Date of Award

2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Gerd A. Blobel

Second Advisor

Paul M. Lieberman

Abstract

Chromatin architecture and transcription are intricately linked. Perturbation of chromatin organization is known to cause miswiring of the genome with dire biological consequences. CCCTC-binding factor (CTCF) is a multi-functional DNA-binding protein that engages in both aspects in a highly context-dependent manner, but it remains to be explored to what extent the two activities are intertwined. To answer this question, we took a loss of function approach and assessed changes in genome organization and nascent transcription dynamics in an auxin-induced degron (AID) system. The first aspect of our exploration reveals key architectural properties of CTCF binding sites (CBSs). We show that CTCF at different CBSs is highly variable in resistance to degradation. The most persistent CBSs are associated with strong architectural features. Single-molecule tracking experiments further link chromatin residence time to CTCF degradation kinetics, suggesting that prolonged chromatin binding may underlie key architectural functions of CTCF. The second part addresses CTCF’s transcriptional functions. By combining acute degradation of CTCF and nascent transcription measurements, we find that CTCF does not present significant barriers to elongating RNA Polymerase II (Pol II) in the gene body. On the other hand, CTCF suppresses antisense but not sense transcription at hundreds of divergent promoters. Genome editing, chromatin conformation studies, and high-resolution transcript mapping revealed that precisely positioned CTCF directly suppresses the initiation of antisense transcripts (uasTrx), in a manner independent of its chromatin architectural function. Primary transcript RNA FISH uncovered that CTCF regulates uasTrx burst fraction rather than intensity and that sense/antisense co-bursting is highly disfavored, suggesting CTCF-regulated competition for transcription initiation. Overall, CTCF shapes the transcriptional landscape in part by suppressing upstream antisense transcription. In sum, the works in this thesis captures the contextual, functional and mechanical heterogeneity of CTCF and provides mechanistic insights into its functions as a chromatin organizer and transcription factor.

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