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Title: The interplay of global chromosomal organisation, promoter-enhancer interactions and transcription
Author: Thiecke, Michiel
ISNI:       0000 0004 9354 0857
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2020
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All somatic cells within an organism contain the same genetic material, yet they display pronounced differences in function and morphology. Precise control of gene expression is of fundamental importance to allow cells to properly develop, maintain homeostasis, and respond to external stimuli. The first step in gene expression is transcription, which starts at the core promoter region. While core promoters are crucial for transcriptional initiation, they are insufficient for establishing complex tissue- and condition-specific gene expression patterns in multicellular organisms. Additional transcriptional control elements, such as gene enhancers, are required for this, with many such elements localising considerable distances away from their target promoters. Enhancers commonly convey their regulatory signals to target promoters by forming physical contacts with them through three-dimensional DNA looping, underpinning the importance of chromosomal organisation in transcriptional control. In recent years, the emergence of chromosome conformation capture and related methodologies has dramatically increased our understanding of chromosomal organisation. In particular, high-throughput Hi-C analyses across cell types have led to the identification of spatial genomic structures, including Topologically Associating Domains (TADs). In parallel, high-resolution versions of these technologies (such as 5C, CHiA-PET, HiChIP and Capture Hi-C) have detected multitudes of novel looping interactions, including connections between promoters and enhancers. The interplay between precise regulatory interactions, the higher-order chromosomal organisation, and their joint contribution to transcriptional control is incompletely understood and is the focus of this work. In the first part of this work, I take advantage of high-resolution Promoter Capture Hi-C (PCHi-C) data to investigate the localisation of promoter interactions with respect to TAD boundaries in human primary blood cells and cell-cycle synchronised HeLa cells. I show that the majority of promoter interactions originate at, and are constrained by TAD boundaries. However, a minority of promoter interactions appear to cross TAD boundaries in all analysed cell types. Furthermore, I identify genes with multiple TAD-boundary crossing interactions per promoter and present evidence that these interactions may be supported by transcriptional machinery. These results suggest a role for transcriptional machinery in shaping promoter interactions in a TAD independent manner. In the second part of this work, I investigate promoter interaction rewiring upon perturbations of architectural proteins. For this analysis, I use PCHi-C data from HeLa cells, in which cohesin or CTCF are rapidly depleted using Auxin-induced degradation. I show that promoter interactions that are lost, maintained, or gained upon cohesin depletion possess distinct distance profiles and relate to TAD organisation in markedly different ways. I demonstrate that promoter-interacting regions that are lost upon cohesin depletion associate with architectural proteins, while those that are maintained or gained show characteristics of enhancers. Finally, I show evidence for a functional role of cohesin-mediated interactions in transcriptional regulation. Collectively, this work reveals the interplay between TADs, promoter interactions and transcription, while suggesting that promoter interactions may be supported by TAD independent mechanisms.
Supervisor: Spivakov, Mikhail ; Corcoran, Anne Sponsor: MRC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Promoter interactions ; CTCF ; SCC1 ; Cohesin ; Topologically Associating Domains ; TADs ; PCHiC ; Promoter Capture Hi-C ; ATAC-seq ; ChIP-seq ; RNA-seq ; SLAM-seq ; RT-PCR ; qPCR ; Genome organisation ; Spatial genome ; Epigenetics ; Transcriptional regulation ; Transcriptomics ; Transcription ; Cell cycle synchronisation ; Targeted protein degradation ; Rapid protein degradation ; Auxin degron ; Sequencing ; Bioinformatics ; Data analysis ; Statistical analysis ; Regression ; LASSO regression ; Logistic regression ; Ordinal logistic regression ; Genome wide ; HeLa ; Haematopoietic cells ; BRD4