GSBS Dissertations and Theses

ORCID ID

0000-0002-6602-6241

Publication Date

2019-07-12

Document Type

Doctoral Dissertation

Academic Program

Interdisciplinary Graduate Program

Department

Program in Systems Biology

First Thesis Advisor

Job Dekker

Keywords

Hi-C, 3D interactions stability

Abstract

Development and application of genomic approaches based on 3C methods combined with increasingly powerful imaging approaches have enabled high-resolution genome-wide analysis of the spatial organization of chromosomes in genome function. In this thesis, I first describe an updated protocol for Hi-C (Hi-C 2.0), integrating recent improvements that significantly contribute to the efficient and high-resolution capture of chromatin interactions. Secondly, I present an assessment of the epigenetic landscape and chromosome conformation around the MYC gene in acute myeloid leukemia (AML) cells before and after small molecule, AI-10-49, treatment. MYC is up-regulated upon inhibition of the RUNX1 repressor by the fusion oncoprotein CBFβ-SMMHC. Treatment of AML cells with AI-10-49 blocks the RUNX1-CBFβ-SMMHC interaction, restoring RUNX1 at MYC regulatory elements. We demonstrate that the established loop is maintained and exchange between activating and repressive chromatin complexes at the regulatory elements, rather than altered chromatin topology, mediates disruption of target gene expression.

Finally, Hi-C interaction maps represent the population-averaged steady-states. To understand the forces that promote and maintain the association of loci with specific sub-nuclear structures genome-wide, we developed liquid chromatin Hi-C. Detection of intrinsic locus-locus interaction stabilities and chromatin mobility are enabled by fragmenting chromosomes prior to fixation and Hi-C, thus removing strong polymeric constraints. Nuclear compartmentalization was found to be stable for average fragment lengths are 10-25 kb while fragmentation below 6kb led to a gradual loss of spatial genome organization. Dissolution kinetics of chromatin interactions vary widely for different domains and are analyzed in detail in the final chapter of this thesis., with lamin-associated domains being most stable, and speckle-associated loci most dynamic.

DOI

10.13028/x96d-xg55

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Available for download on Saturday, August 28, 2021

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