Combined Deficiency of Tet1 and Tet2 Causes Epigenetic Abnormalities but Is Compatible with Postnatal Development

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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...

Section: Discussionmentioning

confidence: 99%

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

Jin

Wang

et al. 2013

125

115

“…A variable combined immunodeficiency of unknown cause is the most prominent abnormality, and development of affected individuals is essentially normal; the facial dysmorphia characteristic of the disorder is mild in nature. The TET1 and TET2 proteins, which oxidize the methyl group of m 5 C, have been attributed important roles in genome demethylation, but mice that lack both proteins are largely viable and fertile (32). This would not be the case if TET1 and TET2 play important roles in programmed DNA demethylation.…”

Section: Genetic Evidencementioning

confidence: 99%

Notes on the role of dynamic DNA methylation in mammalian development

Bestor

Edwards

Boulard

2014

207

166

It has been nearly 40 y since it was suggested that genomic methylation patterns could be transmitted via maintenance methylation during S phase and might play a role in the dynamic regulation of gene expression during development [Holliday R, Pugh JE (1975) Science 187(4173):226-232; Riggs AD (1975) Cytogenet Cell Genet 14(1):9-25]. This revolutionary proposal was justified by "... our almost complete ignorance of the mechanism for the unfolding of the genetic program during development" that prevailed at the time. Many correlations between transcriptional activation and demethylation have since been reported, but causation has not been demonstrated and to date there is no reasonable proof of the existence of a complex biochemical system that activates and represses genes via reversible DNA methylation. Such a system would supplement or replace the conserved web of transcription factors that regulate cellular differentiation in organisms that have unmethylated genomes (such as Caenorhaditis elegans and the Dipteran insects) and those that methylate their genomes. DNA methylation does have essential roles in irreversible promoter silencing, as in the monoallelic expression of imprinted genes, in the silencing of transposons, and in X chromosome inactivation in female mammals. Rather than reinforcing or replacing regulatory pathways that are conserved between organisms that have either methylated or unmethylated genomes, DNA methylation endows genomes with the ability to subject specific sequences to irreversible transcriptional silencing even in the presence of all of the factors required for their expression, an ability that is generally unavailable to organisms that have unmethylated genomes. Structure of Genomic Methylation PatternsThe addition of a fifth base (5-methylcytosine or m 5 C) increases the maximum potential information content of DNA from 2 bits per base pair to 2.32 bits; the addition of naturally occurring oxidized forms of m 5 C (5-hydroxymethycytosine, 5-formylcytosine, and 5-carboxylcytosine) increases the information content still further, although m 5 C is much more abundant than the oxidized derivatives. The assembled and annotated fraction of the human genome contains ∼29 million CpG dinucleotides, each of which can exist in the methylated or unmethylated state.

“…1 compensation from Tet1, Tet2, or both. Even the double deletion of Tet1 and Tet2 has only minor consequences: a significant fraction of Tet1/Tet2 doubly deficient mice survive to adulthood, whereas the remainder succumb late in embryogenesis or shortly after birth (20). This relatively mild embryonic phenotype could reflect the potential involvement of Tet3, which is up-regulated compared with control (CTL) in embryonic stem (ES) cells, embryonic day 13.5 (E13.5) embryos, and adult brain and lung of Tet1/2 DKO mice (20).…”

mentioning

confidence: 99%

Simultaneous deletion of the methylcytosine oxidases Tet1 and Tet3 increases transcriptome variability in early embryogenesis

Kang

Lienhard

Pastor

et al. 2015

101

Dioxygenases of the TET (Ten-Eleven Translocation) family produce oxidized methylcytosines, intermediates in DNA demethylation, as well as new epigenetic marks. Here we show data suggesting that TET proteins maintain the consistency of gene transcription. Embryos lacking Tet1 and Tet3 (Tet1/3 DKO) displayed a strong loss of 5-hydroxymethylcytosine (5hmC) and a concurrent increase in 5-methylcytosine (5mC) at the eight-cell stage. Single cells from eight-cell embryos and individual embryonic day 3.5 blastocysts showed unexpectedly variable gene expression compared with controls, and this variability correlated in blastocysts with variably increased 5mC/5hmC in gene bodies and repetitive elements. Despite the variability, genes encoding regulators of cholesterol biosynthesis were reproducibly down-regulated in Tet1/3 DKO blastocysts, resulting in a characteristic phenotype of holoprosencephaly in the few embryos that survived to later stages. Thus, TET enzymes and DNA cytosine modifications could directly or indirectly modulate transcriptional noise, resulting in the selective susceptibility of certain intracellular pathways to regulation by TET proteins.DNA methylation | cholesterol biosynthesis | TET methylcytosine oxidases | 5-hydroxymethylcytosine | 5hmC