PRDM14 promotes active DNA demethylation through the Ten-eleven translocation (TET)-mediated base excision repair pathway in embryonic stem cells

Abstract

Ten-eleven translocation (TET) proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). 5fC and 5caC can be excised and repaired by the base excision repair (BER) pathway, implicating 5mC oxidation in active DNA demethylation. Genome-wide DNA methylation is erased in the transition from metastable states to the ground state of embryonic stem cells (ESCs) and in migrating primordial germ cells (PGCs), although some resistant regions become demethylated only in gonadal PGCs. Understanding the mechanisms underlying global hypomethylation in naive ESCs and developing PGCs will be useful for realizing cellular pluripotency and totipotency. In this study, we found that PRDM14, the PR domaincontaining transcriptional regulator, accelerates the TET-BER cycle, resulting in the promotion of active DNA demethylation in ESCs. Induction of Prdm14 expression transiently elevated 5hmC, followed by the reduction of 5mC at pluripotency-associated genes, germlinespecific genes and imprinted loci, but not across the entire genome, which resembles the second wave of DNA demethylation observed in gonadal PGCs. PRDM14 physically interacts with TET1 and TET2 and enhances the recruitment of TET1 and TET2 at target loci. Knockdown of TET1 and TET2 impaired transcriptional regulation and DNA demethylation by PRDM14. The repression of the BER pathway by administration of pharmacological inhibitors of APE1 and PARP1 and the knockdown of thymine DNA glycosylase (TDG) also impaired DNA demethylation by PRDM14. Furthermore, DNA demethylation induced by PRDM14 takes place normally in the presence of aphidicolin, which is an inhibitor of G1/S progression. Together, our analysis provides mechanistic insight into DNA demethylation in naive pluripotent stem cells and developing PGCs.

MBD3L2 promotes Tet2 enzymatic activity for mediating 5-methylcytosine oxidation

“…There is some evidence that Tet2 activity can be enhanced by other factors. For example, it has been reported that PRDM14 induces DNA demethylation at that germlinespecific genes Piwil2 and Slc25a3, meanwhile, PRDM14 can also enhance the binding of Tet2 at or near the TSS of these genes (Okashita et al, 2014). Recent studies have shown that histone H3 peptide induces the activation of de novo DNA methyltransferase by removal of Dnmt3a-mediated autoinhibition, which stimulates a configuration change Wang et al, 2013).…”

Altering TET dioxygenase levels within physiological range affects DNA methylation dynamics of HEK293 cells

“…2,41 The transcriptional regulator PRDM14 interacts with TET1 and TET2 and enhances their recruitment to target loci promoting active DNA demethylation in embryonic stem cells. 43 Another example of differential TET interaction is the transcriptional repressor REST that interacts specifically with the neuronal isoform of TET3, but neither with other TET3 nor TET1 or TET2 proteins. 44 In contrast to the moderate TET1 overexpression in our study, Jin and colleagues 30 overexpressed TET1 at very high levels and also identified up-and downregulated genes.…”

Minimal role of base excision repair in TET-induced global DNA demethylation in HEK293T cells

“…Additionally, TET1-CD-induced demethylation events have a different genomic distribution from those induced by full length TET1 or other TET family members primarily through DNA replication-based passive pathway in the HEK293T cell model, suggesting the predominance of passive pathway in all TET-induced global demethylation processes. By contrast, the reported locus-specific DNA demethylation events were always achieved through active demethylation10,16,32 . Thus, passive and active demethylation pathways selectively function in TET-induced global and locus-specific DNA demethylation.…”