Discovery of Novel Readers for Interpretation of the Epigenome

Histone modifications, also known as covalent histone marks, such as acetylation, methylation, phosphorylation, ubiquitination and citrullination play critical role in chromatin regulation and maintenance. The maintenance and regulation of chromatin structure control different biological processes such as transcription, cell fate, DNA repair and recombination. Due to its important role in regulating vital biological processes, deregulation in the maintenance of histone marks have been shown to contribute to the onset and progression of certain diseases, including cancer, and neurodegenerative disorders. Therefore, a complete understanding of the mechanistic basis of histone modification mediated regulation of our genome has remained a critical objective of the post human genome era. Chromatin maintenance toolbox includes peptide–binding protein modules (often called readers) that interpret or read histone marks by binding to them. The binding provides anchorage of the maintenance machinery proteins onto the chromatin for initiating signaling cascades. For this reason, the discovery of a wide variety of readers that bind site-specific histone marks has dominated the quest for unraveling the mechanistic details of histone mediated signaling. Recent reports show that readers also bind histones bearing no modification (unmodified histones) for anchorage and the xiv introduction of covalent histone marks at such sites disrupt anchorage. Thus, interplay between binding of unmodified histones and introduction of histone marks also provide a means of regulating the signal. However, less is known about this mechanism as only few unmodified histone readers are known. Hence, a thorough understanding of all regulatory mechanisms of histone mediated signaling will remain incomplete without completing the inventory of protein modules that anchor onto unmodified histones and details of their functional roles. This dissertation therefore, focused on discovering peptide-binding domains of proteins in the human genome that interact with unmodified histones. Our overall results show that we have identified novel readers (KDM5B-PHD1 and BAZ1A-PHD) of histone H3 specifically anchoring on H3Arg2 residue. A binding assay that can conveniently quantify binding affinity was developed here. The assay can rapidly check for striking peptide binding behavioral differences between paralogs (KDM5B and KDM5D) and homologs. We observed binding differences between paralogs that provide a starting point to further probe differences in their genome wide anchorage/localization and hence, their differential role in gene regulation. For its distinct mode of peptide binding learnt here, KDM5B-PHD1 can be exploited as a site-specific diagnostic reagent