High Resolution Mapping and Functional Analysis of the Methylome in Arabidopsis (mCIP, MBD)
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ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
PROVIDER: GSE5094 | GEO | 2006/08/30
SECONDARY ACCESSION(S): PRJNA95343
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 naïve 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 (“Mirror-Image” 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
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Project description:Integration of the bacterial T-DNA into the plant genome by virulent agrobacteria causes crown gall development on many plant species. Plant tumor development shares fundamental similarities with mammalian cancer progression despite obvious differences in initiation of tumor development. For neoplastic growth in mammals, DNA methylation changes are known to be essential. The role of epigenetic modifications for plant tumor development is addressed here. Genome-wide comparison of methylation profiles of Arabidopsis crown galls and tumor-free tissue revealed 2,876 annotated genes that were affected by differential methylation. Thereof, 1,822 genes were hit by hypermethylated regions and 1,100 genes overlapped with hypomethylated regions (sum of hyper-and hypomethylated genes is higher than the number of affected genes because one gene may contain several differentially methylated regions [DMRs]). DMRs found to be methylated only in tumor-free tissue covered 275 kb of the genome, whereas those methylated only in crown galls total to 560 kb. Contrary to the globally hypermethylated tumor genome, promoter regions of protein coding genes appeared to be rather hypomethylated. In contrast, mammalian cancer cells are associated with global hypomethylation and local hypermethylation of gene promoters. In summary, while aberrant DNA methylation in mammals increases malignacy of the cancer phenotype, our results indicate that methylation events in the plant genome rather confine tumor growth. All 15,431 mCIP-enriched regions reported in the paper are contained in the supplementary BED files.
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