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: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.

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.

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Comprehensive evaluation of genome-wide 5-hydroxymethylcytosine profiling approaches in human DNA [HumanMethylation450]

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