In eukaryotic cells, a key regulatory step in target gene expression is the recognition of the core promoter by the TFIID complex. TFIID is composed of the TATA-box binding protein (TBP) and TBP-Associated Factors (TAFs) (Goodrich and Tjian, 2010; Hochheimer and Tjian, 2003; Naar et al., 2001). To understand gene regulation at the core promoter, I have been focusing on one component of the core promoter recognition complex-TAF3 that was originally identified as a subunit of the TFIID complex in HeLa cells (Gangloff et al., 2001). It was later found that, while other TFIID subunits are destroyed during myogenesis, TAF3 is selectively retained in myotubes in a specialized complex with TBP-related factor 3, TRF3 (Deato and Tjian, 2007) and the sub-nuclear localization of TAF3 serves as another potential mechanism to influence transcription during myogenesis (Yao et al., 2011). Intriguingly, TAF3 recognizes trimethylated histone H3 lysine 4 (H3K4me3) (Vermeulen et al., 2007), which is associated with actively transcribed genes and also with silent developmental genes that are poised for activation upon ES cell differentiation (Mikkelsen et al., 2007).

In our recent studies, surprisingly it was found that TAF3 is highly enriched in ES cells. More importantly, during in vitro EB formation, which mimics early embryogenesis, TAF3 protein levels were selectively reduced. However, the levels of other canonical TAFs and TBP remained stable. Functional and gene expression analysis revealed that high levels of TAF3 are dispensable for ES cell self-renewal but are required for endoderm lineage differentiation and to prevent premature specification of neuroectoderm and mesoderm. Genome-wide binding studies by ChIP-seq found that unlike other canonical TFIID subunits such as TBP and TAF1 that bind only promoters, TAF3 targets both promoters and distal enhancer-like sites in ES cells.

Importantly, TAF3 sites were further clustered into four distinct classes based on the context of co-localization with other factors. Briefly, as well as TAF3, class 1 regions are promoter regions enriched for TFIID, Pol II and H3K4me3. Their proximity (< 1kb) to the TSS of known genes confirms that they are predominantly TFIID-bound promoters. By contrast, Class 2 regions have low levels of TFIID and H3K4me3 but are enriched for Oct4/Nanog/Sox2 and mediator components. Class 3 regions are specifically enriched for TAF3, CTCF and cohesin subunits. Finally, class 4 regions are not enriched for any of the factors we considered besides TAF3. Regions from the last three Classes are generally far away (~10's kb) from the TSS.

Further studies revealed that genes down-regulated upon TAF3 depletion are selectively associated with regions enriched for TAF3, CTCF and cohesin (Class 3) and TAF3 only (Class 4), while no such association was seen for genes up-regulated upon TAF3 depletion. Thus, it's likely that class 3 and class 4 regions are required for the efficient expression of genes in their vicinity and depletion of TAF3 interferes with this function. Indeed, our parallel biochemical experiments confirmed a direct protein-protein interaction between that TAF3 and CTCF. This novel interaction was further shown to mediate DNA looping between promoter distal sites and core promoters to regulate proper transcription activation. Notably, we also found that the class 2 binding regions are enriched around up-regulated genes upon TAF3 depletion, consistent with a mechanism of transcription repression by TAF3 in association with Oct4/Nanog/Sox2. Together, our data support the model that TAF3 orchestrates a complex long-distance chromatin interaction network that safeguards the finely-balanced transcriptional programs underlying ES pluripotency.

The regulation of eukaryotic gene expression is critical for proper cell homeostasis and development and relies largely on appropriate initiation of transcription. Transcriptional regulators, such as sequence-specific transcription factors, coactivators, general transcription factors, and chromatin remodelers, are often expressed in a cell-type specific manner to drive cell fate decisions and developmental transitions. Recently, the XPC DNA repair factor was identified as the Stem Cell Coactivator (SCC) complex, a key transcriptional coactivator required to assist OCT4 and SOX2 in driving the expression of key pluripotency genes in embryonic stem cells.

Chapter 1 provides an introduction to mechanisms of eukaryotic transcriptional regulation, eukaryotic DNA repair, and the involvement of the XPC/SCC complex in both of these capacities.

In chapter 2, I describe the first structures of the human apo and DNA-bound XPC holo-complex, as solved by electron microscopy. Comparison of the apo and DNA-bound structures identified key regions that become locally disordered upon engagement with DNA. Using a combination of sequence homology, computational docking of a partial homolog crystal structure into the EM density, and mapped interaction domains, I present a predictive model illustrating regions of key contacts on the XPC/SCC complex.

In chapter 3, I describe the involvement of largely non-specific RNA but not DNA or heparin in mediating the interaction between SOX2 and SCC in a dose-dependent fashion, a novel mode of potentially RNA-mediated transcriptional regulation. I provide evidence that there are little or no sequence or structural requirements for the RNA I also show that while this interaction is RNA-dependent, direct contacts can be formed between SCC and SOX2, suggesting that RNA is stabilizing and possibly even multimerizing existing protein-protein contacts between SCC and SOX2.

In summary, the findings described in this dissertation provide a structural and biochemical framework for understanding the molecular and cellular mechanisms of SCC-driven gene regulation.

The exquisite structural and functional complexity of the mammalian body requires the exactingly precise spatial and temporal regulation of gene expression. The most critically regulated step of which is the transcription of DNA to RNA by the elaborate interplay of sequence-specific activators and repressors, chromatin modifiers, coactivators, the basal machinery and RNA polymerase II. Until recently, developmental stage and tissue-specific gene expression patterns were believed to be regulated primarily by the combinatorial actions of DNA binding activators and repressors, while the core machinery was held to be invariant. New evidence suggests that significant differences in coactivator and core promoter recognition complex composition between cell types and developmental time points may facilitate the global changes in gene expression programs necessary to confer functional diversity. Because of its critical role in vertebrate development and homeostasis, the role of coactivator and core promoter recognition complex switching in the developing liver was examined.

This work shows that mouse liver progenitors or hepatoblasts contain significant levels of the canonical core promoter recognition complex TFIID, including the TATA binding protein and multiple associated factors or TAFs, and the canonical coactivator complex Mediator. These complexes and their constitutive proteins are significantly downregulated in adult hepatocytes. Furthermore, the promoters of several TFIID components become enriched for repressive chromatin marks in adult hepatocytes, and an in vitro model of liver development recapitulates the downregulation of these complexes observed in vivo. In contrast, expression of the TBP-related factor TRF3, the TAF7 paralogue TAF7l, and TAF13 is maintained or induced upon hepatic differentiation. Additionally, these proteins associate with high molecular weight complexes in vitro where they are enriched at hepatocyte-specific promoters, and their depletion attenuates hepatic gene induction. Together these observations support a model wherein liver development and the induction of hepatic gene expression requires the down regulation of TFIID and its replacement with one or more complexes likely containing TRF3, TAF7l and TAF13.

Embryonic stem cells (ESCs), like all tissues, largely rely on precise transcriptional and epigenetic regulation for proper cell specification and differentiation. General transcription factors, transcriptional regulators, coactivators, and chromatin remodelers are often coordinated and expressed in a cell-type specific manner to ensure the integrity of this gene expression network. However, how ESCs are able to tightly regulate gene expression and remain highly malleable to external developmental cues is still an open biological question. The XPC DNA repair complex was recently identified as one of a set of critical stem cell coactivators (SCC) involved in driving robust OCT4/SOX2-dependent expression of pluripotent genes in ESCs. This dissertation will focus on the molecular mechanisms of the XPC complex and other SCCs in controlling cell identity and fate in pluripotent stem cells.

Chapter 1 will provide an introduction to the transcriptional and epigenetic regulation in pluripotent stem cells. Specifically, it will highlight the core transcriptional network, the mechanisms of reprogramming, and barriers to gene expression (e.g. DNA methylation). Chapter 2 will describe a novel role of the XPC DNA repair complex in regulating DNA methylation in both somatic and pluripotent cells, in part through regulating the activity of thymine DNA glycosylase, a major player in active DNA demethylation. Chapter 3 will describe the identification and characterization of two additional stem cell coactivators: the DKC1 ribonucleoprotein and ABCF1. As it is with the case of the XPC DNA repair complex, DKC1 and ABCF1 are multifunctional proteins with unanticipated roles in pluripotent gene transcription.

In summary, this dissertation will highlight the role of versatile proteins in pluripotent stem cells and their involvement in transcriptional regulation and beyond. By strengthening our understanding of the fundamental molecular mechanisms employed by pluripotent stem cells, we may gain a better insight into how cell identity is specified.

In the 43 years since the first observation of nucleosome core particles as beads on a string, our understanding of the relationship between nucleosome structure and its function in living cells has made giant strides. Despite this progress, many questions remain unanswered regarding the role of nucleosomes in transcription initiation, and their interaction with the core transcriptional machinery. Here, I present my progress on addressing the role of TAF1 recognition of acetylated histone H4 tails, and its effect on transcription initiation and start site selection. Preliminary results suggest that H4 acetylation may play a role in start site selection.

One remarkable advancement in technology that will undoubtedly help answer remaining questions in chromatin biology is the repurposing of the prokaryotic CRISPR-Cas system to edit eukaryotic genomes with exquisite precision. Ironically, a tool that may shed light on complex eukaryotic chromatin interactions was evolved in a prokaryotic environment. My work with in vitro nucleosomes positioned me to address whether Cas9 is capable of engaging nucleosomal DNA. Both in vitro and in vivo analyses done in collaboration with Max Horlbeck and Jonathan Weissman at UCSF suggest that nucleosomes pose a strong barrier to Cas9.

Regulation of gene expression is fundamental to many biological processes such as lineage specification during embryonic development or metabolic responses to environmental cues. Classic in vitro biochemical experiments have been successful at identifying various components that are necessary for controlling gene expression, such as transcription factors, and co-activators as well as components of the machinery that regulate RNA processing, stability and degradation. However, detailed mechanistic understanding of gene regulatory mechanisms for many specific genes and gene networks remains unknown. Recently, many labs have attempted to couple mass spectrometry with purification methods that enrich for a specific genomic locus to identify potential novel regulators associated with specific chromosomal targets of interest. Here, I describe the development of a novel dCAS9 mediated purification method for proteins associated with specific cis-regulatory chromatin elements. First, I established the proof of principle by identifying specific human telomere associated proteins. I then further advanced this “reverse-ChIP” strategy by applying it to the de novo identification of novel regulators linked to the Drosophila histone gene locus.

Chapter 1 provides an overview of what is currently known and unknown about gene regulation at the Drosophila histone cluster as well as a summary of recent advances in the field of “reverse-ChIP” techniques. Chapter 2 describes my development of the dCAS9 mediated purification method for proteins specifically associated with targeted chromatin elements, including the purification and identification of proteins bound to human telomeres. Chapter 3 details how I’ve adapted the dCAS9 mediated purification method to identify novel regulators of the Drosophila histone cluster.

Gene expression is critical for the development, patterning, and homeostasis of the organism. Precise temporal and spatial regulation of gene expression at the level of transcription requires a large network of sequence-specific factors, general transcription factors, co-factors, and epigenetic regulators. Malignancies of specific tissues often arise from perturbation of various gene expression levels. Hematopoiesis is one of the most sensitive biological processes to mis-regulation of transcription. To generate all blood cell types from embryonic development throughout the lifetime of the organism, hematopoiesis requires an intricate balance between the maintenance of a permanent stem cell pool and differentiation of multi-potent stem cells into cell types with unique functions. To generate a terminally differentiated, functional immune cell, multiple lineage-restricting steps are involved, with each governed by a specific transcription program. Therefore, gene expression regulation in hematopoietic differentiation is particularly important for an organism to properly develop, maintain oxygen transport to all tissues, and fight against infections. Furthermore, because of detailed understanding of how to isolate cells at different stages and lineages of hematopoietic differentiation, it provides an important model to study the development and differentiation of other adult tissues.

Hematopoietic stem cells can be driven to differentiate along three main lineages: myeloid, erythroid, and lymphoid. Despite the discoveries of several transcription factors for specific lineages of hematopoietic differentiation, understanding the gene expression program that allow stem cells to make the decision to initiate lymphoid development still remains incomplete. For example, how is the preinititation complex of transcription (PIC) recruited to the gene promoters? Additionally, how are interactions, if any, coordinated among various sequence-specific factors that were identified via gene-by-gene knockout (KO) approaches?

To form the PIC at any gene promoter, transcription factor (TF) IIA, B, D, E, F, and H, and RNA polymerase II (Pol II) must coordinate their promoter-binding and enzymatic activities. TFIID, especially, is important for promoter recognition. As a multi-subunit complex containing TATA-box binding protein (TBP) and 13-14 TBP-associated factors (TAFs), TFIID binds to sequences in the proximal promoter and allows the recruitment of other TFs and Pol II. Previously thought to be invariant from one cell type to another, recently tissue-specific roles for certain TAFs have been uncovered. TAF4B is one of the first TAFs found to have cell-specific expression, since it was identified in human B cells {Dikstein:1996wk}, though a role for its function in hematopoiesis has remained elusive. I used a Taf4b KO mouse line to study its function in both myeloid and lymphoid differentiation. I found that Taf4b KO mice were able to generate myeloid and lymphoid progenitors as well as their wild-type (WT) littermates. Furthermore, both of these types of progenitors from Taf4b KO mice can terminally differentiate into mature cells as well as those from WT mice. Finally, TAF4B-null cells are as competent as heterozygous cells (equivalent to WT in terms of Taf4b expression) to reconstitute the hematopoietic compartment of lethally irradiated mice in all cell lineages tested. In conclusion, TAF4B is dispensable in both myeloid and B cell differentiation. This could be due to TAF4B's high sequence homology with TAF4A. Alternatively, TAF4B can play a role in fine-tuning expression levels of certain B cell or myeloid-specific genes, together with another transcription factor, which cannot be uncovered in a KO mouse approach. I have made a TAF4B-specific polyclonal antibody that can be used to identify its transcriptional targets, as well as identify any potential interaction partners.

Though the basal machinery does not seem to play a role in hematopoietic lineage determination, sequence-specific factors have long been implicated in this process. A study using an inducible hematopoietic-specific KO mouse line found that myocyte enhancer factor 2c (MEF2C) is necessary for multi-potent progenitors to differentiate into the lymphoid lineage {StehlingSun:2009df}. Through a candidate approach, I have identified early B cell factor 1 (EBF1) to be a specific interacting partner of MEF2C. Together, they co-occupy and functionally co-activate many B cell specific genes. When MEF2C is depleted in mice, the animals had reduced B cell gene expression as well as increased myeloid gene expression, consistent with MEF2C's role as a lineage fate regulator. I have identified and confirmed several B cell-specific genes that are co-regulated by EBF1 and MEF2C through a genome-wide survey of their binding via chromatin immunoprecipitation followed by exonuclease treatment and deep-sequencing (ChIP-exo). Furthermore, I found that p38 MAPK is the pathway through which MEF2C is phosphorylated and activated to drive B cell differentiation. When phosphorylated, MEF2C prefers to bind its co-activator EBF1, and not its co-repressor HDAC7. Taken together, the results presented in this thesis elucidated the mechanism of activation, binding partners, and downstream targets by which MEF2C is able to regulate lymphoid-specific differentiation. This study contributes to understanding how transcriptional regulation of genes can drive progenitor cells to differentiate down a particular lineage, and provide a novel mechanism for a transcription repressor to switch to an activator during cellular differentiation.