Abstract
DNA demethylation is characterized by the loss of methyl groups from 5-methylcytosine, and this activity is involved in various biological processes in mammalian cell development and differentiation. In particular, dynamic DNA demethylation in the process of somatic cell reprogramming is required for successful iPSC generation. In the present study, we reported the role of Rad50 in the DNA demethylation process during somatic cell reprogramming. We found that Rad50 was highly expressed in pluripotent stem cells and that Rad50 regulated global DNA demethylation levels. Importantly, the overexpression of Rad50 resulted in the enhanced efficiency of iPSC generation via increased DNA demethylation, whereas Rad50 knockdown led to DNA hypermethylation, which suppressed somatic cell reprogramming into iPSCs. Moreover, we found that Rad50 associated with Tet1 to facilitate the DNA demethylation process in pluripotent reprogramming. Therefore, our findings highlight the novel role of Rad50 in the DNA demethylation process during somatic cell reprogramming.
Highlights
DNA methylation is a major gene regulatory mechanism that is essential for mammalian development[1,2]
Mouse iPSCs and mouse embryonic stem cells (ESCs) were maintained on 0.2% gelatin-coated plates in culture medium consisting of DMEM/F12 supplemented with 15% FBS (Invitrogen), 1 mM L-glutamine (Invitrogen), 1% nonessential amino acids (Invitrogen), 1% penicillin-streptomycin, 0.1 mM β-mercaptoethanol (Sigma), LIF (Millipore), and with or without doxycycline at 37 °C
Rad[50] binding to methylated Oct[4] promoter To identify novel proteins that might be involved in demethylating DNA in cells that are in a pluripotent state, we initially attempted to detect proteins that bind to the methylated Oct[4] promoter in mouse ESCs
Summary
DNA methylation is a major gene regulatory mechanism that is essential for mammalian development[1,2]. 5-methylcytosine (5mC) is a major epigenetic modification that is generated by the addition of a methyl group to cytosine nucleotides in DNA by DNA methyltransferase (DNMT) family members (DNMT1, DNMT2, DNMT3A, DNMT3B, and DNMT3L)[3,4]. Over 98% of DNA methylation occurs in the CpG island of somatic cells. There is no DNA methylation in embryonic stem cells (ESCs)[4]. DNA demethylation is the process of removing methyl groups from the cytosines of DNA6. Demethylation of cytosines in DNA can be achieved through either passive or active mechanisms[6,7].
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