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Genome-Wide and Multi-Scale Mapping of DNA Supercoiling in Differentiating Human Stem Cells


Type

Thesis

Change log

Authors

Perez, Consuelo 

Abstract

The molecular programme of every cell is encoded in its genome. Human cells must organise and compact their genetic material at multiple levels to accommodate the two-meter-long DNA within the confined nuclear space of six microns. Chromatin folding is not only a cellular need, but is also crucial for gene regulation, and coordinates the precise rewiring of gene expression programmes in every cell type during developmental transitions. Therefore, our genome works in a dynamic rather than a static manner, allowing feedback between function and structure. However, how exactly the genome reaches a functional equilibrium in a particular cell type and the mechanisms for maintaining such balance remain incompletely understood.

Increasing understanding of the genome topology derived from mathematical descriptions, in silico and in vitro studies, to single-molecule approaches and functional experiments in cells, has revealed that the free energy stored as twist in the DNA molecule can be used to facilitate key biological processes. Accordingly, changes in the winding of DNA, known as supercoiling, were shown to impact gene expression and chromosome architecture. Nonetheless, its intrinsic dynamic and multifaceted nature, together with experimental limitations, have hindered the study of the timing and factors orchestrating supercoiling formation and dissolution across human chromosomes.

This thesis explores the cellular processes responsible for introducing DNA supercoiling, and its remodelling by topoisomerase enzymes, and addresses how supercoiling underlies chromatin contacts and genome organisation during cell fate transitions. Initially, we mapped DNA supercoiling dynamics genome wide by adapting bTMP-seq (Naughton et al., 2013, Achar et al., 2020) to high-throughput sequencing. This approach offered increased resolution compared to previous studies and allowed us to interrogate the entire human genome. By then integrating this dataset with a chromatin conformation capture Hi-C map, we studied DNA supercoiling across multiple scales: from kilobase genes, through larger-scale topologically associating domains, to megabase epigenomic compartments. Crucially, we observed the formation of supercoiling domains throughout the genome and revealed their temporal remodelling due to transcriptional activity and topoisomerase-mediated relaxation across cell differentiation.

In summary, our multi-omic study revealed widespread supercoiling dynamics in differentiating human stem cells, which likely contribute to the chromatin environment for gene regulation during developmental transitions, in conjunction with previously well-described chromatin features.

Description

Date

2023-10-08

Advisors

Sale, Julian

Keywords

genome organisation, human embryonic stem cells, supercoiling

Qualification

Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge