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Estrogens exert numerous actions throughout the human body, targeting healthy tissue while also enhancing the proliferative capacity of breast cancers. Estrogen signaling is mediated by the estrogen receptor (ER), which binds DNA and ultimately affects the expression of adjacent genes. Current understanding of ER-mediated transcriptional regulation is mostly limited to genes whose transcript levels increase following estrogen exposure, though recent studies demonstrate that direct down-regulation of estrogen-responsive genes is also a significant feature of ER action. We hypothesized that differences in cis-regulatory DNA was a factor in determining target gene expression and performed computational and experimental studies to test this hypothesis. From our in silico analyses, we show that the binding motifs for certain transcription factors are enriched in cis-regulatory sequences adjacent to repressed target genes compared to induced target genes, including the motif for RUNX1. In silico analyses were tested experimentally using dual luciferase reporter assays, which indicate that several ER binding sites are estrogen responsive. Mutagenesis of transcription factor motifs (for ER and RUNX1) reduced the response of reporter gene. Further experiments demonstrated that co-recruitment of ER and RUNX1 is necessary for repression of gene expression at some target genes. These findings highlight a novel interaction between ER and RUNX1 and their role in transcriptional repression in breast cancer.

High­-throughput sequencing has become pervasive in all facets of genomic analysis. I developed computational methods to analyze high­-throughput sequencing data and derive biological conclusions in two research areas -- transcriptional regulation in mammals and evolution of virus under immune pressure. To investigate transcriptional regulation, I integrated data from multiple experiments performed by the ENCODE consortium. First, my analysis revealed that Transcription Factors (TFs) prefer to bind GC-­rich, histone­-depleted regions. By comparing in vivo and in vitro nucleosome dynamics, I observed that while histones have an innate preference for binding GC-­rich DNA, TF binding overrides this preference and produces a negative correlation between GC content and histone enrichment. In the next project, I found that the binding events of multiple TFs co-­occur at genomic regions enriched in activating histone marks that are typically associated with gene enhancers and promoters, suggesting that these regions may be enhancers or have TSS-­distal transcription. Lastly, I used supervised machine ­learning techniques to train histone enrichment signals and sequence features to predict transcriptional enhancers to be validated in mouse-­transgenic assays. In a post­-clinical trial exploratory analysis of Hepatitis C Virus (HCV), I traced the evolutionary path of the envelope proteins E1 and E2 in HCV-infected liver transplant patients, in response to a novel antibody. I developed a systematic amino acid­-level analysis pipeline that quantifies differences in amino acid frequencies in each position between two time points. Upon applying this method across all positions in the E1/E2 region and comparing pre-­liver­-transplant and post­-viral­-rebound time points, mutations in two positions emerged as being key to antibody evasion. Both these mutations--N415K/D and N417S--were in the epitope targeted by the antibody, but surprisingly, did not co­-occur. In post­-rebound viral genomes that contain the N417S mutation but retain the wild-­type variant at 415, N-­linked glycosylation of 415 is another possible escape mechanism. Using the same analysis pipeline, I also identified additional candidate escape mutations outside the epitope, which could be potential therapeutic targets.

Transcription factors (TFs) control gene expression by binding to highly specific DNA sequences in gene regulatory regions. This TF binding is central to control myriad biological processes. Indeed, transcriptional dysregulation has been associated with many diseases such as autoimmune diseases and cancer. In this thesis, I studied the transcriptional regulation of cytokines and gene transcriptional dysregulation in cancer. Cytokines are small proteins produced by immune cells that play a key role in the development of the immune system and response to pathogens and inflammation. I mined three decades of research and developed a user-friendly database, CytReg, containing 843 human and 647 mouse interactions between TFs and cytokines. I analyzed CytReg and integrated it with phenotypic and functional datasets to provide novel insights into the general principles that govern cytokine regulation. I also predicted novel cytokine promoter-TF interactions based on cytokine co-expression patterns and motif analysis, and studied the association of cytokine transcriptional dysregulation with disease. Transcriptional dysregulation can be caused by single nucleotide variants (SNVs) affecting TF binding sites (TFBS). Therefore, I created a database of altered TFBS (aTFBS-DB) by calculating the effect (gain/loss) of all possible SNVs across the human genome for 741 TFs. I showed how the probabilities to gain or disrupt TFBSs in regulatory regions differ between the major TF families, and that cis-eQTL SNVs are more likely to perturb TFBSs than common SNVs in the human population. To further study the effect of somatic SNVs in TFBS, I used the aTFBS-DB to develop TF-aware burden test (TFABT), a novel algorithm to predict cancer driver SNVs in gene promoters. I applied the TFABT to the Pan-Cancer Analysis of Whole Genomes (PCAWG) cohort and identified 2,555 candidate driver SNVs across 20 cancer types. Further, I characterized these cancer drivers using functional and biophysical assay data from three cancer cell lines, demonstrating that most SNVs alter transcriptional activity and differentially recruit cofactors. Taken together, these studies can be used as a blueprint to study transcriptional mechanisms in specific cellular processes (i.e. cytokine expression) and the effect of transcriptional dysregulation in disease (i.e. cancer).

Comparative study of homologous structures in closely related species allows the identification of changes in gene regulatory mechanisms and their impact on the evolution of developmental processes. Nematodes, the invertebrate roundworms, are well suited for such studies, especially the Caenorhabditis briggsae and its famous cousin C. elegans. These two worms diverged from a common ancestor roughly 30 million years ago, yet appear morphologically almost identical. My Ph.D. thesis has focused on a set of nuclear factors in C. briggsae that negatively regulate cell proliferation to generate the hermaphrodite-specific mating and egg-laying organ, i.e., vulva. To this end, I have taken a two-pronged approach: one, developing resources to facilitate genetic and genomic studies in this species, and two, characterizing the roles of a novel class of genes and known repressors of vulval development. My work has uncovered substantial differences in the underlying genetic networks that regulate vulva formation in C. briggsae and C. elegans. / Thesis / Candidate in Philosophy

Parkinson's disease (PD) is the second most common neurodegenerative disease, yet it has no cure. It is characterized by the loss of dopaminergic neurons and accumulation of dense aggregates, primarily composed of α-synuclein protein. Many causative genes have been identified including SNCA, encoding α-synuclein, and Leucine-rich-repeat kinase 2 (LRRK2). The LRRK2 G2019S mutation is known to cause hyperactive kinase activity, but its cellular functions, including its kinase substrates, remain poorly understood. PD has many risk factors including environmental and genetic modifiers. Polymorphisms in the Embryonic lethal-abnormal vision-like 4 (ELAVL4) gene modify PD age-of-onset or susceptibility. Incidentally, a genetic screen in Drosophila identified an ELAVL homologue as required for LRRK2-induced pathology. Therefore, we hypothesized that LRRK2 phosphorylates ELAVL4 to control phenotypes relevant to PD. We discovered that three neuronal ELAVLs including ELAVL4 (also known as HuD) bind to, and post-transcriptionally regulate mRNA encoding α-synuclein and LRRK2. We also show that LRRK2 phosphorylates HuD and its homologues HuB and HuC. This controls binding of nELAVLs (i.e., HuB, HuC, and HuD) to mRNA and post-transcriptionally regulates mRNA abundance and splicing in the mouse midbrain. In mice, the complex interaction between HuD and Lrrk2 G2019S is associated with motor deficits, dopaminergic neuron loss, and accumulated α-synuclein protein levels. Targets of nELAVLs are also selectively misregulated in iPSC-derived neurons and tissues from PD patients. In a model of PD-relevant inflammation, we also show that the ubiquitously expressed ELAVL homologue, HuR, controls LRRK2 protein levels. We show that mice lacking Lrrk2 are more susceptible to an acute model of dextran sodium sulfate (DSS) chemical-induced colitis. Lrrk2-deficient mice treated with DSS also show accumulated α-synuclein in brain tissue. Using in vitro models and mouse tissue we show that LRRK2 controls HuR binding to RNA probes and to the proinflammatory cytokine Tnfa in colon tissue, and this has implications for intestinal pathology relevant to PD. Together, this suggests that misregulation of ELAVLs may be implicated in neurodegeneration and inflammation observed in Parkinson's disease.

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