Exploring the Roles of cis- and trans-Factors in Modulating Higher Order Chromatin Structure and Gene ExpressionPublic Deposited
Gene expression is tightly regulated at the level of transcription through cooperation between cis-regulatory elements and trans-factors that bind to the regulatory elements. Together, these factors regulate the higher order chromatin structure which establishes domains that organize the genome and coordinate gene expression. However, the molecular mechanisms controlling transcription of individual loci within a topological domain (TAD) are not fully understood. The cystic fibrosis transmembrane conductance regulator (CFTR) gene provides a paradigm for investigating these mechanisms. CFTR occupies a TAD bordered by CTCF/cohesin binding sites within which are cell-type-selective cis-regulatory elements for the locus. We showed previously that intronic and extragenic enhancers, when occupied by specific transcription factors, are recruited to the CFTR promoter by a looping mechanism to drive gene expression. Here we use a combination of CRISPR/Cas9 editing of cis-regulatory elements and siRNA-mediated depletion of architectural proteins to determine the relative contribution of structural elements and enhancers to the higher order structure and expression of the CFTR locus. We found the boundaries of the CFTR TAD are conserved among diverse cell types and are dependent on CTCF and cohesin complex. Removal of an upstream CTCF-binding insulator alters the interaction profile, but has little effect on CFTR expression. Within the TAD, intronic enhancers recruit cell-type selective transcription factors and deletion of a pivotal enhancer element dramatically decreases CFTR expression, but has minor effect on its 3D structure. The focus of the thesis work then shifted to the human epididymis tissue, which plays a critical role in sperm maturation. This tissue is affected in male Cystic Fibrosis (CF) patients and contributes to the high incidence of male infertility. First, the transcriptome of distinct regions of the epididymis was characterized by RNA-seq. The results showed that the caput is functionally divergent from the corpus and cauda, which have very similar transcriptomes. Gene ontology analysis revealed that processes of ion transport, response to hormone stimulus and urogenital tract development are more evident in the caput, while defense response processes are more important in the corpus/cauda. Then the study is focused on a specific transcription factor, Androgen Receptor (AR), which is known to be crucial for male genital tract development. AR cistrome in caput epididymis cells were characterized by ChIP-seq and exhibited a distinct profile comparing to prostate tissues. Transcription factors including RUNX1 and CEBPB were shown to be potential co-factors of AR and contribute to the tissue specific AR binding in the caput epididymis. Finally, ADGRG2 gene was identified as a direct target of AR which plays important roles in the biological function of caput epididymis.
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