Identification of MRG15 as barrier to somatic reprogramming
Epigenetic reprogramming requires depletion of 4 factors ; Oct4, Sox2, Klf-4 and c-Myc. We have 4 groups of samples. 2 group is collected as fibroblasts and it is referred as Day Zero. The other groups are collected at the sixth day of epigenetic reprogramming referred as Day Six. Day 18 of epigenetic reprogramming, %5- 15 of cell population transformed into pluripotent stem cells. At day six this percentage is lower.Our previous studies have showed that inhibition of H3K36me3 mark thusleading to H3K36me2 mark on chromatin) significantly increased reprogramming efficiency. A CRISPR Knock-Out Library was built to identify which of the H3K36me3 mark reader protein can phenocopy absence of K36me3 loss in terms of reprogramming efficiency. MRG15 Knock Out has showed the most similar increase in reprogramming as loss of K36me3 mark. It has been reported that MRG15 is responsible from alternative splicing of some genes. Our hypothesis is; in the presence of H3K36me3 mark MRG15 can bind this mark on chromatin and recruit proteins related to alternative splicing ( eg. PTBP1)and regulate alternative splicing. Overall design: We performed a gene expression profiling analysis and detection of differential alternative splicing analysis using data obtained from RNA-seq of dh1f cells expressing non targeting vector and lenticrisprv2 targeting MRG15. Both groups were collected pre and post OSKM treatment. post-OSKM group were sixth day of reprogramming.