Structural and Biochemical Characterization of Chromatin Enzymes

Open Access
Author:
Henrici, Ryan Christopher
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Song Tan, Thesis Supervisor
  • Teh Hui Kao, Honors Advisor
Keywords:
  • BRCA1
  • PRC1
  • cancer
  • chromatin
  • nucleosome
  • structural biology
  • gene regulation
Abstract:
Every cellular action from growth and division to differentiation and specialization occurs within a complex web of gene regulation and control. The pattern of genes turned on and off in any cell is in part determined by post-translational modifications of chromatin in nucleus of that particular cell. These modifications take on many forms and are catalyzed by special proteins generally classified as chromatin modifying enzymes. One family of these enzymes is the Polycomb Repressive Complex 1 (PRC1), which is involved in gene silencing. Among its important functions, Polycomb Repressive Complexes share a common ubiquitin ligase core which catalyzes histone ubiquitylation. Although the crystal structure of the human PRC1 ubiquitylation module consisting of the E2 ubiquitin conjugating enzyme, UbcH5c, in complex with the PRC1 E3 ubiquitin ligase proteins, Bmi1 and Ring1B, was recently solved, we had no structural information for how PRC1 proteins, or any other chromatin-modifying enzymes, recognize their nucleosomal targets. I have characterized the PRC1 ubiquitylation module in complex with its nucleosome substrate using fluorescent catalytic and binding studies as well as X-ray crystallography. This crystal structure is the first visualization of a chromatin enzyme in action and shows how a multisubunit enzyme can recognize the complex topology of the nucleosome. This structure also provides insights into how the structurally and functionally related tumor suppressor protein BRCA1 may function in the nucleus. Like Ring1B and Bmi1, BRCA1 and its binding partner BARD1 are RING-type E3 ligases that form a ubiquitylation module with UbcH5c to ubiquitylate the nucleosome. My biochemical characterization of this module was confounded by in vitro instability of the BARD1/BRCA1/UbcH5c E2-E3 trimer. However, by leveraging our PRC1 structure and two recent structures of ubiquitylation enzymes, I have determined how ubiquitin plays a pivotal role to enable the BRCA1 ubiquitylation module to bind to the nucleosome. Together, these studies provide new insight into the mechanism of BRCA1 and Ring1B-mediated nucleosome ubiquitylation implicated in cancer cell proliferation and stem cell self-regeneration.