DNA topology and Pol II CTD phosphorylation as stepwise regulators of chromatin architecture in differentiating human stem cells

During differentiation, pluripotent cells must commit to one of many possible lineages. This commitment is mediated by RNA polymerase II (Pol II), which is recruited by transcription factors, guided by enhancers, excluded from heterochromatin, and repositioned through DNA looping. While each of these features of Pol II regulation has been well studied in individual cell types, it remains unclear how a single genome can support hundreds of distinct fates. Combinatorial transcription factor binding explains part of this complexity, but it does not fully account for the diversity of regulatory programs, pointing to additional layers of encoding beyond sequence alone. In this thesis, I establish definitive endoderm differentiation as a model system to investigate how such encoding might operate, focusing on two complementary ideas. The first is that changes in DNA topology reshape the binding landscape for Pol II and its regulators, through supercoiling, non-B DNA formation, or altered loop anchors. The second is that Pol II activity is tuned by differentiation-specific phosphorylation of its C-terminal domain (CTD), which modulates interactions with co-transcriptional regulators and condensates. These mechanisms likely intersect: phosphorylated Pol II may be recruited to negatively supercoiled or secondary structure-forming sites, while negative supercoiling can also act as a readout of Pol II activity. To test whether DNA topology could play such a regulatory role, we mapped negative supercoiling (bTMP-seq) and non-B DNA structures (S1 END-seq) during differentiation. These maps revealed a critical window at the onset of endoderm commitment, 24 hours into the 72-hour time course, when negative supercoiling and secondary structure formation accumulated genome-wide. Although supercoiling was consistently transcription-dependent, its coupling to transcription was transiently amplified during this early window, suggesting that DNA topology is actively regulated during lineage commitment. To probe the relationship between transient supercoiling and Pol II regulation, I mapped genome-wide CTD phosphorylation states and integrated them with topological data. Highly supercoiled sites, often containing G-quadruplex structures, coincided with a transient wave of CTD threonine-4 phosphorylation (T4-P) during early differentiation. These regions showed increased nascent transcription, minimal processed RNA, and reduced topoisomerase recruitment – features consistent with unproductive initiation events that generate torsional stress. Many of these sites occurred in enhancer-like or enhancer-adjacent intergenic loci. To determine whether these supercoiled T4-P sites participate in higher-order chromatin regulation, I analysed their 3D interactions with other genomic features using Hi-C. Strikingly, they aligned with dynamic loop-extrusion anchors associated with enhancer-promoter communication. They also exhibited significant but short-lived protein-binding footprints, and were enriched for motifs of lineage-specifying transcription factors and looping factors. Together, these observations suggest that supercoiled T4-P sites act as transient regulatory elements that facilitate enhancer-promoter interactions. In parallel, Pol II at enhancers acquired distinct CTD modifications that were similarly connected to local DNA topology and higher-order chromatin architecture. In summary, DNA topology and Pol II phosphorylation are dynamically regulated during differentiation, and their interplay links local transcriptional activity with higher-order genome organisation. This coordination supports the stepwise assembly of cell type-specific transcriptional hubs – in which a gene, its enhancer, and novel intergenic elements are scaffolded into a functional regulatory unit – and illustrates a general principle by which genome topology and Pol II modifications together contribute to lineage specification.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2025

Author dc:creator

• Zeller, Andrew

Advisor dc:contributor.advisor

• Sale, Julian

• Pol II CTD
• Negative supercoiling
• Non-B DNA structures
• Genome organisation
• Definitive endoderm differentiation

Licence

• https://www.repository.cam.ac.uk/bitstreams/9fb17e06-4950-4461-a081-a6f5c1cae042/download
• https://creativecommons.org/licenses/by/4.0/

Language dc:language

eng

Author Identifier

0009-0008-8922-7987

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/395632