Browsing by Subject "Enhancer Elements, Genetic"
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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 Molecular Mechanisms and Functions of Estrogen Receptor Enhancers in Hormone-Dependent Gene Expression(2017-11-21) Murakami, Shino; Kim, Tae-Kyung; Kraus, W. Lee; Kliewer, Steven A.; Zhang, Chun-LiTranscription is a fundamental regulatory mechanism of biological processes in a range of physiological and pathological conditions. Transcription enhancers are DNA regulatory elements that regulate the expression of the target genes by accommodating transcription factor (TF) binding through sequence specificity. Estrogen receptor alpha (ERα) belongs to ligand-dependent nuclear receptor superfamily. Upon activation by estrogenic ligands, ERα binds to specific sites on chromatin, and assembles and activates enhancer complexes, which in turn lead to the transcription of target genes. Various molecular events have been associated with enhancer function, including coregulator recruitment, induction of enhancer-enriched histone modifications, nucleosome remodeling, enhancer-promoter chromatin interactions, and transcription activation at the enhancer, as well as the target gene promoter. However, we lack a clear understanding of the order of events, the specific roles of each coregulator and enhancer-enriched chromatin features, and the functional relationships among them. Using ERα in estrogen (E2)-regulated gene transcription as a model in combination with molecular and cellular biology, as well as genomic and computational approaches, my dissertation herein describes a series of studies elucidating the molecular mechanisms and functions of these evens that lead to ERα enhancer activation. Collectively, it demonstrates that (1) ERα enhancer assembly and activation is a dynamic process, (2) the temporally-defined recruitment and activation of key coregulators are required for successful activation of ERα enhancers, and (3) enhancer transcripts (eRNA) mark active enhancers. Lastly, I delineate the development of a new technology, single-cell Global Run-on Sequencing (scGRO-seq), to uncover the link between enhancer activity and target gene transcription at the single-cell level. Single-cell imaging and sequencing technologies have demonstrated the heterogeneous nature of gene expression and enhancer activity in a wide range of biological systems, including clonally-expanded populations of cultured cells. However, our understanding on the molecular basis of heterogeneous gene expression is limited because of a lack of technologies that allow us to simultaneously examine enhancer activity and target gene transcription at the single-cell level. scGRO-seq will overcome this problem by capturing active transcription at the enhancers, which is an indicative of enhancer activity, and at the target gene in the same cells.