Investigating the role of p300/CBP HAT activity in embryonic stem cell pluripotency and differentiation

p300 and CBP are homologous proteins that possess histone acetyltransferase (HAT) activity. Both proteins play critical roles in various cellular processes including transcriptional activation. In this study, we aimed to investigate the effect of p300/CBP HAT inhibition on mouse embryonic stem cell (ESC) pluripotency and differentiation utilising the specific p300/CBP inhibitor, A-485. It was observed that p300/CBP catalytic inhibition reduced the growth of ESCs and elevated the proportion of cells in G1 phase of cell cycle. Transcriptome analysis following p300/CBP HAT inhibition revealed a significant alteration in gene expression at different time points. Transcriptional levels of pluripotency genes including Oct4, Nanog, Sox2, Esrrb among others were robustly downregulated. Interestingly, RNA-Seq analysis showed that several trophectoderm associated genes were upregulated in response to A-485 treatment. Moreover, these observations were accompanied by a reduction in acetylation levels of H3K18, H3K27 and H3K56 which are known substrates for p300/CBP. Assay for transposase-accessible chromatin using sequencing (ATAC-Seq) showed that chromatin accessibility was significantly changed following p300/CBP catalytic inhibition. Notably, the chromatin accessibility was reduced at the super-enhancer elements of pluripotency genes. To assess the effect of p300/CBP HAT inhibition on ESCs differentiation, a novel differentiation system known as gastruloids was utilised. Following p300/CBP inhibition, elongation of gastruloids was significantly blocked while expression of differentiation markers associated with elongated gastruloids was downregulated including Brachyury, Meox1 and Hox genes. Taking together, the data presented in this thesis suggests that p300/CBP HAT activity is indispensable for pluripotency and differentiation of ESCs.