The role of chromatin hubs in the regulation of gene expression and maintenance of genomic integrity

by Magdalena Anna Karpińska
Doctoral thesis
Date of Examination:2024-11-20
Date of issue:2025-03-20
Advisor:Dr. Marieke Oudelaar
Referee:Dr. Marieke Oudelaar
Referee:Prof. Dr. Argyris Papantonis
Persistent Address: http://dx.doi.org/10.53846/goediss-11161

 

 

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Abstract

English

The correct development and functioning of complex organisms rely on precise spatiotemporal regulation of gene expression and on efficient maintenance of their genomic stability. Regulation of gene expression during development is controlled by cis-regulatory elements: promoters, enhancers, and boundary elements. These elements communicate in 3D chromatin structures in the nucleus. Maintenance of genomic stability relies on precise and efficient DNA damage repair. Damaged loci in the nucleus have been shown to form repair foci. Therefore, the regulation of gene expression and DNA repair are both dependent on the 3D organization of the genome in the nucleus. In the majority of current studies, genome structure is investigated with Chromosome Conformation Capture (3C) methods that measure pair-wise contacts in a population of cells. These approaches do not allow for detection of higher-order chromatin structures and therefore impair our understanding of how multiple chromatin elements interact in individual cells. The aim of this dissertation is to understand the function of cooperative multi-way chromatin structures in the context of gene expression and genomic maintenance. In order to do that, I used a recently developed multi-way 3C method called Tri-C, which can detect concurrent interactions between three chromatin fragments derived from single nuclei. In Chapter I of this dissertation, I characterize changes in chromatin architecture at high-throughput throughout stages of lymphoid-to-myeloid differentiation using Tri-C and other state-of-art genomics methods. I observe that acute depletion of the CTCF protein impairs the formation of higher-order structures, but does not have a strong effect on pair-wise enhancer-promoter interactions. This allows me to uncouple the function of pair-wise and higher-order enhancer-promoter interactions and demonstrate that gene regulation is dependent on pair-wise enhancer-promoter interactions, but not on their specific clustering into higher-order chromatin structures. In Chapter II of this dissertation, I characterize the formation of higher-order chromatin structures between induced double-stranded break (DSB) sites using Tri-C. I observe that induced DSBs form higher-order structures between DSBs located in cis, i.e., at the same chromosome, but also in trans, i.e., inter-chromosomally. Correlation of these findings with single-cell binding profiles of repair proteins shows that the formation of multi-way structures ensures efficient, cooperative repair of DSBs. In summary, studies presented in this dissertation advance our understanding of the role of genome structure in correct cell functioning, placing a particular focus on understanding multi-way, potentially cooperative interactions.
Keywords: chromatin architecture; gene regulation; genomic stability
 
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