Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Histone H3 Lysine 4 mono-, di- and trimethyl and CTCF in CD4+CD25+CD45RA+ regulatory and conventional CD4+CD25- T-cells

ABSTRACT: Analysis of Histone H3 Lysine 4 mono-, di- and trimethyl and the boundary protein CTCF in CD4+CD25+CD45RA+ regulatory T-cells and conventional CD4+CD25- T-cells. To investigate regulatory functions or potential new transcription start sites in Treg and Tconv cells, we investigated the associated histone modifications. Mono- and dimethylation of histone 3 lysin 4 (H3K4) were previously shown to mark enhancer regions, whereas H3K4 trimethylation generally associates with transcription start sites. At imprinted loci, binding of the insulator protein CTCF, which restricts or directs enhancer-promoter interactions, is often regulated by DNA-methylation. Therefore we performed ChIP-on-chip experiments (chromatin immunoprecipitation followed by microarray hybridization; samples were amplified with ligation mediated PCR [see label protocol for the procedure] prior to labeling) for mono- di- and trimethylation of histone 3 lysin 4 and of CTCF in expanded Treg and Tconv cells. Keywords: ChIP-on-chip ChIP-on-chip experiments for H3K4 mono-, di- and trimethyl and CTCF in CD4+CD25+CD45RA+ regulatory T-cells and conventional CD4+CD25- T-cells were co-hybridizied with the input. Three biologiacal replicates (rep1-3) were performed for every histone mark, two CTCF (rep1 and rep2).

ORGANISM(S): Homo sapiens

SUBMITTER: Christian Schmidl

PROVIDER: E-GEOD-14234 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Lineage-specific DNA methylation in T cells correlates with histone methylation and enhancer activity.

Schmidl Christian C Klug Maja M Boeld Tina J TJ Andreesen Reinhard R Hoffmann Petra P Edinger Matthias M Rehli Michael M

Genome research 20090603 7

DNA methylation participates in establishing and maintaining chromatin structures and regulates gene transcription during mammalian development and cellular differentiation. With few exceptions, research thus far has focused on gene promoters, and little is known about the extent, functional relevance, and regulation of cell type-specific DNA methylation at promoter-distal sites. Here, we present a comprehensive analysis of differential DNA methylation in human conventional CD4(+) T cells (Tconv ...[more]

PMID: 19494038

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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:Chronic chagasic cardiomyopathy is one of the leading causes of heart failure in Latin American countries, being associated with intense inflammatory response and fibrosis. We have previously shown that bone marrow mononuclear cell (BMC) transplantation improves inflammation, fibrosis and ventricular diameter in hearts of mice with chronic Chagas’ disease. Here we investigated alterations of gene expression in the hearts of chronic chagasic mice submitted or not to BMC therapy. C57Bl/6 mice chronically infected with T. cruzi (6 months) were transplanted with BMC or saline i.v. and sacrificed 2 months later. RNA was extracted from the hearts of normal controls, chagasic and BMC transplanted mice and microarray analysis was performed using MO30k oligonucleotide arrays. Out of the 9390 unigenes quantified in all samples, 1702 had their expression altered in chronic chagasic hearts compared to those of normal mice. Major categories of significantly upregulated genes were related to inflammation, fibrosis and immune responses, while genes involved in mitochondrion function were downregulated. When BMC-treated chagasic hearts were compared to infected mice, 1631 (96%) of the alterations detected in infected hearts were not found, although an additional 109 genes were altered by treatment, indicating a remarkable 84% transcriptomic recovery. Immunofluorescence and morphometric analyses confirmed the effects of BMC therapy in the pattern of inflammatory-immune response and expression of adhesion molecules. Our results demonstrate important immunomodulatory effects of BMC therapy in chagasic cardiomyopathy and indicate potentially relevant factors involved in the pathogenesis of the disease that may provide new therapeutic targets. We compared RNA samples extracted from whole hearts of 4 control, 4 chagasic and 4 BMC-treated chagasic mice by analyzing hybridization to microarrays printed by Duke University (http://www.ncbi.nlm.nih.gove/geo/query/acc.cgi?acc=GPL8938) spotted with MO30k mouse Operon version 3.0 70-mer oligonucleotides. The hybridization protocol (see Soares et al, 2010), the slide type and the scanner settings were uniform throughout the entire experiment to minimize the technical noise. Briefly, 20 ug total RNA extracted in Trizol from each of the twelve samples (individual hearts) was reverse transcribed in the presence of fluorescent Alexa Fluor® 555- and Alexa Fluor®647-aha-dUTPs (Invitrogen, Carlsbad, CA) to obtain labeled cDNA. Red and green labeled samples of biological replicas were then co-hybridized (“multiple yellow” strategy, 22) overnight at 50° C. After washing (0.1% SDS and 1% SSC) to remove the non-hybridized cDNA, each array was scanned at 630V (635 nm) and 580V (532 nm) with GenePix 4100B scanner (Axon Instruments, Union City, CA) and images were primarily analyzed with GenePixPro 6.0 (Molecular Devices, Sunnyvale, CA). Microarray data were processed as described previously (Soares et al, 2010). A gene was considered as significantly up- or down-regulated when comparing four hearts from one condition to those from another if the absolute fold change was >1.5x and the p-vlaue of the Sutdent”s heteroscedastic t-test of equality of the means of the distributions with a Bonferroni-type adjustment for each redundancy group (set of spots probing the same gene) was <0.05.

Dataset Information

Histone H3 Lysine 4 mono-, di- and trimethyl and CTCF in CD4+CD25+CD45RA+ regulatory and conventional CD4+CD25- T-cells

Publications

Lineage-specific DNA methylation in T cells correlates with histone methylation and enhancer activity.

Similar Datasets

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