Nucleosome repositioning links DNA (de)methylation and differential CTCF binding during stem cell development
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE56938 | GEO | 2014/05/07
SECONDARY ACCESSION(S): PRJNA245017
REPOSITORIES: GEO
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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. MNase-seq experiments for analysis of mononucleosomes were conducted as described previously (Teif et al. Nature Struct. Mol. Biol., 2012). In addition, a dinucleosome fraction also was gel purified and sequenced. Briefly, embryonic stem cells from 129P2/Ola mice were cultured in ESGRO complete medium (Millipore), harvested and resuspended in low salt buffer (10 mM Hepes, pH 8, 10 mM KCl, 0.5 mM DTT) at 4 M-BM-0C. After disruption of the cells with a douncer the nuclei were collected by centrifugation and washed once with the MNase Buffer (10 mM TrisM-bM-^@M-^SHCl, pH 7.5, 10 mM CaCl2), resuspended in the MNase Buffer and digested with 0.5 units MNase per microliter (Fermentas) and incubation for 6M-bM-^@M-^S11 minutes at 37 M-BM-0C. The MNase digestion was stopped by putting the samples on ice and adding EDTA to a concentration of 10 mM. After digestion with 0.1 M-BM-5g M-BM-5lM-bM-^@M-^S1 RNase A (Fermentas) and removal of protein by phenol and chloroform extraction, the DNA was ethanol precipitated and the resulting DNA pellet was dissolved in H2O. DNA fragments corresponding to mononucleosomes or dinucleosomes were separated on a 2 % agarose gel using an E-Gel electrophoresis system (Life Technologies). The libraries for sequencing were prepared according to the standard Illumina protocol. High-throughput paired-end sequencing of at least 50 bp read length was performed on the Illumina HiSeq 2000 platform at the DKFZ sequencing core facility in Heidelberg, Germany. We obtained about 150 million nucleosome positions per sequencing reaction. ChIP-seq experiments were conducted as described previously (Teif et al. 2012). For each sample, 1 x 106 cells were cross-linked with 1% PFA and cell nuclei were prepared using a swelling buffer (25 mM Hepes, pH 7.8, 1 mM MgCl2, 10 mM KCl, 0.1% NP-40, 1 mM DTT). Chromatin was sheared to mononucleosomal fragments. After IgG preclearance the sheared chromatin was incubated with 4 M-BM-5g of either H3K9me3 (Abcam ab8898) or H3K4me3 (Abcam ab8580) antibody over night. After washes with sonication (10 mM TrisM-bM-^@M-^SHCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5% N-lauroylsarcosine, 0.1% NaM-bM-^@M-^Sdeoxycholate), high-salt-buffer (50 mM Hepes pH 7.9, 500 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% NaM-bM-^@M-^Sdeoxycholate, 0.1% SDS), lithium buffer (20 mM TrisM-bM-^@M-^SHCl pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% NaM-bM-^@M-^Sdeoxycholate) and 10 mM TrisM-bM-^@M-^SHCl, chromatin was eluted from the protein G magnetic beads and the crosslink was reversed over night. After RNase A and proteinase K digestion, DNA was purified and cloned in a barcoded sequencing library for the Illumina HiSeq 2000 sequencing platform (single reads of 50 bp length).
2014-05-07 | E-GEOD-56938 | biostudies-arrayexpress
Project description:Coordinated changes of DNA (de)methylation, nucleosome positioning and chromatin binding of the architectural protein CTCF play an important role for establishing cell type specific chromatin states during differentiation. To elucidate molecular mechanisms that link these processes we studied the perturbed DNA modification landscape in mouse embryonic stem cells (ESCs) carrying a double knockout (DKO) of the Tet1 and Tet2 dioxygenases. These enzymes are responsible for the conversion of 5-methylcytosine (5mC) into its hydroxymethylated (5hmC), formylated (5fC) or carboxylated (5caC) forms. We determined changes in nucleosome positioning, CTCF binding, DNA methylation and gene expression in DKO ESCs, and developed biophysical models to predict differential CTCF binding. Methylation-sensitive nucleosome repositioning accounted for a significant portion of CTCF binding loss in DKO ESCs, while unmethylated and nucleosome-depleted CpG islands were enriched for CTCF sites that remained occupied. A number of CTCF sites also displayed direct correlations with the CpG modification state: CTCF was preferentially lost from sites that were marked with 5hmC in wild type cells but not from 5fC enriched sites. In addition, we found that some CTCF sites can act as bifurcation points defining the differential methylation landscape. CTCF loss from such sites, e.g. at promoters, boundaries of chromatin loops and topologically associated domains (TADs), was correlated with DNA methylation/demethylation spreading and can be linked to downregulation of neighbouring genes. Our results reveal a hierarchical interplay between cytosine modifications, nucleosome positions and DNA sequence that determines differential CTCF binding and regulates gene expression.
2019-03-25 | GSE114599 | GEO
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
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.
2011-11-24 | GSE31343 | GEO
Project description:The interplay of DNA (de)methylation and the architectural chromatin protein CTCF is an important regulator of cellular differentiation, but molecular mechanisms determining differential CTCF binding are not well understood. Mouse embryonic stem cells (ESCs) contain up to 10% of CpG residues in 5-hydroxymethylated form (5hmC) and smaller amounts in 5-formylated or 5-carboxylated forms (5fC, 5caC). Here, we studied double knock out ESCs (DKO) deficient for TET1 and TET2 enzymes which are responsible for the conversion of 5mC to 5hmC, 5fC and 5caC. For both wild type (WT) and DKO cells, we measured changes in nucleosome positions, CTCF binding, DNA methylation and gene expression. Our integrative data analysis combined with biophysical modelling suggests that both differential (de)methylation and CTCF binding are to a large degree predictable from the DNA sequence. Common CTCF sites that remained bound in DKO cells were significantly enriched at unmethylated CpG islands, while CTCF loss was associated with differential DNA methylation. 5hmCs and 5fCs had the opposite effects in terms of CTCF priming: CTCF was preferentially lost from sites that were marked in WT cells by 5hmCs but not 5fCs. Interestingly, the losses of CTCF at TAD boundaries lead to aberrant spreading of DNA methylation, as well as downregulation of neighbouring genes. Overall, our study clarifies the fundamental mechanism of differential CTCF binding and provides evidence of DNA sequence-encoded temporal chromatin changes during cell differentiation.
2019-03-26 | GSE110460 | GEO
Project description:DNA methylation of C5-cytosine (5mC) in the mammalian genome is a key epigenetic event that is critical for various cellular processes. However, how the genome-wide 5mC pattern is dynamically regulated remains a fundamental question in epigenetic biology. The TET family of 5mC hydroxylases, which convert 5mC to 5-hydroxymethylcytosine (5hmC), have provided a new potential mechanism for the dynamic regulation of DNA methylation. The extent to which individual Tet family members contribute to the genome-wide 5mC and 5hmC patterns and associated gene network remains largely unknown. Here we report genome-wide mapping of Tet1 and 5hmC in mESCs and reveal a mechanism of action by which Tet1 controls 5hmC and 5mC levels in mESCs. In combination with microarray and mRNA-seq expression profiling, we identify a comprehensive yet intricate gene network influenced by Tet1. We propose a model whereby Tet1 controls DNA methylation both by binding to CpG-rich regions to prevent unwanted DNA methyltransferase activity, and by converting the existing 5mC to 5hmC through its enzymatic activity. This Tet1-mediated antagonism of CpG methylation imparts differential maintenance of DNA methylation status at Tet1 target loci, thereby providing a new regulatory mechanism for establishing the epigenetic landscape of mESCs, which ultimately contributes to mESC differentiation and the onset of embryonic development. To determine the genome-wide DNA methylation changes caused by Tet1 depletion in mouse ES cells. Tet1 protein was depleted by specific siRNA treatment. The DNA methylation levels in control and Tet1 siRNA-transfected ES cells were determined by targeted bisulfite sequencing.
2011-04-21 | E-GEOD-28533 | biostudies-arrayexpress
Project description:DNA methylation of C5-cytosine (5mC) in the mammalian genome is a key epigenetic event that is critical for various cellular processes. However, how the genome-wide 5mC pattern is dynamically regulated remains a fundamental question in epigenetic biology. The TET family of 5mC hydroxylases, which convert 5mC to 5-hydroxymethylcytosine (5hmC), have provided a new potential mechanism for the dynamic regulation of DNA methylation. The extent to which individual Tet family members contribute to the genome-wide 5mC and 5hmC patterns and associated gene network remains largely unknown. Here we report genome-wide mapping of Tet1 and 5hmC in mESCs and reveal a mechanism of action by which Tet1 controls 5hmC and 5mC levels in mESCs. In combination with microarray and mRNA-seq expression profiling, we identify a comprehensive yet intricate gene network influenced by Tet1. We propose a model whereby Tet1 controls DNA methylation both by binding to CpG-rich regions to prevent unwanted DNA methyltransferase activity, and by converting the existing 5mC to 5hmC through its enzymatic activity. This Tet1-mediated antagonism of CpG methylation imparts differential maintenance of DNA methylation status at Tet1 target loci, thereby providing a new regulatory mechanism for establishing the epigenetic landscape of mESCs, which ultimately contributes to mESC differentiation and the onset of embryonic development. Tet1 protein was depleted in J1 or E14 mouse ES cells by siRNA or shRNA treatment. Total RNA was purified and used to determine the global gene transcription profiles by microarray assays. The Tet1-regulated genes were identified by comparing the gene expression profiles of control and Tet1-depleted ES cells.
2011-04-21 | E-GEOD-28530 | biostudies-arrayexpress
Project description:DNA methylation of C5-cytosine (5mC) in the mammalian genome is a key epigenetic event that is critical for various cellular processes. However, how the genome-wide 5mC pattern is dynamically regulated remains a fundamental question in epigenetic biology. The TET family of 5mC hydroxylases, which convert 5mC to 5-hydroxymethylcytosine (5hmC), have provided a new potential mechanism for the dynamic regulation of DNA methylation. The extent to which individual Tet family members contribute to the genome-wide 5mC and 5hmC patterns and associated gene network remains largely unknown. Here we report genome-wide mapping of Tet1 and 5hmC in mESCs and reveal a mechanism of action by which Tet1 controls 5hmC and 5mC levels in mESCs. In combination with microarray and mRNA-seq expression profiling, we identify a comprehensive yet intricate gene network influenced by Tet1. We propose a model whereby Tet1 controls DNA methylation both by binding to CpG-rich regions to prevent unwanted DNA methyltransferase activity, and by converting the existing 5mC to 5hmC through its enzymatic activity. This Tet1-mediated antagonism of CpG methylation imparts differential maintenance of DNA methylation status at Tet1 target loci, thereby providing a new regulatory mechanism for establishing the epigenetic landscape of mESCs, which ultimately contributes to mESC differentiation and the onset of embryonic development.
2011-04-21 | GSE28532 | GEO
Project description:DNA methylation of C5-cytosine (5mC) in the mammalian genome is a key epigenetic event that is critical for various cellular processes. However, how the genome-wide 5mC pattern is dynamically regulated remains a fundamental question in epigenetic biology. The TET family of 5mC hydroxylases, which convert 5mC to 5-hydroxymethylcytosine (5hmC), have provided a new potential mechanism for the dynamic regulation of DNA methylation. The extent to which individual Tet family members contribute to the genome-wide 5mC and 5hmC patterns and associated gene network remains largely unknown. Here we report genome-wide mapping of Tet1 and 5hmC in mESCs and reveal a mechanism of action by which Tet1 controls 5hmC and 5mC levels in mESCs. In combination with microarray and mRNA-seq expression profiling, we identify a comprehensive yet intricate gene network influenced by Tet1. We propose a model whereby Tet1 controls DNA methylation both by binding to CpG-rich regions to prevent unwanted DNA methyltransferase activity, and by converting the existing 5mC to 5hmC through its enzymatic activity. This Tet1-mediated antagonism of CpG methylation imparts differential maintenance of DNA methylation status at Tet1 target loci, thereby providing a new regulatory mechanism for establishing the epigenetic landscape of mESCs, which ultimately contributes to mESC differentiation and the onset of embryonic development.
2011-04-21 | GSE28533 | GEO
Project description:DNA methylation of C5-cytosine (5mC) in the mammalian genome is a key epigenetic event that is critical for various cellular processes. However, how the genome-wide 5mC pattern is dynamically regulated remains a fundamental question in epigenetic biology. The TET family of 5mC hydroxylases, which convert 5mC to 5-hydroxymethylcytosine (5hmC), have provided a new potential mechanism for the dynamic regulation of DNA methylation. The extent to which individual Tet family members contribute to the genome-wide 5mC and 5hmC patterns and associated gene network remains largely unknown. Here we report genome-wide mapping of Tet1 and 5hmC in mESCs and reveal a mechanism of action by which Tet1 controls 5hmC and 5mC levels in mESCs. In combination with microarray and mRNA-seq expression profiling, we identify a comprehensive yet intricate gene network influenced by Tet1. We propose a model whereby Tet1 controls DNA methylation both by binding to CpG-rich regions to prevent unwanted DNA methyltransferase activity, and by converting the existing 5mC to 5hmC through its enzymatic activity. This Tet1-mediated antagonism of CpG methylation imparts differential maintenance of DNA methylation status at Tet1 target loci, thereby providing a new regulatory mechanism for establishing the epigenetic landscape of mESCs, which ultimately contributes to mESC differentiation and the onset of embryonic development.
2011-04-21 | GSE28530 | GEO