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Array-based assay detects genome-wide 5-methylcytosine and 5-hydroxymethlycytosine in non-human primates and mice

ABSTRACT: Murine and non-human primates (e.g. rhesus monkeys) represent excellent model systems to study human health and disease. However, use of these model systems for genomic studies is limited, particularly with array-based tools, as most have only been developed to survey the human genome. Here we present the optimization of a widely used human DNA methylation array, designed to detect 5-methylcytosine (5-mC), and show that non-human data generated using the optimized array reproducibly distinguishes tissue types within and between chimpanzee, rhesus, and mouse, with correlations near the human DNA level (R2 > 0.99). Genome-wide methylation analysis, using this approach, reveals 6,102 differentially methylated loci between rhesus placental and fetal tissues with pathways analysis significantly overrepresented for developmental processes. Restricting the analysis to oncogenes and tumor suppressors genes finds 125 differentially methylated loci, suggesting that rhesus placental tissue carries a cancer epigenetic signature. Moreover, we adapted the assay to detect 5-hydroxymethylcytosine (5-hmC) and find highly reproducible 5-hmC levels within human, rhesus, and mouse brain tissue that is species-specific with a hierarchical abundance among the three species (human > rhesus >> mouse). Together, these data show that this array-based methylation assay is generalizable to all mammals for the detection of both 5-mC and 5-hmC, greatly improving the utility of mammalian model systems to study the role of epigenetics in human health, disease, and evolution.

ORGANISM(S): Mus musculus Homo sapiens Pan troglodytes Macaca mulatta

PROVIDER: GSE49177 | GEO | 2014/02/06

SECONDARY ACCESSION(S): PRJNA213232

REPOSITORIES: GEO

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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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Array-based assay detects genome-wide 5-methylcytosine and 5-hydroxymethlycytosine in non-human primates and mice

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