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

Background. In the early and late stages of cell reprogramming to induced pluripotent stem cells (iPSCs) ectopic expression of Oct4, Sox2, Klf4 and Myc (OSKM) aroused two peaks of transcriptional and epigenetic change respectively. However, it was relatively quiet in the intermediate stage. In this paper our aim is to gain insight into the molecular events that occur after the initiation phase of pluripotency induction. Methods. GSE42379 containing 28 gene expression profiles and GSE42477 containing 10 genome binding/occupancy profiles of mouse embryonic fibroblasts (MEF) were downloaded from GEO. These datasets included untreated MEFs, OSKM-induced MEFs progressing and refractory to reprogram at 3, 6, 9, 12 day post-transduction, and iPS cell lines. Differentially expressed genes (DEGs) were identified between different cell lines. The Chip-seq peaks and putative target gene were obtained from GeneProf website. Gene ontology analysis was performed on the Ensemble website. Result. Compared with the progressing cells, the refractory cells obtained more than two times DEGs at 6 day post-transduction, in particular, down-regulated DEGs related to cell cycle, cell adhesion and development were over 4 times of that in progressing cells. The expression of the DEGs which could only be detected in refractory cells at 6 day were traced back to day 3, and we found the expression of the up-regulated DEGs at 3 day were higher in refractory cells, whereas the expression of the down-regulated DEGs at 3 day were lower in refractory cells. The analysis of histone modification in genome-wide and in DEGs showed that during the reprogramming process the increase of bivalent sites in genome were mainly attributed to gaining of H3K27me3 and losing of H3K4me3. Different regulation of H3K27me3 in DEGs was the key to regulate the expression differently in progressing and refractory cells. The expression of chromatin modifiers in the two cell populations were checked and found to be differential regulated at different time point during reprogramming process. Conclusion. Genes related to immune response, cell adhesion, DNA replication and cell cycle in the refractory cells responded to the induction factor earlier than in the progressing cells, which led to excessive conversion rate. We supposed that after initiation phase cellular reprogramming required to undergo a rate-limited step guided by different regulation of chromatin modification.