Dissertation

Biological Applications of Chemically Modified Recombinant Histones

Josephson, Brian

2022

Overview

In eukaryotes, DNA is packaged into repeating units called nucleosomes, which consist of ~150 base pairs of DNA wrapped around eight proteins called histones. These histones are often laden with post-translational modifications (PTMs), and while many PTMs have been shown to have profound effects on gene expression and chromatin organization, most remain uncharacterized. This thesis strives to elucidate the functions of PTM containing histones. Chapter 1 describes a light-driven reaction that site-specifically inserts sidechains of interest into histones via radical addition to previously installed dehydroalanine residues, with a scope encompassing many native and PTM containing sidechains, as well as several reactive functional groups. Chapter 2 utilizes the reaction from Chapter 1, as well as others from our group, in several independent applications: The enzymatic removal of installed histone PTMs is tested, histone methylation and phosphorylation are assessed in the structural context of a nucleosome, PTM processing on fluorinated histone sidechains is tracked by 19F-NMR, and interaction partners are crosslinked to histones bearing electrophilic sidechains. Chapter 3 studies histones in more complex, living systems. The understudied ability of histones to penetrate cell membranes is investigated, revealing the incorporation of extracellular histones into the chromatin of nearby cells, provoking a strong inflammatory and immune response. Furthermore, several light-driven histone modification reactions are attempted in the translucent model organism, the zebrafish. In conclusion, this thesis describes powerful tools for modifying histones and applies them to relevant problems in epigenetics, revealing unknown roles and functions of histones and their PTMs.

• Publisher: ProQuest Dissertations & Theses