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Genome-wide analysis identifies a functional association of Tet1 and Polycomb PRC2 in mouse embryonic stem cells but not in differentiated tissues

ABSTRACT: Recent studies have analyzed the distribution and role of 5-hydroxymethylcytosin (5hmC) in Embryonic Stem Cells (ESC). However, DNA hydroxymethylation occurs also in differentiated cells and it is significantly deregulated in cancer. Here we mapped 5hmC genome-wide profile in pluripotent ES cells in comparison to embryonic and adult differentiated cells. Comparative analysis of 5hmC genomic distribution with respect to gene expression reveals that 5hmC is enriched on the gene body of genes expressed at medium/high level and on TSS of genes not expressed or expressed at low level independently from the cell type. ESC showed a significant enrichment of DNA hydroxymethylation in active enhancers and bivalent promoters with respect to more differentiated cells as well as preferential association of Tet1 and 5hmC with promoter bound by Polycomb Repressive Complex 2 (PRC2). Furthermore, we show that in ESC PRC2 interacts with Tet1 and it is required for Tet1 recruitment to the chromatin and 5hmC deposition at developmental genes. Examination of 5hmC in ESC, MEF, Brain, Liver, shGFP ESC, and shSuz12ESC, and examination of expression in shGFP ESC and shSuz12ESC.

ORGANISM(S): Mus musculus

SUBMITTER: Francesco Neri

PROVIDER: E-GEOD-44566 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Genome-wide analysis identifies a functional association of Tet1 and Polycomb PRC2 in mouse embryonic stem cells but not in differentiated tissues

Project description:Recent studies have analyzed the distribution and role of 5-hydroxymethylcytosin (5hmC) in Embryonic Stem Cells (ESC). However, DNA hydroxymethylation occurs also in differentiated cells and it is significantly deregulated in cancer. Here we mapped 5hmC genome-wide profile in pluripotent ES cells in comparison to embryonic and adult differentiated cells. Comparative analysis of 5hmC genomic distribution with respect to gene expression reveals that 5hmC is enriched on the gene body of genes expressed at medium/high level and on TSS of genes not expressed or expressed at low level independently from the cell type. ESC showed a significant enrichment of DNA hydroxymethylation in active enhancers and bivalent promoters with respect to more differentiated cells as well as preferential association of Tet1 and 5hmC with promoter bound by Polycomb Repressive Complex 2 (PRC2). Furthermore, we show that in ESC PRC2 interacts with Tet1 and it is required for Tet1 recruitment to the chromatin and 5hmC deposition at developmental genes.

2013-08-10 | GSE44566 | GEO

Loss of Tet enzymes compromises proper differentiation of embryonic stem cells

Project description:Tet enzymes (Tet1/2/3) catalyze the conversion of 5-methylcytosine (5mC) to 5-hydroxy-methylcytosine (5hmC) and are dynamically expressed in various embryonic and adult cell types. While loss of individual Tet enzymes or combined deficiency of Tet1/2 allows for embryogenesis, the effect of complete loss of Tet activity and 5hmC marks in development has not been established. To define the role of Tet enzymes and 5hmC in development we have generated Tet1, Tet2 and Tet3 triple knockout (TKO) mouse embryonic stem cells (ESCs) and examined their developmental potential in vitro and in vivo. Combined deficiency of all three Tet enzymes led to complete depletion of 5hmC and impaired ESC differentiation as seen in poorly differentiated TKO embryoid bodies and teratomas. Consistent with impaired differentiation, TKO ES cells exhibited limited contribution to the chimeric embryos and could not support embryonic development in tetraploid complementation assays. Gene expression profiles and genome wide methylome analyses of TKO embryoid bodies revealed promoter hypermethylation and deregulation of genes implicated in embryonic development and differentiation. These findings suggest a requirement for Tet and 5hmC-mediated DNA demethylation in proper regulation of gene expression during differentiation of embryonic stem cells and development. Methylation patterns in tissue samples from a series of wt and Tet1/Tet2 DKO embryos, neonates and adults were generated using ethylated DNA immunoprecipitation with antibodies against 5mC (MeDIP) and 5hmC (hMeDIP) followed by deep sequencing.

2014-04-17 | E-GEOD-55049 | biostudies-arrayexpress

Transcriptional regulation of Tet1 during development

Project description:5-hydroxymethylcytosine (5hmC) is a recently discovered epigenetic modification that is lost in human cancers. Formation of 5hmC is catalysed by the Ten eleven translocation (TET) proteins that mediate the sequential oxidation of 5-methylcytosine (5mC) to 5hmC, leading to eventual DNA demethylation. Several mechanisms can lead to loss of 5hmC in cancers, including mutations in IDH or TET2 genes. However, little is known about the role of TET proteins and 5hmC in adult cells. Here, we report that TET1 downmodulation is required to permit adult cells to proliferate. TET1 is rapidly downmodulated in proliferating primary cells and in regenerating liver. TET1 silencing accelerates cell cycle progression while its constitutive expression inhibits cell growth. TET1 is a negative regulator of cell proliferation and it is regulated during development in tissue specific manner. These findings enlarge our knowledge on how one epigenetic modification such as the DNA hydroxymethylation mediated by TET1 is a key player on the control of cell proliferation. Examination of 5hmC in MEF at passage 0 and at passage 5.

2015-08-04 | E-GEOD-57250 | biostudies-arrayexpress

Combined deficiency of Tet1 and Tet2 is compatible with development but leads to epigenetic instability

Project description:Tet enzymes (Tet1/2/3) convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Tet1 and Tet2 mediate 5hmC generation in mouse embryonic stem cells (ESCs) and various embryonic and adult tissues. To investigate the effects of combined deficiency of Tet1 and Tet2 on pluripotency and development, we have generated Tet1 and Tet2 double knockout (DKO) ESCs and mice. DKO ESCs were depleted of 5hmC, but remained pluripotent with subtle defects in differentiation and changes in gene expression. Double mutant embryos and chimeras exhibited mid-gestation defects and postnatal DKO mice displayed partially penetrant neonatal lethality and stochastic perturbation of imprinting. Viable DKO animals developed normally to adulthood but had reduced 5hmC level, increased 5mC level and lacked 5hmC in germ cells. Nevertheless, DKO mice of both sexes were fertile with females having smaller ovaries and reduced fertility. Our data suggest that both Tet1 and Tet2 contribute to 5hmC levels during development. Their combined loss does not block differentiation and embryogenesis, but leads to partially penetrant embryonic and perinatal abnormalities and compromised viability. Moreover, the presence of substantial levels of 5hmC in DKO embryos and adult mice suggests a significant contribution of Tet3 in hydroxylation of 5mC during development. Methylation patterns in tissue samples from a series of wt and Tet1/Tet2 DKO embryos, neonates and adults were generated using methylated DNA immunoprecipitation with antibodies against 5mC (MeDIP) and 5hmC (hMeDIP) followed by deep sequencing.

2013-02-01 | E-GEOD-42396 | biostudies-arrayexpress

Control of 5mC and 5hmC by TET dioxygenases in human embryonic carcinoma cells [MBD-Seq]

Project description:The TET family of dioxygenases catalyze conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), but their involvement in establishing normal 5mC patterns during mammalian development and their contributions to aberrant control of 5mC during cellular transformation remains largely unknown. We depleted TET1, TET2, and TET3 by siRNA in a pluripotent embryonic carcinoma cell model and examined the impact on genome-wide 5mC and 5hmC patterns. TET1 depletion yielded widespread reduction of 5hmC, while depletion of TET2 and TET3 reduced 5hmC at a subset of TET1 targets suggesting functional co-dependence. TET2 or TET3-depletion also caused increased 5hmC, suggesting they play a major role in 5hmC removal. All TETs prevent hypermethylation throughout the genome, a finding dramatically illustrated in CpG island shores, where TET depletion resulted in prolific hypermethylation. Surprisingly, TETs also promote methylation, as hypomethylation was associated with 5hmC reduction. TET function was highly specific to chromatin environment: 5hmC maintenance by all TETs occurred at polycomb-marked chromatin and genes expressed at moderate levels; 5hmC removal by TET2 is associated with highly transcribed genes enriched for H3K4me3 and H3K36me3. Importantly, genes prone to hypermethylation in cancer become depleted of 5hmC with TET deficiency, suggesting the TETs normally promote 5hmC at these loci, and all three TETs are required for 5hmC enrichment at enhancers, a condition necessary for expression of adjacent genes. These results provide novel insight into the division of labor among TET proteins and reveal an important connection of TET activity with chromatin landscape and gene expression. Methylation and hydroxymethylation profiling by affinity-based high throughput sequencing

2014-06-14 | E-GEOD-51902 | biostudies-arrayexpress

Dnmt3L-dependent regulation of DNA methylation promotes stem cells differentiation toward primitive germinal cells [ChIP-seq]

Project description:The de novo DNA methyltransferase 3-like (Dnmt3L) is a catalytically inactive DNA methylase that has been previously shown to cooperate with Dnmt3a and Dnmt3b to methylate DNA. Dnmt3L is highly expressed in mouse embryonic stem cells (ESC) but its function in these cells is unknown. We here report that Dnmt3L is required for the differentiation of ESC into primordial germ cells (PGC) through activation of the homeotic gene Rhox5. By genome-wide analysis we found that Dnmt3L is a positive regulator of methylation at gene bodies of housekeeping genes and a negative regulator of methylation at promoters of bivalent genes. We demonstrate that Dnmt3L interacts with the Polycomb PRC2 complex in competition with the DNA methyltransferases Dnmt3a and Dnmt3b to maintain low the methylation level at H3H27me3 regions. Thus, in ESC, Dnmt3L counteracts the activity of de novo DNA methylases to keep low the level of DNA methylation at developmental gene promoters. Examination of Ezh2 ChIP-Seq in shGFP and shDnmt3L ESC. Examination of Dnmt3L ChIP-Seq in shGFP and shSuz12 ESC.

2013-09-27 | E-GEOD-49178 | biostudies-arrayexpress

Combined loss of Tet1 and Tet2 promotes B-cell, but not myeloid malignancies in mice

Project description:TET1/2/3 are methylcytosine dioxygenases regulating cytosine hydroxymethylation in the genome. Tet1 and Tet2 are abundantly expressed in HSC/HPCs and implicated in the pathogenesis of hematological malignancies. Tet2-deletion in mice causes myeloid malignancies, while Tet1-null mice develop B-cell lymphoma after an extended period of latency. Interestingly, TET1 and TET2 were often concomitantly down-regulated in acute B-lymphocytic leukemia. Here, we investigated the overlapping and non-redundant functions of Tet1/Tet2 in HSC maintenance and development of hematological malignancies using Tet1/2 double knockout (DKO) mice. DKO and Tet2-/- HSC/HPCs had overlapping and unique 5hmC and 5mC profiles and behaved differently. DKO mice exhibited strikingly decreased incidence and delayed onset of myeloid malignancies compared to Tet2-/- mice and in contrast developed lethal B-cell malignancies. Transcriptome analysis of DKO tumors revealed expression changes in many genes dysregulated in human B-cell malignancies, such as LMO2, BCL6 and MYC. These results highlight the critical roles of TET1 or TET2 individually and their cross-talks in the pathogenesis of hematological malignancies. Given the role of Tet proteins in 5mC oxidation, we employed a previously established chemical labeling and affinity purification method coupled with high-throughput sequencing (hMe-Seal) to profile the genome-wide distribution of 5hmC, as well as methylated DNA immunoprecipitation (MeDIP) coupled with high-throughput sequencing (MeDIP-seq) to profile 5mC using BM LK cells purified from young WT, Tet2-/- and DKO mice (6-10 wks old).

2015-11-12 | E-GEOD-73611 | biostudies-arrayexpress

Nucleosome repositioning links DNA (de)methylation and differential CTCF binding during stem cell development

Project description:During differentiation of embryonic stem cells, chromatin reorganizes to establish cell type specific expression programs. Here, we have dissected the linkages between DNA methylation (5mC), hydroxymethylation (5hmC), nucleosome repositioning and binding of the transcription factor CTCF during this process. By integrating MNase-seq and ChIP-seq experiments in mouse embryonic stem cells (ESC) and their differentiated counterparts with biophysical modeling, we find that the interplay between these factors depends on their large-scale genomic context. The mostly unmethylated CpG islands have a reduced nucleosome occupancy and are enriched in cell type-independent binding sites for CTCF. The few remaining methylated CpG dinucleotides are preferentially associated with nucleosomes. In contrast, outside of CpG islands most CpGs are methylated and their average density oscillates so that it is highest in the linker region between nucleosomes. Outside CpG islands binding of TET1, an enzyme that converts 5mC to 5hmC, is associated with labile, MNase-sensitive nucleosomes. Such nucleosomes are poised for eviction in ESCs and become stably bound in differentiated cells where the level of TET1 and 5hmCs goes down. In particular, this has a dramatic effect at variable CTCF sites enriched outside CpG islands, that are occupied by CTCF in ESCs but loose CTCF during differentiation. To rationalize this cell type dependent targeting of CTCF binding in competition with the histone octamer, we have developed a quantitative biophysical model, which allowed predicting differences in CTCF binding due to TET1/5hmC/5mC-dependent nucleosome repositioning.

2014-05-07 | GSE56938 | GEO

5-hydroxymethylcytosine: the sixth DNA base

Project description:We performed a meta analysis of publicly available TET1, 5mC, 5hmC and genome wide bisulfite profiling data mostly from mouse embryonic stem cells (ESC). Genome wide chromatin immunoprecipitation combined with deep sequencing (ChIP-seq) has revealed binding of the TET1 protein at CpG-island (CGI) promoters and at bivalent promoters. We show that TET1 also coincides with DNAseI hypersensitive sites (HS). Presence of TET1 at these THREE locations suggests that it may play a dual role: an active role at CpG-islands and DNAseI hypersensitive sites and a repressive role at bivalent loci. In line with the presence of TET1, significant enrichment of 5hmC but not 5mC is detected at bivalent promoters and DNaseI HS. Surprisingly, 5hmC is not detected or present at very low levels at CGI promoters notwithstanding the presence of TET1 at these loci. Our meta analysis suggest that asymmetric methylation is present at CA- and CT-repeats in the genome of some human ESC. Examination of the distribution of 5-methylcytosine and 5-hydroxymethylcytosine in the genome of mouse embryonic stem cells.

2011-11-24 | E-GEOD-31343 | biostudies-arrayexpress

Astrocytes Tet1 regulates neuronal development and cognition through modulating GluA1 in mice

Project description:Tet (Ten eleven translocation) mediated 5-hydroxymethylcytosine (5hmC) is highly enriched in the neuronal system and is involved in diverse biological processes and diseases. However, the function of 5hmC in astrocytes remains completely unknown. In the present study, we show that Tet1 deficiency alters astrocytes morphology and impairs neuronal function. Specific deletion of Tet1 in astrocytes impairs learning and memory ability of mice. Using 5hmC high-throughput DNA sequencing and RNA sequencing, we found the distribution feature of 5hmC in astrocytes and that Tet1 deficiency induces DNA hydroxymethylation regions (DhMRs) and alters gene expression. Mechanistically, we found that Tet1 deficiency leads to the abnormal Ca2+ signaling by regulating the expression of GluA1, which can be rescued by ectectopic expression of GluA1. Collectively, our findings suggest that Tet1 plays important functions by regulating astrocytes Ca2+ signaling.

2021-11-17 | GSE165370 | GEO

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