Understanding how loss of histone H3 lysine 36 trimethylation enhances somatic cell reprogramming

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) using a set of transcription factors (OCT4, SOX2, KLF4 and c-MYC). However, reprogramming of somatic cells to iPSCs suffers from a very low efficiency indicating the presence of barriers against reprogramming. Epigenetic factors regulating post-translational modifications of DNA and histones are among such barriers of reprogramming. In a preliminary study, SETD2 which is the only enzyme catalyzing histone H3 lysine 36 tri-methylation (H3K36me3) in mammals was shown to be a barrier of reprogramming. H3K36me3 is a histone mark deposited throughout gene bodies of actively transcribed genes. In this thesis, I investigated the molecular mechanisms of how H3K36me3 loss enhances iPSC generation. I proposed several hypotheses to understand how H3K36me3 loss increases reprogramming efficiency. First, by analyzing previously generated RNA-sequencing data, I considered four transcription factors (CITED2, EBF3, SMAD3 and FOSL1) downregulated by SETD2 knockdown as candidate SETD2-downstream genes. I tested whether overexpression of these transcription factors could reverse the increased reprogramming phenotype of SETD2 knockdown. I showed that none of these transcription factors could alone decrease the increased reprogramming efficiency by SETD2 knockdown. In a second line of experiments, I carried out chromatin immunoprecipitation (ChIP) for active H3K36me3 and repressive H3K27me3 marks, which are deposited in a mutually exclusively manner on the tails of histone H3. I analyzed the distribution of H3K27me3 on the genes downregulated upon SETD2 knockdown to investigate whether H3K36me3 loss could lead to H3K27me3 deposition. However, ChIP-qPCR results did not indicate a reciprocal relationship between these two marks on the selected genes such as SMAD3, LPAR1, EBF3 and SFRP1. Lastly, I performed a CRISPR/Cas9-based knockout screen targeting all known H3K36me3 readers in mammals to define the roles of these readers in somatic cell reprogramming. H3K36me3 readers are involved in diverse cellular processes such as alternative splicing, DNA repair, transcription elongation and DNA and histone methylation. Therefore, I aimed to find which H3K36me3 reader could phenocopy SETD2 depletion in reprogramming. CRISPR-based screen demonstrated that PSIP1, MRG15, MSH6, MSL3, NSD2 and NSD3 are barriers against reprogramming as their knockout led to increased reprogramming efficiency. These H3K36me3 readers were validated as barriers to reprogramming and MRG15, the H3K36me3 reader associated with regulation of alternative splicing was chosen for function-related assays. Therefore, I analyzed the effect of SETD2 knockdown on alternative splicing of key factors known to be significant for reprogramming with the goal of understanding whether SETD2 inhibition promotes reprogramming by regulating alternative splicing. Through RT-qPCR analysis, I observed that both SETD2 knockdown and MRG15 knockout individually led to a significant increase in the pluripotent cell-specific variant of MBD2 expression. Taken together, these findings identified the factors serving as barrier against somatic cell reprogramming through reading trimethylation of H3K36 residue and alternative splicing of MBD2 gene as an underlying mechanism for enhanced reprogramming by H3K36me3 loss.