5-hydroxymethylcytosine is associated with enhancers and gene bodies in human embryonic stem cells.
ABSTRACT: Hydroxymethylcytosine (5hmC) was recently found to be abundantly present in certain cell types including embryonic stem cells. The function of 5hmC is poorly understood. Here we have generated a genome-wide map of 5hmC in human embryonic stem cells (hESCs) by hydroxymethyl-DNA immunoprecipitation followed by massively parallel sequencing (hmeDIP-seq). We found that 5hmC is enriched over enhancers as well as gene bodies, suggesting a potential role of 5hmC in gene regulation. Consistent with localization of 5hmC at enhancers, 5hmC was significantly enriched in histone modifications associated with enhancers such as H3K4me1 and H3K27ac. 5hmC was enriched in other protein-DNA interaction sites such as OCT4 and NANOG binding sites. Furthermore we found that 5hmC regions tend to be GC-skewed (excess G over C on one strand of DNA). These findings suggest that 5hmC may be targeted to certain genomic regions based both on gene expression and sequence composition. Overall design: 2 experiments, 2 controls
INSTRUMENT(S): Illumina Genome Analyzer II (Homo sapiens)
Project description:Hydroxymethylcytosine (5hmC) was recently found to be abundantly present in certain cell types including embryonic stem cells. The function of 5hmC is poorly understood. Here we have generated a genome-wide map of 5hmC in human embryonic stem cells (hESCs) by hydroxymethyl-DNA immunoprecipitation followed by massively parallel sequencing (hmeDIP-seq). We found that 5hmC is enriched over enhancers as well as gene bodies, suggesting a potential role of 5hmC in gene regulation. Consistent with localization of 5hmC at enhancers, 5hmC was significantly enriched in histone modifications associated with enhancers such as H3K4me1 and H3K27ac. 5hmC was enriched in other protein-DNA interaction sites such as OCT4 and NANOG binding sites. Furthermore we found that 5hmC regions tend to be GC-skewed (excess G over C on one strand of DNA). These findings suggest that 5hmC may be targeted to certain genomic regions based both on gene expression and sequence composition. 2 experiments, 2 controls
Project description:On mouse tiled microarrays (GPL17802), we profile 5hmC in WT mouse livers and brains following fragment enrichment by one of three affinity based techniques: i- antibody (HmeDIP), ii- Chemical capture (hMeSeal) and iii-JBP-1 protein affinity (JBP). HmeDIP and hMeSeal both return similar patterns of 5hmC whilst JBP enriched patterns (carriedo out on brain only) resemble background noise. 5hmC is largely found to reside in the bodies of active genes as well as being enriched over enhancer and promoter proximal regions. WT C57BL/6 mice aged 30-32 days old at time of organ removal. Tissues taken and frozen in liquid nitrogen prior to DNA extraction. Genomic DNA was sonicated (Bioruptor, Diagenode) to produce DNA fragments ranging in size from 200 to 1,000 bp, with a mean fragment size of around 300 bp. i- HmeDIP: antibody enrichment carried out as previously reported by Thomson et al 2013 (doi:10.1093/nar/gkt232). ii- HmeSeal: Enrichment based on the hMe-Seal based techniques described by Song et al 2010 (doi: 10.1038/nbt.1732) and marketed by Active motif under the name hydroxymethyl-collector kit. iii- JBP-1 affinity purification: Enrichment carried out using the Quest 5hmC DNA enrichment kit marketed by Zymo research.
Project description:We carry out hydroxymethyl DNA immunoprecipitation (hmeDIP) prior to sequencing on the Ion Proton semiconductor sequencing platform to demonstate the strategy as an alternative method to Illumina sequencing. Overall design: Genome 5hmC patterns were generate on the Ion Proton seqencer. 2x mouse liver, 1x MEF, 1x iPSC datasets generated
Project description:H3K36me3 (ChIp-ChIp), H3K4me3 (ChIp-ChIp), H3K27me3 (ChIp-ChIp), 5mC (MIRA) and 5hmC (hMeDIP) profiles were analyzed in neural progenitor cells (NPC) and neurons by using Nimblegen Mouse ChIP-chip 2.1M Economy Whole-Genome Tiling - 4 Array Set. In order to compare two different techniques of 5hmC profiling, we performed 5hmC profiling with Hydroxymethyl Collector™ Kit (Active Motif) method and hybridized it on mouse Chr7 fragment (Nimblegen). As an independent experiment, 5hmC profiling was performed by using hMeDIP method and hybridized on mouse Chr7 fragment (Nimblegen). After MIRA enrichment and genome amplification, DNA was hybridized on mouse Chr7 fragment (Nimblegen). Analysis of epigenetic changes during neural differentiation due to comparisson of epigenetic patterns in neural progenitor cells versus neurons.
Project description:Enhancers are developmentally-controlled transcriptional regulatory regions whose activities are modulated through histone modifications or histone variant deposition. Here, we show by genome-wide mapping that the newly discovered DNA modification 5-hydroxymethylcytosine (5hmC) is dynamically associated with transcription factor binding to distal regulatory sites during neural differentiation of mouse P19 cells as well as during adipocyte differentiation of mouse 3T3-L1 cells. Functional annotation reveals that regions gaining 5hmC are associated with genes expressed either in neural tissues when P19 cells undergo neural differentiation or in adipose tissue when 3T3-L1 cells undergo adipocyte differentiation. Furthermore, distal regions gaining 5hmC together with H3K4me2 and H3K27ac in P19 cells behave as differentiation-dependent transcriptional enhancers. Identified regions are enriched in motifs for transcription factors regulating specific cell fates like Meis1 in P19 cells and PPARgamma in 3T3-L1 cells. Accordingly, a fraction of hydroxymethylated Meis1 sites were associated with a dynamic engagement of the 5mC hydroxylase Tet1. In addition, kinetic studies of cytosine hydroxymethylation of selected enhancers indicated that DNA hydroxymethylation is an early event of enhancer activation. Hence, acquisition of 5hmC in cell-specific distal regulatory regions may represent a major event of enhancer progression toward an active state and participate in selective activation of tissue-specific genes Genome-wide 5hmC distribution was determined using hMeDIP-seq. Cells used in this study are P19.6 mouse embryonal carnicoma cells and P19.6 cells treated for 48 hours with 1µM all-trans retinoic acid (RA), as well as 3T3-L1 cells and 3T3-L1 derived adipocytes differentiated with dexamethasone, insulin and IBMX (differentiation cocktail - DC). Individual hMeDIP samples from P19.6 ord 3T3-L1 cells were pooled for library preparation. Libraries were prepared and sequenced at the IBL sequencing facility (Lille, France) with an Illumina Genome Analyser II.
Project description:Through the analysis of mouse liver tumours promoted by distinct routes (DEN exposure alone, DEN exposure plus non-genotoxic insult with phenobarbital and non-alcoholic fatty liver disease); we report that the cancer associated hyper-methylated CGI events in mice are also predicated by silent promoters that are enriched for both the DNA modification 5-hydroxymethylcytosine (5hmC) and the histone modification H3K27me3 in normal liver. During cancer progression these CGIs undergo hypo-hydroxymethylation, prior to subsequent hyper-methylation; whilst retaining H3K27me3. A similar loss of promoter-core 5hmC is observed in Tet1 deficient mouse livers indicating that reduced Tet1 binding at CGIs may be responsible for the epigenetic dysregulation observed during hepatocarcinogenesis. Consistent with this reduced Tet1 protein levels are observed in mouse liver tumour lesions. As in human, DNA methylation changes at CGIs do not appear to be direct drivers of hepatocellular carcinoma progression in mice. Instead dynamic changes in H3K27me3 promoter deposition are strongly associated with tumour-specific activation and repression of transcription. Our data suggests that loss of promoter associated 5hmC in diverse liver tumours licences DNA methylation reprogramming at silent CGIs during cancer progression. We carry out 5-hydroxymethylation DNA immunoprecipitation (hmeDIP) prior to sequencing Ion Proton P1 to report on the genome-wide 5hmC patterns. Heterozygote pairs of Tet1 B6;129S4-Tet1tm1.1Jae/J mice were bought from The Jackson Laboratory (Maine USA). Heterozygotes were interbred to produce homozygous knock out males with colony mate wild type controls. Genome-wide 5hmC patterns were generated by hydroxymethyl-DNA immuoprecipitation (hmeDIP) followed by genome wide sequencing on the Ion Proton P1 sequencer.
Project description:Prenatal exposure to neurotoxicants such as lead (Pb) may cause stable changes in the DNA methylation (5mC) profile of the fetal genome. However few studies have examined its effect on the DNA de-methylation pathway, specifically the dynamic changes of the 5-hydroxymethylcytosine (5hmC) profile. Therefore, in this study, we investigate the relationship between Pb exposure and 5mC and 5hmC modifications during early development. To study the changes in the 5hmC profile, we use a novel modification of the Infinium™ Human methylation 450K assay (Illumina, Inc.), which we named HMeDIP-450K assay, in an in vitro human embryonic stem cell model of Pb-exposure. We model Pb-exposure associated 5hmC changes as clusters of correlated, adjacent CpG sites, which are co-responding to Pb. We further extend our study to look at Pb-dependent changes in high density 5hmC regions in umbilical cord blood DNA from 48 mother-infant pairs from the Early Life Exposure in Mexico to Environmental Toxicants (ELEMENT) cohort. For our study, we randomly selected UCB from 24 male and 24 female children from the 1st and 4th quartiles of Pb levels. Our data show that Pb-associated changes in the 5hmC and 5mC profiles can be divided into sex-dependent and sex-independent categories. Interestingly, differential 5mC sites are better markers of Pb-associated sex-dependent changes compared to differential 5hmC sites. In this study we identified several 5hmC and 5mC genomic loci, which we believe might have some potential as early biomarkers of prenatal Pb-exposure. Human Methylation 450K array coupled with 5-hydroxymethylcytosine (5hmC) IP or HMeDIP-450K array, was used to determine the high density 5hmC profile of 46 Umbilical cord blood samples from the ELLEMENT cohort. HM450K array, without IP was used to determine the 5-methylcytosine(5mC) plus 5hmC profile. The 5hmC region detected from the HMeDIP-450K array was subtracted from the HM450K(without IP) to get putative high density 5mC regions.
Project description:Methylation at the 5-position of cytosine is a well-studied epigenetic pathway. In addition to 5-methylcytosine (5mC), substantial amounts of 5-hydroxymethylcytosine (5hmC) also referred to as the 6th DNA base, have been detected in certain tissues, most notably the brain. However, the genomic distribution of this cytosine modification is unknown. Here, we have developed an immunoprecipitation technique (5hmC-IP) to examine the occurrence of 5hmC in human DNA from brain frontal lobe tissue. The distribution of 5hmC was compared to that of 5mC. We show that 5hmC is more selectively targeted to genes than is 5mC. 5hmC is found at promoters and is particularly enriched in intragenic regions (gene bodies) but is largely absent from non-gene regions. 5hmC peaks at transcription start sites did not correlate with gene expression levels or H3K4me3 peaks. However, presence of 5hmC in gene bodies was more positively correlated with gene expression levels than was presence of 5mC. Promoters of testis-specific genes showed strong 5mC peaks in brain DNA but were almost completely devoid of 5hmC. Our data provide a first overview of the genomic distribution of 5hmC in human brain and will set the stage for further functional characterization of this novel DNA modification. Overall design: Comparison between 5hmC and 5mC profiles
Project description:DNA hydroxymethylation (5hmC) represents a new layer of epigenetic regulation in addition to DNA methylation (5mC). The genome-wide patterns of 5hmC distribution in many tissues and cells have recently been revealed by hydroxymethylated DNA immunoprecipitation (hMeDIP) followed by high throughput sequencing or tiling arrays. However, the DNA hydroxymethylome data generated by the conventional hMeDIP-seq method can not be used for direct quantitative comparison across different samples. In this study, we report a new quantitative hMeDIP-seq method, which uses barcode technology to label different DNA samples and performs hMeDIP-seq for multiple samples in one reaction system. Using this new method, we profiled and compared the DNA hydroxymethylomes of mouse ES cells (ESCs) and mouse ESCs-derived neural progenitor cells (NPCs). 5hmC levels around TSS in either ESCs or NPCs had negative correlation with gene expression levels，while 5hmC levels at gene body regions had different correlation with gene expression, depending on cell types. We identified differential hydroxymethylated regions (DHMRs) by comparing the 5hmC density of all 5hmC peaks between ESCs and NPCs. Many selected DHMRs (e.g., Ankrd23, Hist1h2aa, Fbxw7, and Epha2) were validated by hMeDIP-qPCR. Furthermore, we analyzed the relationship between the alteration of DNA hydroxymethylation and the gene expression change during neural differentiation, and our data suggest that both up- and down-regulated genes exhibited dramatic decrease in 5hmC during neural differentiation while the alteration of 5hmC had weak positive correlation with the changes in gene expression. Taken together, our data demonstrate that quantitative hMeDIP-seq is a powerful approach for genome-wide comparison of DNA hydroxymethylation across multiple samples. Importantly, the DHMRs between ESCs and NPCs uncovered in this study may provide clues for further investigation of the function of 5hmC in gene regulation and neural differentiation. Comparision of the genome-wide distribution of 5hmC in mouse embryonic stem cells and mouse ES-derived neural progenitor cells.
Project description:To assess how DNA modification patterns are influenced by myelopoesis, we have mapped regions enriched for 5mC or 5hmC throughout the genome during promyelocyte to granulocyte differentiation in the HL-60 cell model. Overall design: MeDIP-seq and hMeDIP-seq were performed on undifferentiated and 1.5% DMSO differentiated HL-60 cells.