MicroRNA-26a targets ten eleven translocation enzymes and is regulated during pancreatic cell differentiation

Abstract

Ten eleven translocation (TET) enzymes (TET1/TET2/TET3) and thymine DNA glycosylase (TDG) play crucial roles in early embryonic and germ cell development by mediating DNA demethylation. However, the molecular mechanisms that regulate TETs/TDG expression and their role in cellular differentiation, including that of the pancreas, are not known. Here, we report that (i) TET1/2/3 and TDG can be direct targets of the microRNA miR-26a, (ii) murine TETs, especially TET2 and TDG, are down-regulated in islets during postnatal differentiation, whereas miR-26a is up-regulated, (iii) changes in 5-hydroxymethylcytosine accompany changes in TET mRNA levels, (iv) these changes in mRNA and 5-hydroxymethylcytosine are also seen in an in vitro differentiation system initiated with FACS-sorted adult ductal progenitor-like cells, and (v) overexpression of miR-26a in mice increases postnatal islet cell number in vivo and endocrine/acinar colonies in vitro. These results establish a previously unknown link between miRNAs and TET expression levels, and suggest a potential role for miR-26a and TET family proteins in pancreatic cell differentiation.T en eleven translocation (TET) enzymes and thymine DNA glycosylase (TDG) are implicated in active DNA demethylation (1-3). The three TET family enzymes oxidize 5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine (5hmC), and subsequently to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) (1, 2, 4, 5). TDG, a base excision repair glycosylase, replaces 5fC and 5caC with an unmodified cytosine via DNA repair (5, 6). Despite these advances, the molecular mechanisms underlying TETs/ TDG regulation are still not known. In addition, although recent data suggest a role of TET and 5hmC in embryonic stem cells and primordial germ cells (2, 7-12), evidence for enzymatic demethylation by TET enzymes during differentiation of cells of later stages, such as the postnatal and adult stem cells of various organs including pancreas, remains very limited (13-16).MicroRNAs (miRNAs) are an abundant class of small, highly conserved noncoding RNAs that bind the 3′-untranslated regions (UTRs) of protein-coding genes to suppress gene expression. Accumulating data have demonstrated that miRNAs are critical for many developmental and cellular processes, including organogenesis and differentiation (17). However, the role of miRNAs in TET expression and active DNA demethylation remains unclear.Three major cell lineages exist in the adult pancreas-duct, acinar, and endocrine cells. The endocrine pancreas is composed of several hormone-releasing cells, including the insulin-secreting beta cells and glucagon-secreting alpha cells. Many transcription factors are known to control pancreas development (18). For example, the expression of pancreatic and duodenal homeobox 1 (Pdx1) in embryonic foregut region induces pancreas commitment (19,20), and those early progenitor cells have the potential to give rise to all three pancreatic lineages (21,22). Subsequent activation of another transcription factor, neurogenin 3 (N...

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