A multitude of mechanisms are involved in regulating gene expression, with the dynamic reorganization of the chromatin network known to be of critical importance. Histone H4, an essential component of the nucleosome, holds a key position in chromatin processes. Recent research has established histone H4 as a novel disease gene family, with mutations leading to reduced viability and disruption of DNA damage repair process in Saccharomyces cerevisiae and disruption of zebrafish development through introduction of H4 mutations. A mutation involving the 91st lysine residue on the Histone H4 protein has been shown to have the most detrimental effects. We hypothesize that mutation of other lysines in the histone H4 protein may also disrupt important sites of post-translational modifications, leading to an altered chromatin state. Our project aim is to determine the impact of mutating key lysine residues of the H4 protein. We have established variant H4 expressing stable cell lines, using an inducible Flp-In™ T-REx™ system, that enables control over protein expression. Immunostaining and RT-qPCR studies show equivalent expression of variant on induction vs endogenous H4, with clear nuclear localization. Alterations in global RNA polymerase progression, observed through EU incorporation were observed for some variants. Alterations in chromatin accessibility and PTM patterns were explored using ATAC-Seq and CUT&RUN, combining the data to associate chromatin accessibility with the altered chromatin state observed for the lysine mutations.
Together, the results allow us to establish an understanding as to how mutating key lysine residues of Histone H4 influences mechanisms important for cellular function in turn enabling a greater comprehension of how these might be altered in development and disease.