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Methylation profiling of differentially expressed regions between C57BL/6 and BALB/c in bone marrow derived macrophages

ABSTRACT: DNA-methylation is a vital epigenetic mark that participates in establishing and maintaining chromatin structures and in regulating gene transcription during mammalian development and cellular differentiation. Inter-individual differences in methylation patterns may represent a major source of phenotypic variation, however, the determinants, inheritance, extent, and consequences of such differences are poorly understood. Here we have analysed methylation profiles of immune cells from two inbreed mouse strains (C57BL/6 & BALB/c) that represent prototypic models for Th1- or Th2-dominated immune responses. Using a methyl-CpG immunoprecipitation approach, genomes were fractionated into methylated and unmethylated genome pools and separately analysed at 180 genomic regions (covering 28 Mb of the mouse genome) that were selected based on differential gene expression between both mouse strains. Differentially methylated regions are detected by analyzing array probes for diametrically opposed enrichment behaviour between both hybridizations (e.g. a region that is relatively enriched in the unmethylated pool of BALB/c and shows reverse enrichment behaviour in the methylated pool is considered hypomethylated in BALB/c). The integrated analysis of hypo and hypermethylation profiles allowed the identification of several hundred differentially methylated regions, but also uncovered regions that were duplicated in one strain, contained single nucleotide polymorphisms or micro- and macro-deletions. Keywords: MCIp-on-Chip; comparative genomic hybridization

ORGANISM(S): Mus musculus

PROVIDER: GSE14463 | GEO | 2009/08/10

SECONDARY ACCESSION(S): PRJNA111459

REPOSITORIES: GEO

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Project description:We have previously developed an approach that fractionates genomic DNA fragments depending on their CpG density (methyl-CpG-immunoprecipitation, MCIp), and adapted this approach to identify regions that are differentially methylated in the two closely related regulatory T-cells (Treg cells) and conventional T-cells (Tconv cells). Because Treg cells naturally occur at a relatively low frequency, we used a previously established protocol to expand Treg cells from a stable naÃ¯ve Treg population that is characterized by the co-expression of CD4, CD25 and CD45RA. We separated gDNA of both expanded T cell lineages (Tregexp and Tconvexp) into unmethylated (CpG) and methylated pools (mCpG) using MCIp and compared cell type-specific differences in DNA methylation by co-hybridization of the two umethylated or the two methylated DNA subpopulations of Treg and Tconv, respectively, to these locus-wide custom tiling arrays. As enriched DNA-fragments from a cell type in the methylated fraction should be depleted in the unmethylated fraction, the signal intensities in CpG pool and mCpG pool hybridizations should complement themselves (âMirror-Imageâ approach) and thereby allow the identification of differentially methylated regions (DMR). Because we expected to find lineage-specific methylation differences with greater probability in regions associated with differential transcriptional activity, we limited our analysis to gene loci that showed cell type-specific gene expression in Treg versus Tconv cells plus a handful of control regions that were equally expressed in both cell types. The microarray used in this study covered 12 megabases of the human genome and contained 69 regions (with a median size of 100.000 kb) and 128 proximal promoter regions and 181 genes, which included a number of well known and functionally relevant genes like CD40LG, IFNG, FOXP3, IL2RA and CTLA4. Keywords: MCIp-on-chip; comparative genomic hybridization With MCIp gDNA from Treg or Tconv cells was separated into hypo- and hypermethylated pools. On each array, wheather the two hypomethylated fractions- one from Treg, the other from Tconv cells- or the two hypermethylated fractions were cohybridized. Two biological replicates.

Project description:Methylation of DNA at CpG dinucleotides represents one of the most important epigenetic mechanisms involved in the control of gene expression in vertebrate cells. In this report, we conducted high-throughput nucleosome reconstitution experiments on 572 KB of human DNA and 668 KB of mouse DNA that was unmethylated or methylated in order to investigate the effects of this epigenetic modification on the positioning and stability of nucleosomes. The DNA loci from both species contained the genes that encode the serum albumin family, the MYC protein, and the tumor suppressor p53. The results demonstrated that a small subset of nucleosomes positioned by nucleotide sequence was sensitive to methylation where the modification increased the affinity of these sequences for the histone octamer. The features that distinguished these nucleosomes from the bulk of the methylation-insensitive nucleosomes were an increase in the frequency of CpG dinucleotides and a unique rotational orientation of CpGs such that their minor grooves tended to face toward the histones in the nucleosome rather than away. We propose that these features serve to enhance the affinity of methylated DNA for the histone octamer and that this effect could be involved in gene regulatory mechanisms such as silencing. These methylation-sensitive nucleosomes were preferentially associated with exons as compared to introns while unmethylated CpG islands near transcription start sites became enriched in nucleosomes upon methylation. The results of this study suggest that the effects of DNA methylation on nucleosome stability in vitro can recapitulate what has been observed in the cell and provide a direct link between DNA methylation and the structure and function of chromatin. For the human data, there were 3 unmethylated nucleosome replicates, 2 methylated nucleosome replicates, an unmethylated MNase control, and an unmethylated sonicated control. For the mouse data, there were 2 unmethylated nucleosome replicates, 2 methylated nucleosome replicates, an unmethylated MNase control, a methylated MNase control, and an unmethylated sonicated control.

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 is produced as part of the ENCODE project. The track displays the methylation status of specific CpG dinucleotides in the given cell types as identified by the Illumina Infinium Human Methylation 450 Bead Array platform (http://www.illumina.com/products/methylation_450_beadchip_kits.ilmn). In general, methylation of CpG sites within a promoter causes silencing of the gene associated with that promoter. The Infinium Human Methylation 450 platform uses bisulfite treated genomic DNA to assay the methylation status of more than 450,000 CpG sites covering all designatable RefSeq genes, including promoter, 5' and 3' regions, without bias against those lacking CpG islands. Additionally, the assay includes CpG islands and shores, CpG sites outside of CpG islands, non-CpG methylated sites identified in human stem cells, differentially methylated sites identified in tumor versus normal (multiple forms of cancer) and across several tissue types, CpG islands outside of coding regions, miRNA promoter regions, and disease-associated regions identified through GWAS. Detailed information for the CpG targets is in an CSV formatted spreadsheet in the supplemental directory (http://hgdownload-test.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeHaibMethyl450/supplemental/). For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Cells were grown according to the approved ENCODE cell culture protocols (http://hgwdev.cse.ucsc.edu/ENCODE/protocols/cell). Genomic DNA was isolated from each cell line with the QIAGEN DNeasy Blood & Tissue Kit according to the instructions provided by the manufacturer. DNA concentrations and a level of quality of each preparation was determined by fluorescence with the Qubit Fluorometer (Invitrogen). Genomic DNA was treated with sodium bisulfite, converting unmethylated cytosines of CpG dinucleotides into uracils; methylated cytosines did not get converted. After bisulfite treatment, the methylation status of a site was assayed by single base-pair extension with a Cy3 or Cy5 labeled nucleotide on oligo-beads specific for the methylated or unmethylated state. The bisulfite conversion reaction was done using the Zymo Research EZ-96 DNA Methylation Kit (http://www.zymoresearch.com/product/ez-96-dna-methylation-kit-d5003). One step of the protocol was modified. During the incubation, a 30 second 95oC denaturing step every hour was included to increase reaction efficiency as recommended by the Illumina Infinium Human Methylation27 protocol. The bead arrays were run according to the protocol provided by Illumina (http://hgdownload-test.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeHaibMethyl450/supplemental/wgEncodeHaibMethyl450IlluminaProtocol.pdf). A beta value was calculated for each CpG target with Illumina's Bead Studio software with the Methylation Module v3.2. Beta-value = intensity value from the methylated bead type/(intensity values from the methylated + intensity value from unmethylated bead types + 100). The data was then quality-filtered using p-values. Beta values with p-value greater than 0.01 are considered to fall below the minimum intensity and threshold are displayed as "NA". Any beta value equal to or greater than 0.6 was considered fully methylated. Any beta value equal to or less than 0.2 was considered to be fully unmethylated. Beta values between 0.2 and 0.6 were considered to be partially methylated. Score in the bed files is beta value x 1000

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Methylation profiling of differentially expressed regions between C57BL/6 and BALB/c in bone marrow derived macrophages

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