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Microrna-26A Targets Ten Eleven Translocation Enzymes and Is Regulated During Pancreatic Cell Differentiation

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Microrna-26A Targets Ten Eleven Translocation Enzymes and Is Regulated During Pancreatic Cell Differentiation MicroRNA-26a targets ten eleven translocation enzymes and is regulated during pancreatic cell differentiation Xianghui Fua,1, Liang Jina,1, Xichun Wanga, Angela Luoa, Junkai Hua,b, Xianwu Zhenga, Walter M. Tsarkc, Arthur D. Riggsa,b,2, Hsun Teresa Kua,b,2,3, and Wendong Huanga,b,2,3 aDepartment of Diabetes and Metabolic Diseases Research, bThe Irell and Manella Graduate School of Biological Sciences, and cTransgenic Mouse Core, Beckman Research Institute of City of Hope, Duarte, CA 91010 Contributed by Arthur D. Riggs, September 18, 2013 (sent for review June 16, 2013) Ten eleven translocation (TET) enzymes (TET1/TET2/TET3) and Expression of TETs and the genomic content of 5hmC vary thymine DNA glycosylase (TDG) play crucial roles in early embry- across tissues (13, 14). Interestingly, TET2 and TET3 are highly onic and germ cell development by mediating DNA demethylation. expressed in the murine adult pancreas (2), yet the pancreas has However, the molecular mechanisms that regulate TETs/TDG ex- lower genomic 5hmC levels than other adult tissues derived from pression and their role in cellular differentiation, including that endoderm, including liver and lung (4), suggesting dynamic DNA of the pancreas, are not known. Here, we report that (i) TET1/2/3 demethylation during pancreas development. Additionally, the and TDG can be direct targets of the microRNA miR-26a, (ii) murine miR-26 family is unique to vertebrates (24) and correlates with TETs, especially TET2 and TDG, are down-regulated in islets during the emergence of the pancreas during evolution (25). Therefore, postnatal differentiation, whereas miR-26a is up-regulated, (iii) we have begun to explore the potential involvement of miR-26a/ changes in 5-hydroxymethylcytosine accompany changes in TET TET in pancreas development. mRNA levels, (iv) these changes in mRNA and 5-hydroxymethylcy- Although the existence of adult pancreatic progenitor cells in tosine are also seen in an in vitro differentiation system initiated vivo remains debatable (26–29), we recently identified rare v progenitor cells in the adult pancreas that possess extensive self- with FACS-sorted adult ductal progenitor-like cells, and ( ) over- CELL BIOLOGY renewal and differentiation capacities in vitro (30). These adult expression of miR-26a in mice increases postnatal islet cell number + + in vivo and endocrine/acinar colonies in vitro. These results estab- progenitor cells, which are CD133 Sox9 , are enriched in the lish a previously unknown link between miRNAs and TET expres- ductal cell fraction and can activate Pdx1 and Ngn3 expression sion levels, and suggest a potential role for miR-26a and TET family before terminal differentiation into endocrine-like cells (30). proteins in pancreatic cell differentiation. Whether TETs, TDG, and miRNAs may affect differentiation of pancreatic cell lineages has not been tested. Here, we report that epigenetics | DNA methyaltion | 5hmC | posttranscriptional regulation | TETs and TDG are direct targets of miR-26a and the expression stem cell of TETs and miR-26a change in opposite directions during in vivo and in vitro pancreatic cell differentiation. en eleven translocation (TET) enzymes and thymine DNA Results Tglycosylase (TDG) are implicated in active DNA demethyla- – TETs and TDG Are Direct Targets of miR-26a. miRNA target genes tion (1 3). The three TET family enzymes oxidize 5-methylcytosine are likely to have relatively long and conserved 3′-UTRs (31). (5mC) in DNA to 5-hydroxymethylcytosine (5hmC), and sub- We noticed that TETs, as well as TDG, have long, evolutionarily sequently to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) conserved 3′-UTRs (Figs. S1 and S2), so we used the TargetScan (1, 2, 4, 5). TDG, a base excision repair glycosylase, replaces 5fC and5caCwithanunmodified cytosine via DNA repair (5, 6). De- Significance spite 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 Ten eleven translocation (TET) enzymes are recently identified primordial germ cells (2, 7–12), evidence for enzymatic demethy- proteins that mediate DNA demethylation, but their regulation lation by TET enzymes during differentiation of cells of later is still unknown. This paper indicates that a microRNA, miR-26a, stages, such as the postnatal and adult stem cells of various organs is able to directly target TETs and then modulate 5-hydrox- including pancreas, remains very limited (13–16). ymethylcytosine levels. Furthermore, regulation of TETs by MicroRNAs (miRNAs) are an abundant class of small, highly miR-26a is shown to promote pancreatic cell differentiation conserved noncoding RNAs that bind the 3′-untranslated regions both in vitro and in vivo. These results highlight a link between (UTRs) of protein-coding genes to suppress gene expression. a miRNA, DNA demethylation, and stem/progenitor cell dif- Accumulating data have demonstrated that miRNAs are critical ferentiation. It also suggests a potential approach to increase for many developmental and cellular processes, including organo- the efficiency of generating pancreatic endocrine cells in vitro genesis and differentiation (17). However, the role of miRNAs in through modulating miR-26a. TET expression and active DNA demethylation remains unclear. Three major cell lineages exist in the adult pancreas—duct, Author contributions: X.F., L.J., A.D.R., H.T.K., and W.H. designed research; X.F., L.J., X.W., acinar, and endocrine cells. The endocrine pancreas is composed A.L., J.H., and X.Z. performed research; X.Z. and W.M.T. contributed new reagents/ana- of several hormone-releasing cells, including the insulin-secreting lytic tools; X.F., L.J., A.D.R., H.T.K., and W.H. analyzed data; and X.F., L.J., A.D.R., H.T.K., beta cells and glucagon-secreting alpha cells. Many transcription and W.H. wrote the paper. factors are known to control pancreas development (18). For The authors declare no conflict of interest. example, the expression of pancreatic and duodenal homeobox 1 1X.F. and L.J. contributed equally to this work. (Pdx1) in embryonic foregut region induces pancreas commit- 2To whom correspondence may be addressed. E-mail: [email protected], [email protected], ment (19, 20), and those early progenitor cells have the potential [email protected]. to give rise to all three pancreatic lineages (21, 22). Subsequent 3H.T.K. and W.H. contributed equally to this work. activation of another transcription factor, neurogenin 3 (Ngn3), This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. restricts the lineage potential to endocrine cells (21, 23). 1073/pnas.1317397110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1317397110 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 algorithm (32) to search for miRNAs that could potentially 2A), we found that expression of the endogenous TETs and TDG regulate TETs and TDG. Strikingly, the miR-26 family (miR-26a was inhibited by miR-26a overexpression (Fig. 2B) and enhanced and miR-26b) has at least three putative target binding sites in by miR-26 knockdown (Fig. 2C). TET2 was the most responsive TETs and one in TDG, in both the human and mouse genomes and TDG the least responsive to miR-26a regulation, which (Fig. 1A and Fig. S1). This putative regulation is also present in correlates with the number of miR-26a target sites within their zebrafish, suggesting strong conservation (Fig. 1A and Fig. S1). 3′-UTRs (Fig. 1A). Consistently, levels of TET1, TET2, and Notably, miR-26 target sites are preferentially located near both TDG protein were decreased in miR-26a–overexpressing cells, ends of the 3′-UTR of TETs (Fig. S1), implying effective tar- but increased in miR-26a–depleted cells (Fig. 2 D and E). geting (33). Moreover, some miR-26 target sites are close to- Because all three TET proteins can convert 5mC to 5hmC, we gether (within 40 nt), which falls within the optimal distance for investigated whether miR-26a affects 5hmC levels. Dot blot analysis cooperative regulation (33, 34) (Fig. S1). of global 5hmC levels in genomic DNA revealed that miR-26a To directly test whether miR-26a targets TETs and TDG, we overexpression reduced 5hmC levels, whereas miR-26 depletion cloned the 3′-UTRs of TET1, TET2, TET3, and TDG down- significantly increased its levels (Fig. 2F). In contrast, global 5mC stream of a luciferase reporter, and cotransfected these reporter levels were not affected by miR-26a overexpression or depletion constructs along with miRNA precursors into the human cell line (Fig. S4), consistent with the previous report that small hairpin RNA HEK293T. Coexpression of miR-26a was found to effectively (shRNA)-mediated knockdown of TET1 had no obvious effect on down-regulate luciferase expression in constructs with these 5mC levels (2). The levels of 5hmC in HEK293 cells are less than 3′-UTRs (Fig. 1B). Overexpression of miR-181a, an miRNA that 0.5% of total 5mC (4), so the changes in level of 5hmC that we see does not have a putative target site in these 3′-UTRs, showed no would not be expected to affect global 5mC levels. Collectively, these repression on luciferase expression (Fig. S3). Mutations in the data demonstrate that miR-26a can directly regulate TETs/TDG. seed sequence of the predicted miR-26a binding sites within TET2 (Fig. 1C) or TDG (Fig. 1D) abolished the inhibitory miR-26a/TET Is Potentially Involved in Differentiation and Proliferation effects of miR-26 on luciferase expression, indicating TET2 and of the Developing Pancreas in Vivo. The mouse and human genomes TDG are directly regulated by miR-26a. harbor three distinct miR-26 loci (miR-26a-1, miR-26a-2, and miR-26b), which renders genetic loss-of-function analysis difficult. miR-26a Modulates Levels of TETs/TDG and 5hmC in HEK293T Cells.
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