Browsing by Subject "Promoter Regions, Genetic"
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Item Aberrant DNA Methylation and Cancer: A Global Analysis of Promoter Hypermethylation in Human Lung Cancers(2006-12-20) Shames, David S.; Minna, John D.Tumor-acquired alterations in DNA methylation include both genome-wide hypomethylation and locus specific hypermethylation. Global loss of DNA methylation destabilizes chromatin architecture, augments genomic instability, and may reactivate repetitive element expression. Promoter hypermethylation often coincides with loss of heterozygosity at the same loci, and together these events can result in loss of function of the gene in tumor cells. The "rules" governing which genes are methylated during the pathogenesis of individual cancers are unknown; however, it is known that certain genes are methylated with high frequency in selected tumors, whereas others are methylated across most types of tumors. The objective of the work described below was to use global profiling platforms (RNA and DNA) to identify epigenetically modulated genes that may be involved in cancer pathogenesis and bring these to the point where they could be developed as targets for diagnostic and treatment strategies. Using a global expression profiling approach and pharmacological inhibition of the DNA methyltransferases, 132 genes were identified that have 5' CpG islands, are induced from undetectable levels by 5-aza-2'-deoxycytidine (5-aza) in multiple non-small cell lung cancer cell lines, and are expressed in untreated immortalized human bronchial epithelial cells. Methylation analysis of a subset (45/132) of these promoter regions in primary lung cancer (N=20) and adjacent non-malignant tissue showed that 31 genes had acquired methylation in the tumors, but did not show methylation in normal lung or peripheral blood cells. Promoter methylation of eight of these genes were studied in breast cancers (N=37), colon cancers (N=24), and prostate cancers (N=24) along with counterpart non-malignant tissues. We found that seven loci were frequently methylated in both breast and lung cancers, with four showing extensive methylation in all four epithelial tumors. The data presented below suggest that while tumors differ in their molecular genetic phenotypes and pathogenesis, there may be underlying similarities in the pathways they follow toward malignancy. Some of these similarities may be reflected in the methylation programs tumor cells engage, which in turn, provides an opportunity to exploit for therapeutic applications and diagnosis. The approaches described herein entail a systematic and reproducible method to identify novel methylation markers in a variety of cancers, and the results of these studies provide a basis for developing a generic set of methylation markers for early detection screening across common epithelial cancers.Item Congenital Heart Defect-Associated Enhancers Shape Human Cardiomyocyte Lineage Commitment(August 2023) Armendariz, Daniel Alejandro; Xu, Jian; Hon, Gary C.; Johnson, Jane E.; Munshi, Nikhil; Wu, JunAdvancements in whole genome sequencing have identified thousands of disease-associated variants which land within enhancer boundaries. As enhancers play critical roles in orchestrating gene networks throughout development, variants which disrupt enhancer function have been shown to contribute to developmental defects. However studying enhancer variants within a developmental context has been limited by a few key challenges. First, thousands of enhancer variants have been identified which could be causal for disease. Thus, a high-throughput approach is necessary to feasibly interrogate these elements. Second, enhancers function in a cell-type specific and spatiotemporal manner to regulate target gene expression. Perturbation of these enhancers thus requires an in vitro model that can phenocopy the lineage and context in which they are active. Addressing these points, I first identify 25 putative cardiac enhancers harboring variants identified in patients with congenital heart defects (CHD). Using a CRISPRi repression system, I perturb these putative enhancers in human embryonic stem cells (hESC) followed by differentiation towards cardiomyocytes (CM). This allows for the study of enhancer activity throughout the specification of the vital muscle cells of the cardiac system. I then perform single-cell RNA sequencing to identify diverse CM cell populations and assess the impact of enhancer perturbations on lineage specification. My analysis revealed 16 enhancers of known cardiac genes which, when perturbed, result in deficient CM differentiation. Genetic knockouts of two enhancers near TBX5 phenocopied the single-cell data and revealed enrichment of early CM populations resulting from depletion of later stages. My thesis provides a framework for single-cell enhancer screens within a developmental context and provides support for the biological relevance of the approach. I expect that the throughput of this methodology and the ease at which it can be adapted towards diverse developmental systems will provide an invaluable tool for future studies.Item Enzymatic Disassembly of Promoter Bound 7SK snRNP Drives Transcription Elongation of HIV and Cellular Genes(2015-11-19) McNamara, Ryan Philip; Cobb, Melanie H.; D'Orso, Iván; Conrad, Nicholas; Kahn, JeffreyGene expression of the human immunodeficiency virus (HIV) and cellular primary responsive genes (PRG’s) is regulated at the step of transcription elongation. Shortly after transcription initiation, RNA Polymerase II (Pol II) pauses and it only enters into productive elongation after inducible transcription factors (TF’s) recruit the P-TEFb kinase to phosphorylate Pol II in response to stimuli. To ensure tight regulation of this process, the majority of P-TEFb is held in a catalytically inactive form, reversibly bound to the 7SK small nuclear ribonucleoprotein (snRNP). In the absence of stimuli, the 7SK snRNP resides in both the nucleoplasm and promoter regions. However, an understanding of how TF’s capture P-TEFb from the 7SK snRNP at the promoter and the mechanism and purpose of localizing the 7SK snRNP to promoters has been largely unexplored. It was therefore my goal to biochemically and functionally characterize this pathway through the use of both the HIV encoded TF Tat and cellular TF’s such as nuclear factor kappa b (NF-κB). Detailed throughout this dissertation, I present the novel findings that HIV Tat and NF-κB function to recruit the PPM1G phosphatase to their targeted promoters, which dephosphorylates P-TEFb and triggers its release from the 7SK snRNP. Additionally, this extraction of P-TEFb from the 7SK snRNP occurs at promoters through the transcriptional regulator KAP1, which physically tethers the snRNP to promoters genome wide. Recruitment of the 7SK snRNP complex occurs after transcription initiation, allowing P-TEFb to be directly positioned for rapid extraction by TF’s upon stimuli and transferred onto the paused Pol II. The enzymatic uncoupling of P-TEFb from the promoter bound 7SK snRNP enables rapid Pol II elongation and gene expression in response to stimuli (for PRG’s) or in the presence of Tat (for HIV). Ultimately, these findings indicate that inducible transcription programs can rapidly respond to environmental cues through the localized positioning of elongation factors at promoters. Moreover, these findings illustrate that HIV has evolved to hijack a cellular gene expression program, thus leading to viral takeover of the host and progression of AIDS.