Use this URL to cite or link to this record in EThOS:
Title: A single-molecule view of CTCF-mediated DNA looping
Author: Bloznelyte, Kotryna
ISNI:       0000 0004 7658 0005
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2018
Availability of Full Text:
Access from EThOS:
Access from Institution:
Genome folding is a key aspect of nuclear processes in eukaryotic cells, underlying proper gene expression, orderly DNA replication and genomic stability. CTCF and cohesin have emerged as two key architectural proteins that mediate long-range DNA interactions and are essential for proper genome folding. Both proteins are found enriched at the base of chromatin loops and the borders of self-interacting chromatin domains, which are a major structural and functional feature of genome organisation in interphase. CTCF binding sites at the base of chromatin loops show a strong preference for convergent orientation along the linear DNA sequence. The mechanistic features underlying the role of CTCF in loop formation are incompletely understood. In particular, it is unclear how CTCF binding directionality controls loop formation. There is growing support from computational and experimental studies for the loop extrusion model, which proposes that a cohesin-based complex extrudes DNA loop until it is blocked by encountering convergently oriented CTCF sites. This thesis focuses on investigating molecular features of CTCF-DNA interaction to gain insight into how CTCF executes its functions in genome folding, and touches on the role of cohesin in this context. In particular, we investigated whether CTCF binding induces a bend in DNA, which might provide a mechanism for biasing loop formation or halting loop extrusion. We did not detect bending in single molecule imaging assays. However, we discovered that CTCF protein alone can induce DNA loop formation in vitro. The frequency of these looping events did not depend on the orientation of the CTCF binding sites, suggesting that additional factors are required to impose the preference for convergence in vivo. To investigate this further, we erformed mass spectrometry analysis to identify interaction partners of DNA-bound CTCF, and generated tools to study the particular contribution of cohesin towards CTCF-based DNA looping with single molecule fluorescence microscopy.
Supervisor: Merkenschlager, Matthias ; Rueda, David Sponsor: Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral