Molecular Mechanism Of Transcriptional Factors Mediated Passive DNA Demethylation

DNA methylation is an important epigenetic modification,and it plays a critical role in cell differentiation,embryonic development,pathology and other processes.Reprogramming of differentiated cells to pluripotent embryonic stem cell-like cells(iPS)is of great application value in pathological understanding of many disease and regenerative medicine.Cell reprogramming is associated with a global alteration of DNA methylation,including extensive DNA demethylation of pluripotency-related genes,DNA demethylation can be accomplished through active demethylation and passive demethylation.Active DNA demethylation is an TET-mediated oxidative demethylation process in which TET proteins progressively oxidize 5-methylcytosine(5mC)to 5-hydroxymethylcytosine(5hmC)and further oxidized forms,which are recognized by TDG and underwent base-excision repair pathway leading to the removal of methyl groups.Passive DNA demethylation is achieved through blocking DNA maintenance methylation during DNA replication,followed by an additional round of DNA replication.At present,little is known on the molecular mechanisms of passive DNA demethylation.Here we investigated whether the classical Yamanaka(OCT4,SOX2,KLF4,C-MYC)and NANOG could initiate DNA passive demethylation and,if does,the underlying mechanisms.By in vitro binding assays,we observed that C-MYC and NANOG can bind specific hemimethylated DNA sequences but poorly fully methylated DNA.We also find that C-MYC binds hemimethylated DNA competitively with UHRF1,where as NANOG can markedly inhibit the hemimethylated DNA-binding activity of UHRF1.In addition,we obserbed that OCT4,SOX2 and KLF4 can also compete with UHRF1 for binding hemimethylated DNA.Using Co-IP assay,we discovered that four factors and NANOG have weak interactions with UHRF1.These results indicated that these transcription factors especially NANOG may contribute to replication dependent passive DNA demethylation in vivo.In addition,we used DNMT3A/3B-double knockout Hela cells to conduct 5mC immunostaining assay and found that ectopic expression of NANOG or OCT4 can reduce the levels of global DNA methylation,whereas ectopic expression of SOX2 or KLF4 do not appear to affect global DNA methylation.By immunostaining we also observed that ectopic expression of NANOG and OCT4 reduced cellular levels of DNMT1.These findings suggest that the ESC key transcription factors may promote DNA passive demethylation through at least two different approches:inhibiting UHRF1 binding of hemimethylated DNA and downregulation of DNMT1 proteins.