Browsing by Subject "Transcription"

Addition and removal of phosphate is an important post-translational modification involved in cellular signaling. The enzymes responsible for removing this phosphorylation mark, called phosphatases, play a vital role in the cellular decision making processes. In this work we discuss two discoveries, a novel enzyme for a known signaling function involving control of transcription and a novel target for an important cellular stress response enzyme.

In the first project we sought to determine a novel enzyme responsible for dephosphorylating the C-terminal domain of RNA polymerase II. This domain serves as a vital signaling platform for transcription of mammalian genes, with the ability to recruit cofactors that bind to specific patterns of phosphorylation throughout its repeating amino acid sequence. Using a functional assay for phosphatase activity at the Thr4 position we biochemically isolated the unknown enzyme and identified it as PP1 and validated its function in vitro and in vivo.

The second phosphatase studied in this dissertation is MESH1—a mammalian ortholog of the bacterial stringent response protein SpoT that dephosphorylates ppGpp. Because ppGpp is absent in mammalian cells MESH1 lacks a viable target. We established NADPH as a substrate of MESH1 biochemically and corroborated these results by determining the substrate bound structure. Our results reveal a novel regulatory role of MESH1 in a pathway that resembles the bacterial stringent response.

Abstract

Listeria monocytogenes is a gram-positive soil saprophytic bacterium that is capable of causing fatal infection in humans. The main virulence regulator PrfA, a member of the Crp/FNR family of transcriptional regulators, activates the expression of essential proteins required for host cell invasion and cell-to-cell spread. The mechanism of PrfA activation and the identity of its small molecule coactivator have remained a mystery for more than 20 years, but it is hypothesized that PrfA shares mechanistic similarity to the E. coli cAMP binding protein, Crp. Crp activates gene expression by binding cAMP, increasing the DNA binding affinity of the protein and causing a significant DNA bend that facilitates RNA polymerase binding and downstream gene activation. Our data suggests PrfA activates virulence protein expression through a mechanism distinct from the canonical Crp activation mechanism that involves a combination of cysteine residue reduction and glutathione (GSH) binding.

Listeria lacking glutathione synthase (ΔgshF) is avirulent in mice; however virulence is rescued when the bacterium expresses the constitutively active PrfA mutant G145S. Interestingly, Listeria expressing a PrfA mutant in which its four cysteines are mutated to alanine (Quad PrfA), demonstrate a 30-fold decrease in virulence. The Quad and ΔgshF double mutant strains are avirulent. DNA-binding affinity, measured through fluorescence polarization assays, indicate reduction of the cysteine side chains is sufficient to allow PrfA to binds its physiological promoters Phly and PactA with low nanomolar affinity. Oxidized PrfA binds the promoters poorly.

Unexpectedly, Quad also binds promoter DNA with nanomolar affinity, suggesting that the cysteines play a role in transcription efficiency in addition to DNA binding. Both PrfA and Quad bind GSH at physiologically relevant and comparable affinities, however GSH did not affect DNA binding in either case. Thermal denaturation assays suggest that Quad and wild-type PrfA differ structurally upon binding GSH, which supports the in vivo difference in infection between the regulator and its mutant.

Structures of PrfA in complex with cognate DNA, determined through X-ray crystallography, further support the disparity between PrfA and Crp activation mechanisms as two structures of reduced PrfA bound to Phly (PrfA-Phly30 and PrfA-Phly24) suggest the DNA adopts a less bent DNA conformation when compared to Crp-cAMP- DNA. The structure of Quad-Phly30 confirms the DNA-binding data as the protein-DNA complex adopts the same overall conformation as PrfA-Phly.

From these results, we hypothesize a two-step activation mechanism wherein PrfA, oxidized upon cell entry and unable to bind DNA, is reduced upon its intracellular release and binds DNA, causing a slight bend in the promoter and small increase in transcription of PrfA-regulated genes. The structures of PrfA-Phly30 and PrfA-Phly24 likely visualize this intermediate complex. Increasing concentrations of GSH shift the protein to a (PrfA-GSH)-DNA complex which is fully active transcriptionally and is hypothesized to resemble closely the transcriptionally active structure of the cAMP-(Crp)-DNA complex. Thermal denaturation results suggest Quad PrfA is deficient in this second step, which explains the decrease in virulence and implicates the cysteine residues as critical for transcription efficiency. Further structural and biochemical studies are on-going to clarify this mechanism of activation.

The cell division cycle is the process in which the entirety of a cell's contents is duplicated completely and then equally segregated into two identical daughter cells. The order of the steps in the cell cycle must be followed with fidelity to guarantee two viable cells. Understanding the regulatory mechanisms that control cell-cycle events remains to be a fundamental question in cell biology. In this dissertation, I explore the mechanisms that coordinate and regulate cell-cycle progression in the budding yeast, Saccharomyces cerevisiae.

Cell-cycle events have been shown to be triggered by oscillations in the activity of cyclin dependent kinases (CDKs) when bound to cyclins. However, several studies have shown that some cell-cycle events, such as periodic transcription, can continue in the absence of CDK activity. How are periodic transcription and other cell-cycle events coupled to each other during a wild-type cell cycle? Currently, two models of cell-cycle regulation have been proposed. One model hypothesizes that oscillations in CDK activity controls the timing of cell-cycle events, including periodic transcription. The second model proposes that a transcription factor (TF) network oscillator controls the timing of cell-cycle events, via proper timing of gene expression, including cyclins. By measuring global gene expression dynamics in cells with persistent CDK activity, I show that periodic transcription continues. This result fits with the second model of cell-cycle regulation. Further, I show that during a wild-type cell cycle, checkpoints are responsible for arresting the bulk of periodic transcription. This finding adds a new layer of regulation to the second model, providing a mechanism that coordinates cell-cycle events with a TF network oscillator. Taken together, these data provide further insight into the regulation of the cell cycle.

The genomic DNA is bound by a myriad of proteins to form the chromatin inside the nucleus of the cell. The proteins can bind to the genome in different combinations leading to a combinatorial explosion in the number of possible chromatin configurations. The differences in the chromatin configurations for the same genome sequence give rise to distinct cell types. Likewise, cells of the same type also undergo changes in chromatin configurations under different environmental conditions. Key changes to the occupancy profile of the chromatin may dictate changes in gene regulation or vice versa. Therefore, it is important to decipher the chromatin occupancy profiles of the genome and understand how these configurations are related to the transcription of genes.

In this dissertation, we analyze chromatin using chromatin accessibility data sets, particularly MNase-seq and ATAC-seq, that describe the protein-bound and unbound regions of the genome. We first describe a state-space model that uses chromatin accessibility data to jointly infer the occupancy profile of hundreds of proteins binding to the genome. We apply our model to the yeast genome to study the occupancy profile of transcription factors and nucleosomes. We further extend our model to study chromatin dynamics of yeast cells subjected to cadmium stress. In doing so, we identify genomic regions exhibiting changes in the occupancy profile of transcription factors and nucleosomes. Upon comparing with available gene expression data we find that key changes in chromatin configuration occur around gene bodies that are differentially regulated during cadmium stress. Our analyses highlight how specific changes to the occupancy profiles relate to gene expression.

Building upon the interrelatedness of chromatin and transcription, we describe a regression-based approach that predicts transcription from chromatin accessibility data sets. We find that the chromatin accessibility in specific parts of the genome is highly correlated to gene expression. These genomic regions are potential regulatory regions that can lie far away from the gene body and interact with the genes due to the looping of the DNA. Our model identifies these regulatory regions in a gene-specific manner that helps us further understand the connections between chromatin and transcription.

The cell cycle is a series of ordered events that culminates in a single cell dividing into two daughter cells. These events must be properly coordinated to ensure the faithful passage of genetic material. How cell cycle events are carried out accurately remains a fundamental question in cell biology. In this dissertation, I investigate mechanisms orchestrating cell-cycle events in the yeast, Saccharomyces cerevisiae.

Cyclin dependent kinase (CDK) activity is thought to both form the fundamental cell-cycle oscillator and act as an effector of that oscillator, regulating cell-cycle events. By measuring transcript dynamics over time in cells lacking all CDK activity, I show that transcriptional oscillations are not dependent on CDK activity. This data indicates that CDKs do not form the underlying cell-cycle oscillator. I propose a model in which a transcription factor network rather than CDK activity forms the cell-cycle oscillator. In this model, CDKs are activated by the periodic transcription of cyclin genes and feedback on the network increasing the robustness of network oscillations in addition to regulating cell-cycle events.

I also investigate CDK-dependent and -independent mechanism regulating the duplication of the yeast centrosome, the spindle pole body (SPB). It is critical for the formation of a bipolar spindle in mitosis that the SPB duplicates once and only once per cell cycle. Through a combination of genetic and microscopic techniques I show that three distinct mechanisms regulate SPB duplication, ensuring its restriction to once per cell cycle.

Together, the data presented in this dissertation support a model in which CDKs, periodic transcription, and a TF-network oscillator are all important cell-cycle regulatory mechanisms that collaborate to regulate the intricate collection of events that constitute the cell cycle.

The regulation of metabolism is one of the key challenges faced by organisms across all domains of life. Despite fluctuating environments, cells must produce the same metabolic outputs to thrive. Although much is known about the regulation of metabolism in the bacteria and the eukaryotes, relatively little is known about the regulation of metabolism in archaea. Previous work identified the winged helix-turn-helix transcription factor TrmB as a major regulator of metabolism in the model archaeon Halobacterium salinarum. TrmB was found to bind to the promoter of 113 genes in the absence of glucose. Many of these genes encode enzymes involved in metabolic processes, including central carbon metabolism, purine synthesis, and amino acid degradation. Although much is known about TrmB, it remains unclear how it dynamically regulates its ~100 metabolic enzyme-coding gene targets, what the effect of transcriptional regulation is on metabolite levels, and why TrmB regulates so many metabolic processes in response to glucose. Using dynamic gene expression and TrmB-DNA binding assays, we found that that TrmB functions alone to regulate central metabolic enzyme-coding genes, but cooperates with various regulators to control peripheral metabolic pathways. After determining the temporal pattern of gene expression changes and their dependence on TrmB, we used dynamic metabolite profiling to investigate the effects of transcriptional changes on metabolite levels and phenotypes. We found that TrmB-mediated transcriptional changes resulted in substantial changes in metabolite levels. Additionally, we showed that mis-regulation of genes encoding enzymes involved in gluconeogenesis in the ΔtrmB mutant strain in the absence of glucose results in low PRPP levels, which cause a metabolic block in de novo purine synthesis that is partially responsible for the growth defect of the ΔtrmB mutant strain. Finally, using a series of quantitative phenotyping experiments, we showed that TrmB regulates the gluconeogenic production of sugars incorporated into the cell surface S-layer glycoprotein. Because S-layer glycosylation is proportional to growth, we hypothesize that TrmB transduces a growth rate signal to co-regulated metabolic pathways including amino acid, purine, and cobalamin biosynthesis. Taken together, our results suggest that TrmB is a global regulator of archaeal metabolism that works in concert with other transcription factors to regulate diverse metabolic pathways in response to nutrients and growth rate.

The late phase of long-term synaptic potentiation (LTP) at glutamatergic synapses, which is thought to underlie the long lasting memory (at least hours), requires gene transcription in the nucleus. However, it remains elusive how signaling initiated at synapses during induction of LTP is transmitted into the nucleus to commence transcription. Using a combination of two-photon glutamate uncaging and a genetically encoded FRET sensor, I found that induction of synapse-specific LTP at only a few (3-7) dendritic spines leads to pronounced activation of extracellular signal-regulated kinase (ERK) in the nucleus and downstream phosphorylation of transcription factors, cAMP-response element-binding protein (CREB) and E26-like protein-1 (Elk-1). The underlying molecular mechanism of this nuclear ERK activation was investigated: it seems to involve activation of NMDA receptors, metabotrophic glutamate receptors, and the classical Ras pathway. I also found that the spatial pattern of synaptic stimulation matters: spatially dispersed stimulation over multiple dendritic branches activated nuclear ERK much more efficiently than clustered stimulation within a single dendritic branch. In sum, these results suggest that biochemical signals could be transmitted from individual spines to the nucleus following LTP induction and that such synapse-to-nucleus signaling requires integration across multiple dendritic branches.

The centromere serves as the foundation for the kinetochore and attachment point for spindle microtubules during metaphase. The proper function of this locus is required to ensure chromosome segregation and genomic stability. In humans, repetitive alpha satellite DNA underlies the human centromere region and is organized into specific chromatin domains that are maintained by a complex combination of factors. Although the centromere region is generally thought to be specified epigenetically, some evidence suggests that the underlying DNA sequence is also involved in centromere function. To better define links between alpha satellite and function within the human centromere region, we investigated two attributes of alpha satellite DNA: its transcription into noncoding alpha satellite RNAs and genomic variation within the alpha satellite array. Noncoding transcripts produced from alpha satellite DNA are associated with normal centromere and pericentromere function and evidence from other organisms suggests RNAs from this region are pivotal in the centromere and kinetochore assembly cascade and in maintaining the chromatin environments of the centromere region. However, alpha satellite RNAs have not yet been fully characterized and data reflecting the chromosome-specific nature of alpha satellite arrays is lacking. Additionally, genomic variation within alpha satellite arrays has been linked to reduced centromere protein recruitment and chromosome instability, yet the molecular basis for this is unknown. These gaps in knowledge have stymied our understanding of the role of genomic and epigenetic attributes of alpha satellite that affect function within the human centromere region. Thus, this work aims to functionally characterize the role of alpha satellite transcripts and to determine how genomic variation impacts chromosome stability. Utilizing cytological and molecular techniques that allow the differentiation of alpha satellite RNAs from individual chromosomes and arrays, we have demonstrated that each chromosome produces unique noncoding RNAs that localize in cis to their site of production. Both centromeric and pericentromeric alpha satellite arrays produce noncoding RNAs, but these transcripts are spatially and functionally distinct. Alpha satellite RNAs from the centromere bind at least two key centromere proteins: CENP-A and CENP-C, while alpha satellite RNAs from the pericentromere colocalize with SUV39H1. Centromeric alpha satellite RNAs are required for complete loading of new CENP-A-containing nucleosomes, as well as maintenance of CENP-C levels. Genomic variation affects the origin of alpha satellite transcripts, such that highly variant arrays produce a different set of transcripts than wild type arrays. Further, the long-range organization of variation across the alpha satellite array in unstable chromosomes suggests certain spatial organizations of variation are poor platforms for building a stable centromere and kinetochore. Collectively, these findings implicate alpha satellite RNA and genomic variation and/or the interplay of these two elements as essential factors in the function of the human centromere region.

HIV-1 relies on both viral and cellular host factors for expression of its genome. Tat specific factor 1 (Tat-SF1) was identified as a cellular cofactor required for enhanced transcription of HIV-1 in vitro. Insight into the role of Tat-SF1 in the HIV-1 lifecycle has previously been limited to immunodepletions and in vitro analyses or transient overexpression experiments. Here, we present studies that utilize RNA interference (RNAi) to reevaluate Tat-SF1's role in Tat transactivation and HIV-1 replication in vivo. We report that although Tat-SF1 depletion reduces HIV-1 infectivity, it does not affect Tat transactivation in vivo. However, Tat-SF1 depletion changes the levels of unspliced and spliced RNAs. We propose that Tat-SF1 has a novel role of post-transcriptionally regulating HIV-1 gene expression, possibly through alternative splicing.

The functions of Tat-SF1 in cellular gene expression are not well understood, so we utilized the stable cell lines constructed for our HIV-1 studies to investigate the cellular functions of Tat-SF1. To identify target genes of Tat-SF1, we employed a combination of RNAi and human exon arrays. These arrays, which survey both transcript-level and exon-level changes genome-wide, revealed approximately 1,400 genes with alternative exon usage after Tat-SF1 depletion (p≤0.01). In contrast, 500 genes showed significant transcript-level changes (p≤0.01), all with minimal fold changes. Computational analyses showed that genes with alternative exon usage after Tat-SF1 depletion were over-represented in the insulin signaling and ubiquitin mediated proteolysis biological pathways. Furthermore, there was approximately 2-fold enrichment of Tat-SF1 target genes among previously reported HIV-1 dependency factors. The type of exon choice affected by Tat-SF1 depletion exhibited a strong 5’ bias. Finally, a novel Tat-SF1 binding motif, GACGGG, was found to be over-represented among target genes and may play a functional role in first exon choice. Together, these data are the strongest evidence to date of Tat-SF1 functioning in both transcription and splicing of cellular genes.

Inositol phosphates (IPs) are eukaryotic signaling molecules that play important roles in a wide range of biological processes. IPs are required for embryonic development and patterning, insulin secretion, the regulation of telomere length, proper progression through the cell cycle, and the regulation of ion channels. This work uses the yeast Saccharomyces cerevisiae as a model system for investigating the functions of IPs and focuses on the transcriptional regulation of the gene encoding the secreted mating pheromone MFα2 by the IP kinase Ipk2 (also called Arg82, ArgR3, and IPMK). This work shows that Ipk2 has both kinase-dependent and kinase-independent functions in regulating the transcription of MFα2. Transcription of MFα2 is also dependent upon the integrity of an Mcm1-binding site in its promoter. This is the first description of a role for this binding site in the transcription of MFα2.

In vivo and in vitro screening approaches to identify additional factors associated with MFα2 expression or with IP biology generally are also described. These unbiased approaches provide some valuable insight for further investigations.

CD8+ T cells are associated with long term control of virus replication to low or undetectable levels in a population of HIV+ therapy-naïve individuals known as virus controllers (VCs; <5000 RNA copies/ml and CD4+ lymphocyte counts >400 cells/µl). These subjects' ability to control viremia in the absence of therapy makes them the gold standard for the type of CD8+ T-cell response that should be induced with a vaccine. Studying the regulation of CD8+ T cells responses in these VCs provides the opportunity to discover mechanisms of durable control of HIV-1. Previous research has shown that the CD8+ T cell population in VCs is heterogeneous in its ability to inhibit virus replication and distinct T cells are responsible for virus inhibition. Further defining both the functional properties and regulation of the specific features of the select CD8+ T cells responsible for potent control of viremia the in VCs would enable better evaluation of T cell-directed vaccine strategies and may inform the design of new therapies.

Here we discuss the progress made in elucidating the features and regulation of CD8+ T cell response in virus controllers. We first detail the development of assays to quantify CD8+ T cells' ability to inhibit virus replication. This includes the use of a multi-clade HIV-1 panel which can subsequently be used as a tool for evaluation of T cell directed vaccines. We used these assays to evaluate the CD8+ response among cohorts of HIV-1 seronegative, HIV-1 acutely infected, and HIV-1 chronically infected (both VC and chronic viremic) patients. Contact and soluble CD8+ T cell virus inhibition assays (VIAs) are able to distinguish these patient groups based on the presence and magnitude of the responses. When employed in conjunction with peptide stimulation, the soluble assay reveals peptide stimulation induces CD8+ T cell responses with a prevalence of Gag p24 and Nef specificity among the virus controllers tested. Given this prevalence, we aimed to determine the gene expression profile of Gag p24-, Nef-, and unstimulated CD8+ T cells. RNA was isolated from CD8+ T-cells from two virus controllers with strong virus inhibition and one seronegative donor after a 5.5 hour stimulation period then analyzed using the Illumina Human BeadChip platform (Duke Center for Human Genome Variation). Analysis revealed that 565 (242 Nef and 323 Gag) genes were differentially expressed in CD8+ T-cells that were able to inhibit virus replication compared to those that could not. We compared the differentially expressed genes to published data sets from other CD8+ T-cell effector function experiments focusing our analysis on the most recurring genes with immunological, gene regulatory, apoptotic or unknown functions. The most commonly identified gene in these studies was TNFRSF9. Using PCR in a larger cohort of virus controllers we confirmed the up-regulation of TNFRSF9 in Gag p24 and Nef-specific CD8+ T cell mediated virus inhibition. We also observed increase in the mRNA encoding antiviral cytokines macrophage inflammatory proteins (MIP-1α, MIP-1αP, MIP-1β), interferon gamma (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), and recently identified lymphotactin (XCL1).

Our previous work suggests the CD8+ T-cell response to HIV-1 can be regulated at the level of gene regulation. Because RNA abundance is modulated by transcription of new mRNAs and decay of new and existing RNA we aimed to evaluate the net rate of transcription and mRNA decay for the cytokines we identified as differentially regulated. To estimate rate of mRNA synthesis and decay, we stimulated isolated CD8+ T-cells with Gag p24 and Nef peptides adding 4-thiouridine (4SU) during the final hour of stimulation, allowing for separation of RNA made during the final hour of stimulation. Subsequent PCR of RNA isolated from these cells, allowed us to determine how much mRNA was made for our genes of interest during the final hour which we used to calculate rate of transcription. To assess if stimulation caused a change in RNA stability, we calculated the decay rates of these mRNA over time. In Gag p24 and Nef stimulated T cells , the abundance of the mRNA of many of the cytokines examined was dependent on changes in both transcription and mRNA decay with evidence for potential differences in the regulation of mRNA between Nef and Gag specific CD8+ T cells. The results were highly reproducible in that in one subject that was measured in three independent experiments the results were concordant.

This data suggests that mRNA stability, in addition to transcription, is key in regulating the direct anti-HIV-1 function of antigen-specific memory CD8+ T cells by enabling rapid recall of anti-HIV-1 effector functions, namely the production and increased stability of antiviral cytokines. We have started to uncover the mechanisms employed by CD8+ T cell subsets with antigen-specific anti-HIV-1 activity, in turn, enhancing our ability to inhibit virus replication by informing both cure strategies and HIV-1 vaccine designs that aim to reduce transmission and can aid in blocking HIV-1 acquisition.

In the past decade, a challenge to the canonical model of cell cycle transcriptional control has been posed by a series of high-throughput gene expression studies in budding yeast. Using genetic methods to inhibit or lock the activity of the cyclin-CDK/APC oscillator, these population studies demonstrated that a significant proportion of cell cycle transcription persists in the absence of cyclin-CDK/APC oscillations. To account for these findings, a network of serially activating transcription factors with sources of negative feedback from transcriptional repressors (referred to as a \say{TF network}) was proposed to drive cyclin-CDK/APC independent gene expression.

However, population studies of cell cycle gene expression are limited due to loss of phase synchrony that limits the timescale of measurement of gene expression and due to expression averaging that limits assessment of heterogeneity of expression within the population. To circumvent these limitations I used a single-cell timelapse microscopy approach to assess transcriptional dynamics of cell cycle regulated genes during extended cell cycle arrests in both the Gl/S and early mitosis (metaphase) phases of the cell cycle.

During G1/S arrest, transcriptional dynamics of four cell cycle regulated genes was assessed and activation of out-of-phase cell cycle transcription was observed in two of these genes. Though budding oscillations were observed in G1/S arrested cells, robust transcriptional oscillations were not seen for any of the four genes and budding dynamics were uncoupled from transcriptional dynamics after the first bud emergence. During cell cycle arrest in early mitosis, transcriptional dynamics of ten cell cycle regulated genes was assessed and activation of out-of-phase transcription was observed for four genes. All four genes activated once with canonical ordering but robust oscillations were not observed during mitotic arrest. Together these studies demonstrate activation, but not oscillation, of cell cycle transcription in the absence of cyclin-CDK/APC oscillations.

The androgen receptor (AR) signaling axis is a well-established therapeutic target in prostate cancer, due to its central role in tumor maintenance and progression. Although patients respond initially to androgen deprivation therapies and AR antagonists, they invariably progress to a castration-resistant state. Consequently, there is an unmet need for agents that target the AR signaling axis in a unique manner.

Histone deacetylase (HDAC) inhibitors repress AR signaling and prostate cancer growth in cellular and xenograft models. However, HDAC inhibitors also induce epithelial to mesenchymal (EMT) and neuroendocrine differentiation, both of which are associated with prostate cancer progression and aggressiveness. Given that 18 different HDAC isoforms have been identified in humans, and non-selective or Class I (HDAC1, 2, 3, and 8) HDAC inhibitors have been used in most of these studies, the relative contribution of individual HDAC isoforms to AR transcriptional activity and prostate cancer pathophysiology remains to be elucidated. The overarching goals of this study were to (1) determine the role of individual Class I HDACs in AR transcriptional activity and prostate cancer growth, (2) identify selective HDAC inhibitors that have reduced adverse profiles to the treatment of prostate cancer, and (3) identify potential HDAC-interacting proteins that regulate AR target gene transcription and prostate cancer growth.

Using genetic knockdown studies and pharmacological inhibitors with isoform selectivity, we identified that HDAC3 was required for AR transcriptional activity and proliferation in cellular models of androgen-sensitive and castration-resistant prostate cancer (CRPC). Additionally, we found that RGFP966, an HDAC3-selective inhibitor, attenuated the growth of a xenograft model of CRPC. Furthermore, non-selective HDAC inhibitors induced EMT and neuroendocrine markers in prostate cancer cells, but RGFP966 treatment did not. These studies provide rationale for selective inhibition of HDAC3 for the treatment of CRPC, and could provide an explanation for the lack of success using non-selective HDAC inhibitors in clinical trials for prostate cancer.

We also assessed the role of REV-ERB alpha, an HDAC3-interacting protein, in the regulation of AR transcriptional activity and prostate cancer growth. Using siRNA knockdown studies, REV-ERB inhibitors, and overexpression studies, we concluded that REV-ERB alpha; was required for AR target gene induction and prostate cancer growth, including models of CRPC. These studies also provide rational for targeting REV-ERB alpha; for the treatment of CRPC.

Taken together, these studies identify two novel targets in the HDAC signaling axis for the treatment of prostate cancer: HDAC3 and REV-ERB alpha. Our studies provide greater insight into AR transcriptional regulation and prostate cancer pathophysiology.

Epstein-Barr virus (EBV) is a ubiquitous human pathogen that establishes a lifelong latent infection in over ninety percent of all adult humans worldwide. While typically benign, EBV has been causally associated with a number of human malignancies in the settings of immune suppression, genetic, and/or environmental factors. While a highly successful pathogen based on prevalence, the ability of the virus to immortalize human B cells (a stage of infection thought to be critical for the establishment of latency) is quite poor. We hypothesize that the interactions between the virus and the human host early after infection are ultimately important for the outcome of viral latency establishment. To answer this question we broadly profiled primary human B cells at both early and late times after EBV infection to assay both host mRNA expression and the host-driven response to apoptotic stimuli. We found that EBV infection induces host gene expression signatures early after infection that are functionally distinct from the gene expression program late after infection. These studies also led to the novel discovery that viral gene expression is controlled differently early after infection, including the delayed expression of a viral protein that is critical for the establishment of latency. Furthermore, we have also shown that EBV can use a single viral protein to alter and repress host apoptotic sensitivity in the face of an anti-viral apoptotic response.

Human cells are perpetually receiving and responding to a variety of intrinsic and extrinsic signals. A primary mechanism by which cells carry out these responses is via changes in the regulation of gene expression. Many studies have examined gene regulation in steady state systems, but few have investigated the genomic response to stimuli. Therefore, it is less well understood how cellular stimuli elicit dynamic gene expression responses. Here, we investigate how extracellular stimuli mediate gene expression responses via: 1) Changes in transcription factor configurations at enhancer elements; and 2) Changes in chromatin looping between putative enhancers and their target gene promoters. To study these phenomena, we used glucocorticoid (GC) treatment as a model transcriptional stimulus. This hormone steroid is known to bind to and activate the GC receptor (GR), a ligand-induced transcription factor (TF), and is therefore a highly tractable system for studying stimulus responsive gene regulation. Using this model system, we first used high-resolution TF-binding site mapping approaches to elucidate the genomic binding locations of GR and its associated cofactors. Using these approaches, we found evidence that: 1) The GR binds to the genome as both a monomer and dimer; and 2) The GR binds to the genome with AP-1 in a more relaxed configuration, while it binds FOXA1 in a more constrained configuration. We next interrogated the role of chromatin looping in mediating dynamic transcriptional responses. For this work we used high-throughput genomics methods to assay chromatin conformation across a time course of GC treatment. These studies resulted in several main findings: 1) Chromatin loops do not form in response to stimulus, but are instead pre-formed before GC treatment; 2) Chromatin looping interactions increase between distal GR binding sites and GC-responsive genes; 3) The insulator protein CTCF is depleted at stimulus responsive looping interactions; and 4) GC treatment mediates changes in higher-order chromosome compartmentalization that are concordant with gene expression responses. Together these results provide evidence for a genome topology that is pre-wired to respond to stimulus, and that subsequent transcriptional responses are mediated through GR binding to putative enhancer elements with other TFs, in a variety of genomic binding configurations.

Transcription initiation is a key component in the regulation of gene expression. Recent high-throughput sequencing techniques have enhanced our understanding of mammalian transcription by revealing narrow and broad patterns of transcription start sites (TSSs). Transcription initiation is central to the determination of condition specificity, as distinct repertoires of transcription factors (TFs) that assist in the recruitment of the RNA polymerase II to the DNA are present under different conditions. However, our understanding of the presence and spatiotemporal architecture of the promoter patterns in the fruit fly remains in its infancy. Nucleosome organization and transcription initiation have been considered hallmarks of gene expression, but their cooperative regulation is also not yet understood.

In this work, we applied a hierarchical clustering strategy on available 5' expressed sequence tags (ESTs), and developed an improved paired-end sequencing strategy to explore the transcription initiation landscape of the D.melanogaster genome. We distinguished three initiation patterns: 'Peaked or Narrow Peak TSSs‛, 'Broad Peak TSSs‛, and 'Broad TSS cluster groups or Weak Peak TSSs‛. The promoters of peaked TSSs contained the location specific sequence elements, and were bound by TATA Binding Protein (TBP), while the promoters of broad TSS cluster groups were associated with non-location-specific elements, and were bound by the TATA-box related Factor 2 (TRF2).

Available ESTs and a tiling array time series enabled us to show that TSSs had distinct associations to conditions, and temporal patterns of embryonic activity differed across the majority of alternative promoters. Peaked promoters had an association to maternally inherited transcripts, and broad TSS cluster group promoters were more highly associated to zygotic utilization. The paired-end sequencing strategy identified a large number of 5' capped transcripts originating from coding exons that were unlikely the result of alternative TSSs, but rather the product of post-transcriptional modifications.

We applied an innovative search program called FREE to embryo, head, and testes specific core promoter sequences and identified 123 motifs: 16 novel and 107 supported by other motif sources. Motifs in the embryo specific core promoters were found at location hotspots from the TSS. A family of oligos was discovered that matched the Pause Button motif that is associated with RNA pol II stalling.

Lastly, we analyzed nucleosome organization, chromatin structure, and insulators across the three promoter patterns in the fruit fly and human genomes. The WP promoters showed higher associations with H2A.Z, DNase Hypersensitivity Sites (DHS), H3K4 methylations, and Class I insulators CTCF/BEAF32/CP190. Conversely, NP promoters had higher associations with polII and GAF binding. BP promoters exhibited a combination of features from both promoter patterns. Our study provides a comprehensive map of initiation sites and the conditions under which they are utilized in D. melanogaster. The presence of promoter specific histone replacements, chromatin modifications, and insulator elements support the existence of two divergent strategies of transcriptional regulation in higher eukaryotes. Together, these data illustrate the complex regulatory code of transcription initiation.

During development, morphogenetic processes require the integration of numerous signals to properly shape cells and tissues. These signals are interpreted by cells to induce the precise transcriptional circuitry that controls morphogenesis. In the fly embryonic epidermis, the fly Wnt, wingless (wg), generates naked cuticle zones that separate the ventral denticle belts by repressing expression of shavenbaby (svb), which encodes a transcription factor required for denticle formation. What is not known is how Wg and Svb interact to produce the stereotyped diversity of denticle shapes within each belt. One possibility is that graded levels of Svb, responding to graded levels of Wg signaling, determine denticle shape. Indeed, we found that the svb promoter responds differentially to altered Wg activity levels. However, artificially increasing the levels of ectopic svb does not produce morphologically distinct denticles, suggesting that additional factors are involved. We have discovered that a second Wg-responsive transcription factor, encoded by SoxNeuro (SoxN), cooperates with Svb to shape the denticles. Co-expressing SoxN with svb is sufficient to rescue the morphology of denticles in an ectopic location. Furthermore, embryos that lack Svb activity retain the ability to produce a small number of rounded, reduced ventral denticles, due to SoxN activity. We found that svb;SoxN double mutant embryos secrete cuticles that completely lack ventral denticles and dorsal hairs. We also found that a group of known Svb target genes belonging to the zona pellucida family of proteins are differentially activated by SoxN. Finally, we discovered two novel target genes of SoxN, CG16885 and CG30101, which are expressed in denticle-producing cells and which are regulated independently of Svb. SoxN was shown previously to down-regulate Wg signaling and to promote expression of svb. Here, we propose that SoxN acts with Svb, in an additional, direct role, to promote denticle morphogenesis.

Understanding transcriptional regulation in development and disease is one of the central questions in modern biology. The current working model is that Transcription Factors (TFs) combinatorially bind to specific regions of the genome and drive the expression of groups of genes in a cell-type specific fashion. In organisms with large genomes, particularly mammals, TFs bind to enhancer regions that are often several kilobases away from the genes they regulate, which makes identifying the regulators of gene expression difficult. In order to overcome these obstacles and uncover transcriptional regulatory networks, we used an approach combining expression profiling and genome-wide identification of enhancers followed by motif analysis. Further, we applied these approaches to uncover the TFs important in mammalian sex determination.

Using expression data from a panel of 19 human cell lines we identified genes showing patterns of cell-type specific up-regulation, down-regulation and constitutive expression. We then utilized matched DNase-seq data to assign DNase Hypersensitivity Sites (DHSs) to each gene based on proximity. These DHSs were scanned for matches to motifs and compiled to generate scores reflecting the presence of TF binding sites (TFBSs) in each gene's putative regulatory regions. We used a sparse logistic regression classifier to classify differentially regulated groups of genes. Comparing our approach to proximal promoter regions, we discovered that using sequence features in regions of open chromatin provided significant performance improvement. Crucially, we discovered both known and novel regulators of gene expression in different cell types. For some of these TFs, we found cell-type specific footprints indicating direct binding to their cognate motifs.

The mammalian gonad is an excellent system to study cell fate determination processes and the dynamic regulation orchestrated by TFs in development. At embryonic day (E) 10.5, the bipotential gonad initiates either testis development in XY embryos, or ovarian development in XX embryos. Genetic studies over the last 3 decades have revealed about 30 genes important in this process, but there are still significant gaps in our understanding. Specifically, we do not know the network of TFs and their specific combinations that cause the rapid changes in gene expression observed during gonadal fate commitment. Further, more than half the cases of human sex reversal are as yet unexplained.

To apply the methods we developed to identify regulators of gene expression to the gonad, we took two approaches. First, we carried out a careful dissection of the transcriptional dynamics during gonad differentiation in the critical window between E11.0 and E12.0. We profiled the transcriptome at 6 equally spaced time points and developed a Hidden Markov Model to reveal the cascades of transcription that drive the differentiation of the gonad. Further, we discovered that while the ovary maintains its transcriptional state at this early stage, concurrent up- and down-regulation of hundreds of genes are orchestrated by the testis pathway. Further, we compared two different strains of mice with differential susceptibility to XY male-to-female sex reversal. This analysis revealed that in the C57BL/6J strain, the male pathway is delayed by ~5 hours, likely explaining the increased susceptibility to sex reversal in this strain. Finally, we validated the function of Lmo4, a transcriptional co-factor up-regulated in XY gonads at E11.6 in both strains. RNAi mediated knockdown of Lmo4 in primary gonadal cells led to the down-regulation of male pathway genes including key regulators such as Sox9 and Fgf9.

To find the enhancers in the XY gonad, we conducted DNase-seq in E13.5 XY supporting cells. In addition, we conducted ChIP-seq for H3K27ac, a mark correlated with active enhancer activity. Further, we conducted motif analysis to reveal novel regulators of sex determination. Our work is an important step towards combining expression and chromatin profiling data to assemble transcriptional networks and is applicable to several systems.

If nothing else, the 2019 Coronavirus pandemic has made it abundantly clear that understanding the mechanisms of infectious disease is imperative to the survival of our species. While the last fifty years of developments in molecular biology has accelerated our ability to study microbial pathogens, limitations in pathogen tropism, microbial survival in laboratory conditions, uneven sampling of human cohorts across geographical and socioeconomic lines, and heterogeneous complexity during human infection have limited our ability to study complex mechanisms of human susceptibility to infectious disease. In this work, I build on recent developments in utilizing High-throughput Human in vitro Susceptibility Testing (Hi-HOST) to not only (a) identify novel sites in the human genome that contribute to natural variation in infectious disease susceptibility based on highly quantifiable cellular phenotypes, but (b) use these sites as a springboard to understand the entire, complex host-pathogen interaction. From this perspective, I paired the model pathogen Salmonella enterica and the Hi-HOST system to identify that natural variation in the mammalian gene arhgef26 contributes to susceptibility to Salmonella invasion. I used this finding as a starting point to fully explore the role of ARHGEF26 during infection, redefining its role in invasion, inflammation, and its interaction with host and bacterial proteins during the process. Similarly, I used prior Hi-HOST findings that methionine metabolism influences the host response to Salmonella enterica serovar Typhimurium (S. Typhimurium) as a launching point to investigate the impacts of host and bacterial metabolism on the virulence of S. Typhimurium. I found that the metabolite methylthioadenosine is a potent inhibitor of S. Typhimurium type III secretion, motility, and invasion. Finally, I mechanistically explain some of these findings by linking methionine metabolism to DNA methylation using a novel approach to integrate the Salmonella Typhimurium methylome and transcriptome. In sum, these findings demonstrate the ability for cellular GWAS to serve as a launching point to understand complex host-pathogen interactions.