High-resolution analysis of regulatory interactions and nano-scale chromatin structures
by Abrar Aljahani
Date of Examination:2024-02-26
Date of issue:2024-09-11
Advisor:Dr. Marieke Oudelaar
Referee:Dr. Marieke Oudelaar
Referee:Prof. Dr. Argyris Papantonis
Persistent Address:
http://dx.doi.org/10.53846/goediss-10581
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Abstract
English
Despite having the same genetic blueprint, the development of a complex, multicellular organism from a single fertilized egg results in a wide variety of hundreds distinct cell types, each uniquely contributing to the organism’s intricate composition. This process requires precise decoding of the genetic material in the appropriate spatio-temporal developmental window, which is contingent upon the activity of cis-regulatory elements. These elements, often located far away from each other along the linear genome, must interact, directly or indirectly, within the nucleus’s three-dimensional space, where the genome is organized in a hierarchical manner. The objective of this thesis is to thoroughly examine these interactions under various scenarios to better understand the relationship between genome organization and gene regulation. To accomplish this, we developed Tiled-MCC, an MNase-based Chromosome Conformation Capture (3C) technique. This method has facilitated the generation of the most detailed 2D maps of localized chromatin structures currently available, offering an unprecedented resolution of up to 20 base pairs. In Chapter 1, I present the rationale behind Tiled-MCC and show its ultra-highresolution performance in investigating the roles of Cohesin and CTCF in fine-scale enhancer-promoter interactions and localized nano-scale chromatin structures. I show that Cohesin significantly contributes to the robustness of tissue-specific enhancer-promoter interactions, while CTCF is critical for the precision and specificity of these interactions. At the localized level, Tiled-MCC micro-topologies reveal Cohesin-independent intricate structures, in which CTCF retains its insulating properties, contrary to its loss of insulation at a broader scale when Cohesin is absent. In Chapter 2, I show that the acute depletion of the Mediator complex, alongside a comprehensive analysis of low-and high-resolution methods, underscores the significance of high-resolution techniques in revealing the role of the Mediator complex in maintaining fine-scale enhancer-promoter interactions, a detail previously concealed by the constraints of low-resolution methods. I further provide evidence that in the absence of the Mediator complex, Cohesin occupancy at enhancers is reduced accompanied with increased intragenic CTCF-CTCF interactions, suggesting the redistribution of Cohesin. Tiled-MCC mapping of superenhancer regions reveals Mediator-occupied interacting regions and their engagement with other elements in the same region. In Chapter 3, I present the role of the histone chaperone, FACT, in maintaining localized chromatin structure through employing Tiled-MCC to resolve the patterns of genome folding at the nucleosome level. In the absence of FACT, interactions along active gene bodies are increased, forming compacted and less organized 9 structures. This comes with the combined perturbation of the transcription elongation process as well as RNA Pol II pausing. In Chapter 4, I present a follow-up study on FACT and its association with CTCF and Cohesin. Further investigation of Cohesin and CTCF occupancy in the absence of FACT revealed their significant increased occupancy at active gene bodies, indicative of the exposure of CTCF motifs as a result of reduced competition against nucleosomes due to the loss of the canonical nucleosome structure and enrichment of sub-nucleosomal fragments. In the regions that were analyzed, these changes, however, did not propagate to affect enhancer-promoter interactions and large-scale chromatin structures, suggesting the perseverance of larger-scale chromatin topologies amid local nucleosome perturbations. Given that this thesis primarily concentrates on the development of a highresolution technique employed to closely inspect the roles of four main chromatinassociated factors, the introduction is dedicated to describing the fundamental concepts commonly pertinent to these areas: cis-regulatory elements, the layers of genome organization, and relevant implications of 3C methodologies, while the introduction section of the first three chapters provide an overview of the current knowledge regarding these factors, as well as highlighting the existing gaps in our understanding in terms of their functionality.
Keywords: gene regulation; 3D genome; enhancer-promoter interactions
