Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Genome-wide DNA hydroxymethylation changes are associated with neurodevelopmental genes in the developing human brain

ABSTRACT: 5-hydroxymethylcytosine (5-hmC) is a newly discovered modified form of cytosine that has been suspected to be an important epigenetic modification in neurodevelopment. While DNA methylation dynamics have already been implicated during neurodevelopment, little is known about hydroxymethylation in this process. Here we report DNA hydroxymethylation dynamics during cerebellum development in the human brain. Overall, we find a positive correlation between 5-hmC levels and cerebellum development. Genome-wide profiling reveals that 5-hmC is highly enriched on specific gene regions, including exons and especially the untranslated regions (UTRs), but it is depleted on introns and intergenic regions. Furthermore, we have identified fetus-specific and adult-specific differentially hydroxymethylated regions (DhMRs), most of which overlap with genes and CpG island shores. Surprisingly, during development DhMRs are highly enriched in genes encoding mRNAs that can be regulated by fragile X mental retardation protein (FMRP), some of which are disrupted in autism, as well as in many known autism genes. Our results suggest that 5-hmC-mediated epigenetic regulation may broadly impact the development of the human brain, and its dysregulation could contribute to the molecular pathogenesis of neurodevelopmental disorders. We generated comprehensive genome-wide profiles of 5hmC in human cerebellum.

ORGANISM(S): Homo sapiens

SUBMITTER: Tao Wang

PROVIDER: E-GEOD-40539 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:5-methylcytosine (5-mC) can be oxidized to 5-hydroxymethylcytosine (5-hmC). Genome-wide profiling of 5-hmC thus far indicated 5-hmC may not only be an intermediate form of DNA demethylation but could also constitute an epigenetic mark per se. We describe a cost-effective and selective method to detect both the hydroxymethylation and methylation status of cytosines in more than 1.8 million MspI sites in the human genome. This method involves the selective glucosylation of 5-hmC residues, short-sequence tag generation and high-throughput sequencing. We tested this method by screening H9 human embryonic stem cells and their differentiated embroid body cells, and found that differential hydroxymethylation preferentially occur in bivalent genes during cellular differentiation. Especially, our results support hydroxymethylation can regulate key transcription regulators with bivalent marks through demethylation and affect cellular decision on choosing active or inactive state of these genes upon cellular differentiation. We developed a cost-effective and selective method to detect both the hydroxymethylation and methylation status of cytosines in more than 1.8 million MspI sites in the human genome. In this method, we took advantage of the differential enzymatic sensitivities of the isoschizomers MspI and HpaII. HpaII cleaves only a completely unmodified site, any modification at either cytosine blocks the cleavage, while MspI recognizes and cleaves both 5-mC and 5-hmC, but not the newly discovered 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). Furthermore, beta-glucosyltransferase (beta-GT) can transfer a glucose to the hydroxyl group of 5-hmC and generate beta-glucosyl-5-hydroxymethylcytosine (5-ghmC) that blocks MspI digestion. Thus, either by combining beta-GT treatment with MspI digestion or simply applying MspI/HpaII digestion, short sequence tags generated can be used for inferring hydroxymethylation or methylation status in around 1.8 million cytosine sites in the human genome. We tested this method by screening H9 human embryonic stem cells and their differentiated embroid body cells.

Project description:DNA methylation (5-mC) and hydroxymethylation (5-hmC) are regarded as important epigenetic hallmarks in the carcinogenesis of colorectal cancer by transcriptional regulation. 5hmC is an intermediate during active demethylation and maintains the equilibrium of DNA methylation. Previous studies on DNA methylation don’t differentiate 5-hmC from 5-mC. Here, in order to elucidate the epigenetic mechanisms of carcinogenesis of colorectal cancer, we integrate genome wide levels of 5-mC, 5-hmC and Transcriptional expression. 12 samples, including six colorectal tumor tissues and corresponding normal colonic tissues were recruited after surgery. Genome-wide DNA methylation was determined by methylated DNA immune- precipitation sequencing (MeDIP-seq), and hydroxymethylation by hydroxyl- methylated DNA immune-precipitation sequencing (hMedip-seq). Transcriptional expression was determined by RNA-seq. Group-wise different methylation region (DMR), different hydroxyl methylation region (DhMR) and different expressed gene (DEG) were identified. Epigenetic biomarkers were screened by integrating DMR, DhMR and DEG. We found that a genome-scale distinct hydroxymethylation pattern could be used as epigenetic biomarker for clearly differentiating colorectal cancer from normal tissues. 59249 differentially methylated regions (DMR), 187172 differentially hydroxymethylated region (DhMR) and 948 differentially expressed genes (DEGs) were identified. After cross-matched genes containing DMRs or DhMRs with DEGs, seven genes were screened. Furthermore, hypermethylation of HADHB was persistently found to be correlated with its down-regulation of transcription in CRC, potentially suggesting its role as TSG. The differences of methylation, hydroxymethylation and transcriptional expression in HADHB between cancerous and normal tissues were validated among additional colorectal cancer patients. To further validate this assumption, we also performed functional analysis and found that the expression of HADHB obviously reduced cancer cells migration and invasiveness. This study provided valuable basic data for screening epigenetic biomarkers and elucidated the epigenetic mechanisms of carcinogenesis of colorectal cancer.

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Genome-wide DNA hydroxymethylation changes are associated with neurodevelopmental genes in the developing human brain

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