Binding Affinity and E3 Ligase Activity Differences of Non-Canonical Polycomb Repressive Complex 1 (ncPRC1) Constructs

Judge, Emma Marie

Undergraduate Honors Thesis

Binding Affinity and E3 Ligase Activity Differences of Non-Canonical Polycomb Repressive Complex 1 (ncPRC1) Constructs Public Deposited

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Citeable URL: https://scholar.colorado.edu/concern/undergraduate_honors_theses/br86b504c

Abstract

Polycomb group (PcG) proteins function as chromatin-based transcriptional repressors that are essential for normal gene regulation during development. However, how these systems function to achieve transcriptional regulation remains poorly understood. Here, I work to express and purify three different constructs of the PcG protein complex non-canonical PRC1 (ncPRC1). This study was designed to uncover how the ncPRC1 proteins RYBP and PCGF1 help to recruit the catalytic RING1B subunit to nucleosomes and CpG islands in DNA as well as set a methodological basis for studying the E3 ligase activity of three ncPRC1 protein constructs. The study was conducted by utilizing baculovirus expression systems and various insect cell lines to express the protein complexes. A method for purification of these complexes was established and the final products were used to measure and quantify binding assays as well as an E3 ligase activity assay. The results showed that the ncPRC1 protein PCGF1 is crucial for high affinity binding to nucleosomes, however, its absence does not drastically change the binding affinity of ncPRC1 to CpG DNA. In addition, I showed that ncPRC1 is an active E3 ligase. These results are significant in which they set a methodological basis for studying the E3 ligase activity of all three protein complexes as well as help us to uncover the specific role played by RYBP and PCGF1 subunits in the recruitment and regulation of RING1B E3 ligase activity for transcriptional repression and gene regulation.

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