Identification of MRG15 (MORFL1) as a barrier to somatic cell reprogramming

Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) is specified by the expression of OCT4, SOX2, KLF4, and c-MYC transcription factors. However, reprogramming has a limited success rate, suggesting the presence of barriers to this conversion. Epigenetic mechanisms such as DNA methylation and post-translational modifications on histone tails can hinder reprogramming. Preliminary studies demonstrated that SETD2, H3 lysine 36 tri-methyltransferase hinders cellular reprogramming. A CRISPR/Cas9 screen on H3K36me3-reader proteins yielded promising results in reprogramming efficiency, specifically with MRG15. In this thesis, I investigated the molecular processes behind MRG15's role as a barrier to iPSC production. First, I examined the effect of a SETD2 inhibitor in cellular reprogramming. This inhibitor selectively inhibited SETD2 activity, decreased H3K36me3 levels, and, similar to SETD2 knockdown, increased reprogramming efficiency. Next, I tested MRG15 knockout fibroblasts for reprogramming efficiency, and it increased iPSC generation four-fold. Overexpression of wildtype MRG15 rescued the knockout phenotype, confirming that the increase in reprogramming efficiency is not caused by Cas9 off-target events. I hypothesized that MRG15 acts downstream of SETD2 to block cellular reprogramming. However, double knockout of MRG15 and SETD2 yielded an additive increase in iPSC generation, which suggests MRG15 is not a direct SETD2 downstream effector. Next, I created MRG15 knockout iPSCs and examined OCT4, SOX2, NANOG, and SSEA4 pluripotency markers. Results indicated that iPSC maintenance does not require MRG15. RNA sequencing was performed with MRG15 knockout fibroblast samples, and gene set enrichment analysis demonstrated that MRG15 knockout samples are significantly enriched in pluripotent gene sets. Additionally, MRG15 knock-out upregulated cell cycle-related gene set expression, which may indicate that MRG15 knockout enhances iPSC production through higher proliferation. MRG15 can also be found in the SIN3B/HDAC2 repressive histone deacetylation complex; therefore, in a final set of experiments, I observed that MRG15 knockout led to a four-fold increase in H3K14 acetylation. Taken together, these results demonstrate that MRG15 and SETD2 operate as obstacles to cellular reprogramming through separate mechanisms; inhibiting these factors enhances the currently poor efficiency of generating iPSCs.