex has been implicated in regulation of multiple stages of neural crest development, including neuronal differentiation, differentiation of Schwann cell as well as melanocyte differentiation [68, 79, 93, 143, 144]. The factors regulating the fate choice of the SCP towards a specific lineage are still unclear. Moreover in-vitro culture of melanoblasts as well as SCPs indicates that these cells can be made to differentiate into fairly diverse cell types [22, 23, 35].

SOX10 is a highly versatile factor expressed very early on in neural crest cells and is a critical regulatory factor for expression of genes belonging to both Schwann cells and melanocytes. MITF on the other hand is exclusively up regulated in melanoblasts and suppressed during Schwann cell differentiation[146]. Modulation of MITF expression in melanoblasts and SCPs was found to affect the differentiation of these cell types.

Mechanistic information about the effects of MITF on Schwann cell differentiation was scarce in these above reports.

Melanoblast differentiation into melanocytes involves increased MITF expression, whereas significantly reduced Schwann cell gene expression was observed. Moreover depletion of MITF leads to a robust activation of MPZ suggesting that MITF is inhibitory to Schwann cell gene expression. Further mechanistic insight is provided by our studies

utilizing the transformed fibroblasts, which indicate that MITF inhibits the ability of

SOX10 to transcriptionally activate Schwann cell specific MPZ expression.

Chromatin immunoprecipitation analysis indicates that MITF is physically enriched at

Schwann cell gene targets as the melanoblast progresses towards melanocyte differentiation. MITF is a basic helix-loop-helix leucine zipper transcription factor that recognizes highly specific sequences with respect to melanocyte genes, known as M- boxes. Till date there have been no reports indicating the presence of such sequences at

Schwann cell gene targets and hence further analysis would be essential to understand if

MITF recognizes similar sequences in Schwann cells or whether it binds to any unique sequences.

BRG1 has been shown to be required by SOX10 to activate MPZ expression. ChIP analysis in melanoblasts transfected with siMITF reveals increased association of BRG1 with the MPZ promoter as compared to melanoblasts retaining MITF. Moreover MITF depleted melanoblasts also showed a significant increase in the transcriptionally permissive histone modification H3K4me3. MITF depletion did not affect SOX10 association with the MPZ promoter. This indicates that MITF may repress MPZ transcription by specifically sequestering the SWI/SNF complex thereby reducing the availability of BRG1 to mediate necessary chromatin changes required for gene activation.

Multiple reports in the literature indicate that SOX family factors affect the availability of other factors by sequestering them into complexes, thereby indirectly affecting

transcription [53, 151]. To our knowledge this is the first report indicating a similar phenomenon with respect to the master regulator of melanocyte differentiation MITF.

Further analysis is warranted in order to dissect whether increasing SOX10 levels or whether increased expression of BRG1 rescues MPZ expression in MITF containing cells. Understanding the mode of MITF mediated inhibition of Schwann cell expression may be of therapeutic benefits for patients suffering from pigmentary disorders, where in by transplanting Schwann cell precursors with increased MITF expression can possibly lead to formation of pigmented melanocytes. A study utilizing transplantation of

Schwann cells to treat patients suffering from paralysis is currently FDA approved[29] and thus this concept of transplanting Schwann cells for therapy is quite feasible.

FIGURE LEGENDS

Figure 1: Increased expression of MITF and decreased expression of MPZ occurs in melanoblasts treated with melanocyte differentiation promoting hormone.

The unpigmented immature precursor melanoblasts were incubated in differentiation inducing medium for 48 hours to promote melanocyte differentiation. Quantitative RT-

PCR (qRT-PCR) of MITF (A) and MPZ (B) expression normalized to RPL7 expression.

The data are average of three independent experiments. Standard error bars and statistical significance are shown (* p<0.05)

Figure 2: MITF depletion leads to increased expression of Schwann cell marker MPZ.

Melanoblasts were transfected with either control siRNA or siRNA targeting MITF.

48hours post transfection the cells were incubated in differentiation inducing medium and harvested for protein and RNA quantification 48 hours post incubation in differentiation medium. Western blot analysis indicates efficiency of MITF depletion (A). Tubulin is used as loading control. Quantitative RT-PCR (qRT-PCR) of MPZ expression normalized to RPL7 expression (B). The data are representative of at least four experiments and are the average of two independent experiments performed in triplicate.

Standard error bars and statistical significance are shown (*p<0.05).

Figure 3: MITF inhibits the ability of SOX10 to induce expression of MPZ.

The B22 cell line that expresses dominant negative BRG1 was either infected with a pBabe control vector, pBabe-SOX10, pBabe-MITF, or pBabe-SOX10 together with pBabe-MITF in the presence of absence of tetracycline and then cultured in low serum

media to promote differentiation. Quantitative RT-PCR (qRT-PCR) of MPZ expression normalized to RPL7 expression. This data are representative of at least four experiments and are the average of two independent experiments performed in triplicate. Standard error bars and significance are shown (*p<0.05).

Figure 4: MITF reduces enrichment of BRG1 and active mark H3K4me at MPZ promoter.

Chromatin immunoprecipitations (ChIPs) were performed with control IgG, antisera to

MITF, BRG1, H3K4me and SOX10 on chromatin from melanoblasts transfected with control siRNA or siRNA targeting MITF. Cells were incubated in normal serum media for 48 hours and then a batch of transfected cells were incubated in differentiation inducing medium for 24 hours. Enrichment at the MPZ promoter is relative to control

IgG and normalized to control region SCN2A1. Detection of MITF interactions with

MPZ promoter (A). Detection of BRG1 interactions with MPZ promoter (B). Detection of H3K4me histone modification at the MPZ promoter (C). Detection of SOX10 interactions with MPZ promoter (D). The data are average of two independent experiments performed in triplicate. Standard error bars and statistical significance are shown (*p<0.05).

100

FIGURES

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101

A Fig. 2

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Fig. 3

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104

CONCLUSIONS

 SWI/SNF complexes are downstream modulators of Alpha-MSH mediated

melanocyte differentiation.

 SWI/SNF complex mediate downstream epigenetic modifications required for

Alpha-MSH mediated melanocyte specific gene expression.

 SWI/SNF complex function as transcriptional co-factors for both MITF and

SOX10 in mediating melanocyte specific gene expression.

 MITF and SOX10 independently recruit the SWI/SNF complex to the promoter

and enhancer elements of a melanocyte gene respectively.

 SWI/SNF complex is also utilized by SOX10 in mediating activation of Schwann

cell gene expression.

 SOX10 mediates recruitment of BRG1 containing SWI/SNF complex at the

Schwann cell gene target promoter and intron elements.

 MITF transcriptionally represses Schwann cell gene expression.

 MITF reduces the association of BRG1 with the Schwann cell gene target

promoter

 MITF reduces the enrichment of transcriptionally permissive histone

modification, H3K4me3 at the Schwann cell target gene promoter.

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