Functional Investigation Of Tetl During IPSC Induction Reveals An Important Role Of DNA Methylation And Hydroxymethylation In Reprogramming

During development from a zygote to an adult, unique epigenetic pattern is developed in different cell lines to regulate the characterized expression profiles and differentiation potential. Therefore, epigenetic states including modifications on DNA and histones must be tightly regulated for proper transitions in both programming and reprogramming process. A milestone in somatic cell reprogramming is achieved in 2006. The direct reprogramming of differentiated somatic cells to induced pluripotent stem cells (iPSCs) can be achieved through the overexpression of a set of defined transcription factors such as Oct4 (O), Sox2 (S), Klf4 (K) and c-Myc (M). However the inside molecular mechanism of iPS generation remained unclear, which impeded the further clinical application.Global epigenetic reprogramming of DNA and histones is crucial for the reprogramming process overall, and involves removal of the original somatic cell epigenetic landscape and establishment of a pluripotent stem cell-specific epigenetic landscape instead. DNA methylation has been proposed to function in the silencing of somatic genes and chromatin remodeling during iPSC generation and DNA demethylation appears to play an important role in reactivating pluripotency genes, which are hypermethylated and silenced in somatic cells, particularly in the late stages of the reprogramming process. Recently,5-hydroxymethylcytosine (5-hmC), hydroxylated from 5-methylcytosine (5mC) by Tetl-3 proteins, has been detected in a broad range of cell types and is thought to be involved in active and/or passive DNA demethylation. In our research we attend to get insight of the molecular mechanisms for Tetl-drived DNA demethylation in different system and also the global dynamics of DNA modification remodeling in Tetl-mediated somatic cell reprogramming.We demonstrate that the DNA hydroxylase Tetl facilitates traditional iPSC induction in a hydroxylase-dependent manner. We found that Tetl can act at the Oct4 locus to promote 5mC to 5hmC conversion and facilitates the DNA demethylation and transcriptional reactivation during OSKM iPSC induction. Moreover, Tetl (T) can replace Oct4 and initiate somatic cell reprogramming in conjunction with Sox2, Klf4 and c-Myc. We established the TSKM secondary reprogramming system and found the efficient reactivation of Oct4 is relying on Tetl and 5hmC during TSKM reprogramming. Since the process in TSKM secondary reprogramming is fast, which is as less as 7 days, we further used it to characterize the dynamic profiles of,5mC,5hmC and gene expression during reprogramming. Our analysis revealed that 5mC and 5hmC modifications both increased at an intermediate stage of the process, correlating with a transition in the transcriptional profile. We also found that 5hmC enrichment is involved in the demethylation and reactivation of genes and regulatory regions that are important for pluripotency. Our data indicate that changes in DNA methylation and hydroxymethylation play important roles in genome-wide epigenetic remodeling during reprogramming. The TSKM 2° reprogramming system that we have established will be a valuable tool for further investigation of the mechanisms of epigenetic remodeling involved in somatic cell reprogramming and for developing a systematic understanding of the events that occur in this exciting process.