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Genome-Wide Mapping of Hypomethylated Sites in Human Genomes

ABSTRACT: We have developed a method for mapping unmethylated sites in human genome based on the resistant of TspR1 digested ends to exoIII nuclease degradation. Digestion with TspR1 and methylation-sensitive restriction endonuclease, HpaII, followed by exoIII and single strand DNA nuclease allows the removal of DNA fragments containing unmethylated HpaII sites. We then use array CGH to map the sequences depleted by this procedures in human genomes derived from five human tissues, a primary breast tumor and two breast tumor cell lines. Analysis of methylation patterns of the normal tissue genomes indicates that the hypomethylated sites are enriched in the 5’ end of widely expressed genes including promoter, first exon and first intron. In contrast, genomes of the MCF-7 and MDA-MB-231 cell lines show extensive hypomethylation in the intragenic and intergenic regions whereas primary tumor exhibits intermediate pattern between normal tissue and cell lines. A striking characteristic of tumor genomes is the presence of megabase-sized hypomethylated zones. These hypomethylated zones are associated with large genes, fragile sites, evolutionary breakpoints, chromosomal rearrangement breakpoints, tumor supperessor genes, and with regions containing tissue-specific gene clusters or with gene poor region containing novel tissue-specific genes. Bisulfite sequencing analysis shows a novel mosaic methylation pattern with alternative methylated and unmethylated zones was found in human histone gene clusters in chromosome 6. Correlation with microarray analysis show that genes with hypomethylated sequence 2kb up- or down-stream of transcription start site are highly expressed whereas genes with extensive intragenic and 3’ UTR hypomethylation are silenced. The method described herein can be used for large scale screening of changes in methylation pattern in the genome of interest. Keywords: Genome-Wide Mapping of Hypomethylated Sites in Human Genomes

ORGANISM(S): Homo sapiens

PROVIDER: GSE9015 | GEO | 2007/09/29

SECONDARY ACCESSION(S): PRJNA102505

REPOSITORIES: GEO

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Project description:Genome wide methylation profiling of gliomas is likely to provide important clues to improving treatment outcomes. Restriction enzyme based approaches have been widely utilized for methylation profiling of cancer genomes and will continue to have importance in combination with higher density microarrays. With the availability of the human genome sequence and microarray probe sequences, these approaches can be readily characterized and optimized via in silico modeling. We adapted the previously described HpaII/MspI based Methylation Sensitive Restriction Enzyme (MSRE) assay for use with two-color Agilent 244K CpG island microarrays. In this assay, fragmented genomic DNA is digested in separate reactions with isoschizomeric HpaII (methylation-sensitive) and MspI (methylation-insensitive) restriction enzymes. Using in silico hybridization, we found that genomic fragmentation with BfaI was superior to MseI, providing a maximum effective coverage of 22,362 CpG islands in the human genome. In addition, we confirmed the presence of an internal control group of fragments lacking HpaII/MspI sites which enable separation of methylated and unmethylated fragments. We used this method on genomic DNA isolated from normal brain, U87MG cells, and a glioblastoma patient tumor sample and confirmed selected differentially methylated CpG islands using bisulfite sequencing. Along with additional validation points, we performed a receiver operating characteristics (ROC) analysis to determine the optimal threshold (p â¤ 0.001). Based on this threshold, we identified ~2400 CpG islands common to all three samples and 145 CpG islands unique to glioblastoma. These data provide general guidance to individuals seeking to maximize effective coverage using restriction enzyme based methylation profiling approaches. Five samples: normal human brain DNA, U87MG cell line, glioblastoma tumor, human DNA treated with SssI enzyme used as positive control, and Genomiphied human DNA used as negative control. Two technical replicates were performed on all samples except the negative control.

Project description:Purpose: Generate genome-wide methylation profiles of non-small cell lung carcinomas (NSCLC) and their matching lung tissues for detection of hypermethylated and hypomethylated regions present in the tumors. Methods: MethylCapture followed by next-generation sequencing (Illumina GAIIx) of 7 nsclc tumor samples and paired lung tissues in replicated, plus one cell line, 2 fully artificially methylated and 2 fully artificially unmethylated controls. Normalization of methylation reads based on CpG coupling factor–method. Relative methylation scores (rms) in 500bp non-overlapping windows. 90th percentile of rpm (reads per million) values for all 500bp genome-wide windows, with rpm <1.33 were considered. Distributions of 10bp bins rms values within each 500bp genomic region were compared using both one-sided Student’s t-test and one-sided Wilcoxon rank-sum test. Testing was done separately for hypo- and hypermethylation and p-value threshold of 10-18. Results: MethylCap-seq data revealed strong positive correlation between replicate experiments and between paired tumor/lung samples. 14472 differentially methylated regions (DMR) with non-overlapping 500 bp windows were found. 57 DMRs were present in all NSCLC tumors. 287 were unique for squamous-cell carcinomas and 26 unique for adenocarcinomas. While hypomethylated DMRs did not correlate to any particular functional category of genes, the hypermethylated DMRs were strongly associated with genes encoding transcriptional regulators. Furthermore, subtelomeric regions and satellite repeats were hypomethylated in the NSCLC samples. Conclusion: We provide a resource containing genome-wide DNA methylation maps of NSCLC and their paired lung tissues, and comprehensive lists of known and novel DMRs and associated genes in NSCLC. The DMRs can be in further studies to develop sensitive biological markers for NSCLC, which may enable non-invasive diagnosis and early detection of the disease, and potentially allow histological classification. MethylCap-seq of 7 nsclc tumor samples and paired lung tissues, plus 2 fully methylated and 2 fully unmethylated controls.

Project description:This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Florencia Pauli mailto:fpauli@hudsonalpha.org). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu). This track shows average methylation status in CpG islands. In general, methylation of CpG sites within a promoter causes silencing of the gene associated with that promoter For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf CpG regions were assayed via Methyl-seq, a method developed in the Myers laboratory to measure the methylation status at CpGs throughout the genome. It combines DNA digestion by a methyl-sensitive enzyme HpaII and its methyl-insensitive isoschizomer MspI with the Illumina DNA sequencing platform. The method was first applied in a collaboration with the laboratory of Dr. Julie Baker at Stanford University to study methylation and gene expression changes that occur in human embryonic stem cells before and after differentiation to definitive endoderm. A paper describing the results as well as the method has been submitted for publication [1]. This study profiled genomic DNA and mRNA samples derived from two human embryonic stem cell lines: H9 and BG02. These cells were differentiated into definitive endoderm, embryoid bodies, embryoid body-derived cells, and AFP+ (alpha-fetoprotein positive) hepatocytes. These in vitro samples were profiled with Methyl-seq and compared them with normal tissue samples from 11-week and 24-week fetal liver and adult liver. Methyl-seq assays more than 250,000 methyl-sensitive restriction enzyme cleavage sites, representing more than 90,000 genomic regions. These regions include 35,528 annotated CpG islands, while the remaining 55,084 non-CpG island regions are distributed across the genome in promoters, genes, and intergenic regions. Sequence tags present in MspI libraries but not in HpaII libraries are derived from methylated regions. Conversely, sequence tags that occur in HpaII libraries come from at least partially unmethylated regions. In vitro differentiation: Definitive endoderm precursor cells were generated from H9 hES cells by treating them with activin A. Embryoid bodies (EBs) were generated by growing undifferentitated H9 and BG02 hESCs in suspension. EB-derived cells were obtained by plating clumps of the cells from the EBs. AFP+ fetal hepatocytes were derived from EBs by plating EB cells with FgF, followed by fluorescence activated cell sorting (FACS) to isolate cells expressing the green fluorescent protein (GFP) reporter gene driven from the AFP promoter. Isolation of genomic DNA: Genomic DNA is isolated from biological replicates of each cell line by using the QIAGEN DNeasy Blood & Tissue Kit according to the instructions provided by the manufacturer. DNA concentrations and a level of quality of each preparation is determined by UV absorbance. HpaII and MspI digestions: Cleavage of DNA by restriction endonuclease HpaII is prevented by the presence of a 5-methyl group at the internal C residue of its recognition sequence CCGG. MspI, an isoschizomer of HpaII, cleaves DNA irrespective of the presence of a methyl group at this position. For the MspI library, 5 µg genomic DNA was digested in a 100 µl reaction with 1X NEB Buffer2 and 20 units MspI restriction enzyme and incubated for 18 hr at 37°C. For the HpaII library, 5 µg genomic DNA was digested in a 100 µl reaction with 1X NEB Buffer1 and 20 units HpaII restriction enzyme and incubated for 18 hr at 37°C. Note that in subsequent versions of the Methyl-seq protocol, which will be described later, much lower amounts of genomic DNA were used (1 µg and potentially lower). DNA library construction and sequencing: High-throughput sequencing libraries were generated from DNA fragments of the HpaII or MspI digested genomic DNA according to the protocol posted at the website: http://myers.hudsonalpha.org/content/protocols.html. This approach was recently modified by removing the first PCR amplification step, just prior to the gel electrophoresis size-selection step, which was found to reduce a fragment-size bias in the sequencing libraries. These libraries were sequenced with an Illumina Genome Analyzer (GA2) according to the manufacturer's recommendations. Data analysis: For this analyis, reads that align to human genome sequence version hg19 and contain the 5'-CGG-3' HpaII-cut signature on their 5' end were used. These aligned sequence reads were mapped to CCGG sites predicted in silico on hg19. Sites with four or more Msp1 tags occurring in either the forward or reverse direction were retained for analysis. These "assayable" sites were then grouped with neighboring sites that are within 35-75 bp of each other. Thus, a "region" can be comprised of between 2 and 18 digestion sites that are each within 35-75 bp of another site. Methylated and non-methylated calls were made by using HpaII tag data from all assayable cut sites. For each site across each region, the larger of either the forward read count or reverse read count was used. Regions that have an average of 0 or 1 read per cut site are called methylated, and regions with more than one sequence read per site are called unmethylated.

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Genome-Wide Mapping of Hypomethylated Sites in Human Genomes

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