Mitotic histone H3/H4 modifications and variants

Eukaryotic nuclear DNA is packaged into large structures known as chromatin. This structure`s fundamental unit is the nucleosome, formed by DNA wrapped around the histone proteins H2A, H2B, H3 and H4. A large number of posttranslational histone modifications have been described that are thought to affect most aspects of chromatin physiology. However, genetic analysis of histones is hampered by their requirement for viability, their multiple gene copies and the pleiotropic phenotypes of mutants. Thus, although several mitotic histone modifications are known, no direct roles have been established. Here, I propose a new approach that bypasses these problems by using Xenopus laevis egg extracts that recapitulate most aspects of chromosome physiology. I have developed a new method to deplete H3 and H4 from egg extracts. In add-back experiments, wild type and mutant versions of these histones will be analysed for their contribution to mitosis. Furthermore, I will biochemically analyse chromatin containing specific modification patterns to gain insight into their functional consequences. These experiments will help establish a mechanistic understanding of histone functions ensuring genome integrity during mitosis.