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Epigenetic modification of the mammalian genome by DNA methylation (5-methylcytosine) has a profound impact on chromatin structure, gene expression and maintenance of cellular identity (1). The recent demonstration that members of the Ten-eleven translocation (Tet) family of proteins can convert 5-methylcytosine to 5-hydroxymethylcytosine raised the possibility that Tet proteins are capable of establishing a distinct epigenetic state (2,3). We have recently demonstrated that Tet1 is specifically expressed in murine embryonic stem (ES) cells and is required for ES cell maintenance (2). Using chromatin immunoprecipitation coupled with high-throughput DNA sequencing, here we show in mouse ES cells that Tet1 is preferentially bound to CpG-rich sequences at promoters of both transcriptionally active and Polycomb-repressed genes. Despite an increase in levels of DNA methylation at many Tet1-binding sites, Tet1 depletion does not lead to downregulation of all the Tet1 targets. Interestingly, although Tet1-mediated promoter hypomethylation is required for maintaining the expression of a group of transcriptionally active genes, it is also involved in repression of Polycomb-targeted developmental regulators. Tet1 contributes to silencing of this group of genes by facilitating recruitment of PRC2 to CpG-rich gene promoters. Thus, our study not only establishes a role for Tet1 in modulating DNA methylation levels at CpG-rich promoters, but also reveals a dual function of Tet1 in promoting transcription of pluripotency factors as well as participating in the repression of Polycomb-targeted developmental regulators., The Tet protein family includes three members (Tet1-3), all of which have the capacity to convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in a 2-oxoglutarate- and Fe(II)-dependent manner (2,3). Consistent with [...]

Tet proteins and DNA demethylation The enzymes responsible for methylating DNA, a ubiquitous epigenetic modification, have been extensively studied. Less is known about demethylation. Here, Tet1, an enzyme that catalyses the conversion of 5-methylcytosine of DNA to 5-hydroxyl-methylcytosine, is shown to have a role in mouse embryonic stem-cell maintenance by maintaining the expression of Nanog and preserving the Nanog promoter in a hypomethylated state. This suggests that Tet1 has a role in regulating DNA methylation, as well as in embryonic stem-cell self-renewal and inner cell mass specification. TET1 is an enzyme that catalyses the conversion of 5-methylcytosine of DNA to 5-hydroxymethylcytosine, raising the possibility that it is involved in mediating DNA demethylation. These authors show that Tet1 is involved in mouse embryonic stem cell maintenance and specification of the inner cell mass. It is required to maintain both the expression of Nanog in embryonic stem cells and the Nanog promoter in a hypomethylated state, supporting a role for Tet1 in regulating DNA methylation. DNA methylation is one of the best-characterized epigenetic modifications.sup.1,2,3,4. Although the enzymes that catalyse DNA methylation have been characterized, enzymes responsible for demethylation have been elusive.sup.5. A recent study indicates that the human TET1 protein could catalyse the conversion of 5-methylcytosine (5mC) of DNA to 5-hydroxymethylcytosine (5hmC), raising the possibility that DNA demethylation may be a Tet1-mediated process.sup.6. Here we extend this study by demonstrating that all three mouse Tet proteins (Tet1, Tet2 and Tet3) can also catalyse a similar reaction. Tet1 has an important role in mouse embryonic stem (ES) cell maintenance through maintaining the expression of Nanog in ES cells. Downregulation of Nanog via Tet1 knockdown correlates with methylation of the Nanog promoter, supporting a role for Tet1 in regulating DNA methylation status. Furthermore, knockdown of Tet1 in pre-implantation embryos results in a bias towards trophectoderm differentiation. Thus, our studies not only uncover the enzymatic activity of the Tet proteins, but also demonstrate a role for Tet1 in ES cell maintenance and inner cell mass cell specification., Author(s): Shinsuke Ito [sup.1] [sup.2] , Ana C. D'Alessio [sup.1] [sup.2] , Olena V. Taranova [sup.1] [sup.2] , Kwonho Hong [sup.1] [sup.2] , Lawrence C. Sowers [sup.3] , Yi Zhang [...]

Abstract: The epigenome, which comprises chromatin, associated proteins, and the pattern of covalent modification of DNA by methylation, sets up and maintains gene expression programs. It was originally believed that [...]

Epigenetic modification of the mammalian genome by DNA methylation (5-methylcytosine) has a profound impact on chromatin structure, gene expression and maintenance of cellular identity. The recent demonstration that members of the Ten-eleven translocation (Tet) family of proteins can convert 5-methylcytosine to 5-hydroxymethylcytosine raised the possibility that Tet proteins are capable of establishing a distinct epigenetic state. We have recently demonstrated that Tet1 is specifically expressed in murine embryonic stem (ES) cells and is required for ES cell maintenance. Using chromatin immunoprecipitation coupled with high-throughput DNA sequencing, here we show in mouse ES cells that Tet1 is preferentially bound to CpG-rich sequences at promoters of both transcriptionally active and Polycomb-repressed genes. Despite an increase in levels of DNA methylation at many Tet1-binding sites, Tet1 depletion does not lead to downregulation of all the Tet1 targets. Interestingly, although Tet1-mediated promoter hypomethylation is required for maintaining the expression of a group of transcriptionally active genes, it is also involved in repression of Polycomb-targeted developmental regulators. Tet1 contributes to silencing of this group of genes by facilitating recruitment of PRC2 to CpG-rich gene promoters. Thus, our study not only establishes a role for Tet1 in modulating DNA methylation levels at CpG-rich promoters, but also reveals a dual function of Tet1 in promoting transcription of pluripotency factors as well as participating in the repression of Polycomb-targeted developmental regulators. [ABSTRACT FROM AUTHOR]

A conference paper about genome analysis which reveals Ten-eleven translocation- (Tet) and Thymine DNA glycosylase- (TDG) 5-methylcytosine oxidation dynamics is presented. It states that 5-formylcytosine (5fC)/5-carboxylcytosine (5caC) are removed by TDG and base excision repair (BER) to regenerate unmethylated cytosine. It further states that biological roles of DNA methylation can be recognized by comprehensive mapping of 5-methylcytosine (5mC) oxidation and BER pathway.

Recent studies have demonstrated that the Ten-eleven translocation (Tet) family proteins can enzymatically convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). While 5mC has been studied extensively, little is known about the distribution and function of 5hmC. Here we present a genome-wide profile of 5hmC in mouse embryonic stem (ES) cells. A combined analysis of global 5hmC distribution and gene expression profile in wild-type and Tet1-depleted ES cells suggests that 5hmC is enriched at both gene bodies of actively transcribed genes and extended promoter regions of Polycomb-repressed developmental regulators. Thus, our study reveals the first genome-wide 5hmC distribution in pluripotent stem cells, and supports its dual function in regulating gene expression. [ABSTRACT FROM AUTHOR]

Hundreds of transcription factors (TFs) are expressed in each cell type, but cell identity can be induced through the activity of just a small number of core TFs. Systematic identification of these core TFs for a wide variety of cell types is currently lacking and would establish a foundation for understanding the transcriptional control of cell identity in development, disease, and cell-based therapy. Here, we describe a computational approach that generates an atlas of candidate core TFs for a broad spectrum of human cells. The potential impact of the atlas was demonstrated via cellular reprogramming efforts where candidate core TFs proved capable of converting human fibroblasts to retinal pigment epithelial-like cells. These results suggest that candidate core TFs from the atlas will prove a useful starting point for studying transcriptional control of cell identity and reprogramming in many human cell types., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Paired-like homeodomain transcription factors (PITX) regulate the activity of pituitary hormone-encoding genes. Here, we examined mechanisms through which the family of PITX proteins control murine FSH beta-subunit (Fshb) transcription. We observed that endogenous PITX1 and PITX2 isoforms from murine LbetaT2 gonadotrope cells could bind a highly conserved proximal cis-element. Transfection of PITX1 or PITX2C in heterologous cells stimulated both murine and human Fshb/FSHB promoter-reporter activities, and in both cases, mutation of the critical cis-element abrogated these effects. In homologous LbetaT2 cells, the same mutation decreased basal reporter activity and greatly reduced activin A-stimulated transcription from murine and human promoter-reporters. Transfecting dominant-negative forms of PITX1 or PITX2C or knocking down PITX1 or -2 expression by RNA interference in LbetaT2 cells inhibited murine Fshb transcription, confirming roles for endogenous PITX proteins. Both PITX1 and PITX2C interacted with Smad3 (an effector of the activin signaling cascade in these cells) in coprecipitation experiments, and the PITX binding site mutation greatly inhibited Smad2/3/4-stimulated Fshb transcription. In summary, both PITX1 and PITX2C regulate murine and human Fshb/FSHB transcription through a conserved cis-element in the proximal promoter. Furthermore, the data indicate both common and distinct mechanisms of PITX1 and PITX2C action.