Elucidating dynamics of histone H3 lysine 36 methylation using optogenetics

Chromatin plays fundamental roles in packaging the genome within the nucleus and in regulating biological processes such as transcription and DNA repair. In eukaryotes, DNA is wrapped around octamers of core histone proteins to form nucleosomes, which are further compacted into higher order structures. Histone proteins are chemically modified with post-translational modifications (PTMs), which are added and removed to regulate dynamic cellular processes. Dysregulation of histone PTMs, DNA packaging, and subsequent processes manifests in myriad human diseases. Thus, understanding how PTMs contribute to human biology and disease is paramount. Optogenetics, which harnesses genetically-encoded, light-responsive proteins to obtain precise spatiotemporal control of cellular processes, has emerged as an important tool in the determination of histone PTM establishment, function, and removal. This work aims to design a novel optogenetic tool, improve the function of this and other tools, and apply these tools to study dynamics of histone PTMs. To create a widely generalizable tool to study diverse biological processes, we generated the light induced nuclear exporter (LINX) system based on the photosensitive Avena sativa LOV2 (AsLOV2) domain from the photoreceptor phototropin 1 to control protein localization. We then identified mutations in the LOV2 domain that increase the efficacy of the photoswitch and demonstrated improved function in both the LINX system as well as the previously described light activated nuclear shuttle (LANS) system. We fused LINX to Bre1 (LINX-Bre1), a Saccharomyces cerevisiae (S. cerevisiae) histone E3 ubiquitin ligase responsible for histone H2B monoubiquitylation (H2Bub1), and fused LANS to Set2 (LANS-Set2), the sole histone H3 lysine 36 (H3K36) methyltransferase in S. cerevisiae, to achieve light-dependent control of chromatin modifiers. With these tools we observed the addition and removal of H2Bub1 and H3K36 methylation (H3K36me), conserved transcription-coupled histone PTMs that maintain chromatin integrity. Using LANS-Set2, we found rapid addition and removal of H3K36me, whereas in a strain lacking the H3K36 trimethyl-specific demethylase Rph1, we demonstrated slower turnover of H3K36 trimethylation (H3K36me3). Further, we used LANS-Set2 to examine H3K36me3 dynamics genome-wide. Together, these studies advance our understanding of dynamics of chromatin regulation and establish new optogenetic methods to study these dynamics.Doctor of Philosoph