Building a wiring diagram of the human genome

Abstract/Contents

Abstract

How cells utilize the same DNA to accomplish different functions is a central problem in cell biology. This process is thought to be controlled in large part by hundreds of thousands of small non-coding DNA elements, like enhancers, that turn protein-coding genes on and off across vast genomic distances. The dominant model invoked to explain how these interactions occur is through the formation of DNA loops that bring elements that lie far apart along the DNA polymer into spatial proximity. However, the wiring diagram of enhancer-gene looping, and the mechanisms by which these interactions form, are poorly understood. In this dissertation, I present my graduate work on (i) the comprehensive mapping of DNA looping between regulatory elements genome-wide; (ii) the dynamics of DNA looping in vivo; and (iii) the molecular mechanisms underlying the formation of DNA loops between regulatory elements. First, we develop in situ Hi-C, a DNA-DNA proximity ligation assay in the setting of intact nuclei, which we use to create kilobase resolution 3D maps of the human genome. These maps reveal a partitioning of the genome into roughly 10,000 loops. The loops are bound by the DNA-binding protein CTCF and the cohesin complex at convergently oriented CTCF-bound DNA motifs. Second, in order to explain this "convergent rule", we propose a model of loop formation by DNA extrusion, where the cohesin complex binds at a single locus and tracks in opposite directions along the DNA, extruding a loop, until it halts at barriers including inward-oriented CTCF-bound motifs. We test key predictions of the extrusion model. We show that it correctly predicts the detailed consequences that CRISPR genome-editing of CTCF motifs will have on loops. It also correctly predicts the effects of degrading cohesin: namely, rapid dissolution of loops genome-wide. We show that extrusion is ATP-dependent and moves at speeds of ~0.5-1kb/s. These models and estimates have since been validated, in detail, by numerous laboratories. Finally, we develop intact Hi-C, DNA-DNA proximity ligation in the setting of intact chromatin, which is much more sensitive to the presence and the position of loops. Using intact Hi-C, we comprehensively identify all loci capable of forming loops in the genome, including tens of thousands of enhancers, which we systematically link to their targets and connect to transcriptional consequences. By combining our comprehensive loop anchor annotations with perturbations of anchor associated proteins, we identify a novel mechanism for promoter-enhancer loop formation when cohesin loop extrusion is arrested at RNA polymerase II binding sites. Taken together, this work establishes a mechanistic basis for the formation of regulatory DNA loops in the human genome. It also lays the foundation for the generation of a comprehensive catalog of distal regulatory interactions in an organism.

Description

Type of resource	text
Form	electronic resource; remote; computer; online resource
Extent	1 online resource.
Place	California
Place	[Stanford, California]
Publisher	[Stanford University]
Copyright date	2022; ©2022
Publication date	2022; 2022
Issuance	monographic
Language	English

Creators/Contributors

Author	Rao, Suhas Surya Pilibail
Degree supervisor	Kornberg, Roger D
Thesis advisor	Kornberg, Roger D
Thesis advisor	Greenleaf, William James
Thesis advisor	Kundaje, Anshul, 1980-
Thesis advisor	Lieberman Aiden, Erez
Degree committee member	Greenleaf, William James
Degree committee member	Kundaje, Anshul, 1980-
Degree committee member	Lieberman Aiden, Erez
Associated with	Stanford University, Biophysics Program

Subjects

Genre	Theses
Genre	Text

Bibliographic information

Statement of responsibility	Suhas Rao.
Note	Submitted to the Biophysics Program.
Thesis	Thesis Ph.D. Stanford University 2022.
Location	https://purl.stanford.edu/hg728kg2113

Access conditions

Copyright

© 2022 by Suhas Surya Pilibail Rao

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