Student Theses and Dissertations

Date of Award

2018

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Funabiki Laboratory

Abstract

The major components of mitotic chromosomes including histones, topoisomerase II and condensin are known to compact and shape the chromatin into rod-like chromatids. However, a complete picture of the proteins involved in shaping mitotic chromatin is unsettled. Here, I perform mass spectrometry on chromatin isolated from Xenopus egg extracts. By comparing interphase and M phase, I reveal how the chromatin proteome is affected by the cell cycle. I find that although topoisomerase II associates with chromatin throughout the cell cycle, its catalytic activity is greatly enhanced in mitosis where it can act on nucleosomal substrates. In contrast, condensin is specifically recruited to M phase chromatin and prefers a non-nucleosomal substrate. I show that nearly all proteins involved in nucleosome assembly and remodeling are evicted from mitotic chromosomes, concurrent with a reduction in mitotic nucleosome assembly and discuss a role for these phenomena in shaping mitotic chromosomes. In analyzing my mass spectrometry data, I noticed that subunits of stoichiometric chromatin-bound complexes behaved similarly across a variety of conditions. Using this principle, I identify a novel nucleosome remodeling complex comprising HELLS and CDCA7, two proteins known to be causative for Immunodeficiency, centromeric region instability, facial anomalies (ICF) syndrome, a rare immunodeficiency disease. Consistent with previous literature, HELLS alone fails to remodel nucleosomes, but the HELLS-CDCA7 complex possesses robust nucleosome remodeling activity. CDCA7 is essential for loading HELLS onto chromatin, and CDCA7 harboring patient ICF mutations fails to recruit the complex to chromatin. Finally, I show that the HELLSCDCA7 complex is required for proper mitotic chromosome structure. Together, my study identifies a unique bipartite nucleosome remodeling complex where the functional remodeling activity is split between two proteins. These data lead me to a unifying model for ICF syndrome where all known ICF mutations converge on defective DNA methylation. I propose that HELLS-CDCA7 mediated remodeling of juxtacentromere heterochromatic nucleosomes facilitates DNMT3B-mediated methylation and discuss this model alongside alternative models for ICF syndrome. Finally, I consider the role of CDCA7 paralogs in differentially regulating HELLS, and present an outlook on the future of ICF syndrome research.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

Available for download on Thursday, March 05, 2020

Included in

Life Sciences Commons

Share

COinS