Designing Chromatin Substrates for Structural and Functional Analysis of Regulatory Complexes

Stoner, Ryan

Undergraduate Honors Thesis

Designing Chromatin Substrates for Structural and Functional Analysis of Regulatory Complexes Public Deposited

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Citeable URL: https://scholar.colorado.edu/concern/undergraduate_honors_theses/0k225c365

Abstract

The processes behind cellular differentiation and the maintenance of cellular identity are both thought to be regulated by chromatin modification. Furthering our understanding of these mechanisms could offer invaluable insights into both cancer and development. This study will focus on a specific group of proteins called polycomb repressive complexes, which help to control gene expression through the modifications they exert on chromatin. Polycomb group proteins consist of three families: PRC1 which deposits ubiquitin on H2AK119, PRC2 which mono-, di-, and tri-methylates H3K27, and PR-DUB which acts as a proofreading mechanism, removing misplaced ubiquitin on H2A. The exact mechanism by which PRC1, an E3 ubiquitin ligase, is recruited to and activated on chromatin is poorly understood. To better understand how PRC1 acts on chromatin, chromatin substrates were designed and assembled for experimental analysis. All four histones making up the histone octamer, as well as modified histones capable of crosslinking with ubiquitin, were cloned, expressed, and purified using a wide variety of biochemical techniques. This was used to form the nucleosome core particle which can undergo functional and structural assays in conjunction with PRC1-RYBP. This PRC1-RYBP was found to be active, capable of binding to CpG rich nucleosome sequences and modifying chromatin by ubiquitinating H2A. Preliminary tests that explored the interaction of PRC1-RYBP with the nucleosome found that PRC1-RYBP had a higher affinity for the nucleosome modified with ubiquitin on H2A than for the wild-type nucleosome. This affinity is higher than previously thought, binding much more tightly than other E3 ligases. More work involving PRC1, including additional activity and binding assays along with negative stain and cryo-EM structural analysis will continue to be carried out in the future, providing a deeper look into the critical role that PRCs play in controlling gene expression.

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