Different regulation of chromatin modification guide cellular reprogramming into a rate-limited step after initiation phase

12 Background. In the early and late stages of cell reprogramming to induced 13 pluripotent stem cells (iPSCs) ectopic expression of Oct4, Sox2, Klf4 and Myc 14 (OSKM) aroused two peaks of transcriptional and epigenetic change respectively. 15 However, it was relatively quiet in the intermediate stage. In this paper our aim is to 16 gain insight into the molecular events that occur after the initiation phase of 17 pluripotency induction. 18 Methods. GSE42379 containing 28 gene expression profiles and GSE42477 19 containing 10 genome binding/occupancy profiles of mouse embryonic fibroblasts 20 (MEF) were downloaded from GEO. These datasets included untreated MEFs, 21 OSKM-induced MEFs progressing and refractory to reprogram at 3, 6, 9, 12 day 22 post-transduction, and iPS cell lines. Differentially expressed genes (DEGs) were 23 identified between different cell lines. The Chip-seq peaks and putative target gene 24 were obtained from GeneProf website. Gene ontology analysis was performed on the 25 Ensemble website. 26 Result. Compared with the progressing cells, the refractory cells obtained more than 27 two times DEGs at 6 day post-transduction, in particular, down-regulated DEGs 28 related to cell cycle, cell adhesion and development were over 4 times of that in 29 progressing cells. The expression of the DEGs which could only be detected in 30 refractory cells at 6 day were traced back to day 3, and we found the expression of the 31 up-regulated DEGs at 3 day were higher in refractory cells, whereas the expression of 32 the down-regulated DEGs at 3 day were lower in refractory cells. The analysis of 33 histone modification in genome-wide and in DEGs showed that during the 34 reprogramming process the increase of bivalent sites in genome were mainly 35 attributed to gaining of H3K27me3 and losing of H3K4me3. Different regulation of 36 H3K27me3 in DEGs was the key to regulate the expression differently in progressing 37 and refractory cells. The expression of chromatin modifiers in the two cell populations 38 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.1991v1 | CC-BY 4.0 Open Access | rec: 22 Apr 2016, publ: 22 Apr 2016 2 were checked and found to be differential regulated at different time point during 39 reprogramming process. 40 Conclusion. Genes related to immune response, cell adhesion, DNA replication and 41 cell cycle in the refractory cells responded to the induction factor earlier than in the 42 progressing cells, which led to excessive conversion rate. We supposed that after 43 initiation phase cellular reprogramming required to undergo a rate-limited step guided 44 by different regulation of chromatin modification. 45