Student Theses and Dissertations

Date of Award

2021

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Funabiki Laboratory

Abstract

Mitotic chromosomes are scaled to the cell size to ensure effective chromosome segregation. Recent studies have shown how condensins and DNA topoisomerase II organize the mitotic chromosome. However, the regulation of these factors in maintaining proper chromosome size in different cell types remains a mystery. Here, I investigated the role of the linker histone variant H1.8 in regulating mitotic chromosome structure. I showed that H1.8 suppresses binding of condensins and topo II to mitotic chromatin in Xenopus egg extracts. Using an in vitro reconstitution system, I showed that H1.8 inhibits binding of purified condensins and topo II to nucleosome arrays. I also showed that condensin binding to nucleosome arrays is sensitive to magnesium dependent chromatin compaction. By using direct measurement of chromosome length, I then showed that H1.8 suppresses chromosome length solely through condensin I enrichment on chromatin. I then investigated the organization of Xenopus egg extract chromosomes using chromosome conformation capture technique Hi-C. Using Hi-C analysis, I showed that condensin I organizes both mitotic loops and loop layers of mitotic chromosomes and that H1.8 mediated suppression of condensin I increases both mitotic loop and layer sizes. This analysis also corroborates direct measurements of chromosome length. I also showed that nucleosome depletion results in further reduction in loop and layer sizes over H1.8 depletion. This suggests that chromosome length can be regulated by condensin I binding through competitive inhibition by both nucleosomes and linker histones. Mitotic chromosomes are organized in a rod to increase both physical rigidity of chromosomes and to ensure effective resolution. Using Hi-C data, I observed that both condensins play a role in maintaining chromosome rigidity and subsequently in maintaining chromosome individualization. I then showed that, like sister chromatid resolution, condensin activity drives topo II activity to continuously resolve interchromosomal links in mitosis. Since H1.8 suppresses both condensin and topo II, it suppresses chromosome individualization. I then go on to show that this suppression of chromosome individualization is necessary to maintain spindle integrity. Based on these data, I propose a model where mitotic chromosome length and individualization can be regulated by using linker histone stoichiometry on chromatin as a rheostat. As linker histones are a dynamic component of chromatin that have been shown to have extensive cell cycle dependent phosphorylation, I discuss the possibility that titrating linker histone stoichiometry on chromatin may be used as a mechanism to control the binding of DNA binding proteins in both interphase and mitosis and thus regulate cellular functions.

Comments

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

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