UC San Diego

Throughout mitosis, the cell's genome is faced with drastic structural and transcriptional alterations that it must overcome to faithfully propagate its transcriptional program to the next generation. The cell most likely reestablishes the interphase chromatin organization and gene expression program after mitosis with the help of epigenetic information encoded on the mitotic chromosomes (termed as mitotic bookmarking). A recent study comparing mitotic and interphase histone modifications discovered associations between local histone deacetylation and gene expression patterns, suggesting the acetylation state as a mitotic bookmark. This study hypothesizes that HDAC mediated, mitotic-specific, deacetylation of the nucleosome entering the TSS-associated NDR during mitosis, is bookmarking genes to reactivate earlier than others after mitosis. To test the roles of this local mitotic histone deacetylation, we would need a system that perturbs the suggested “bookmarking” deacetylation pattern in mitosis, to then measure the effects on reactivation kinetics. In this master’s thesis, I am developing and evaluating the potential of a CRISPR-dCas9 system to investigate the roles of mitotic histone deacetylation patterns in the NDR on transcription reactivation regulation.After establishing HeLa-S3 cell models expressing three different fusion proteins, VPR- dCas9 and WT/Mut p300 core-dCas9, I performed mitotic synchronization and ChIP-seq. I was able to probe chimera directed acetylation in mitotically synchronized and unsynchronized cells, looking at differences in activity when targeted to various regions in the NDR of a test gene. Across all the ChIP-seq experiments for both fusion proteins, the results displayed an interesting distinction in acetylation activity between the interphase and mitotic conditions when targeted to a certain region in the NDR of the test gene. While further research is required, my results indicate that the CRISPR-dCas9 system has the potential to specifically perturb local histone acetylation patterns both in interphase and mitosis, and serves as a viable option to study our mitotic bookmarking hypothesis.