Project description:This SuperSeries is composed of the following subset Series: GSE14232: Transcriptome analysis of freshly sorted and expanded regulatory and conventional T cells GSE14233: Detection of differentially methylated regions in CD4+CD25+CD45RA+ regulatory T-cells and conventional CD4+CD25- T-cells GSE14234: Histone H3 Lysine 4 mono-, di- and trimethyl and CTCF in CD4+CD25+CD45RA+ regulatory and conventional CD4+CD25- T-cells Refer to individual Series
Project description:Chromatin structure and function is maintained by dynamic protein-protein and protein-nucleic acid interactions. Histones are a family of proteins that are abundant chromatin constituents and that carry numerous post-translational modifications (PTMs). Histone PTMs mediate a variety of biological activities, including recruitment of enzymatic readers, writers and erasers that modulate protein activities, DNA replication, transcription and repair. Individual histone molecules contain multiple co-existing PTMs some of which exhibit crosstalk, i.e. coordinated or mutually exclusive activities. We here present an integrated experimental and computational approach for systems level molecular characterization of PTMs and PTM crosstalk. Using wildtype and engineered mouse embryonic stem cells with perturbations in the Polycomb Repressive Complex 1 (PRC1, suz12-/-), PRC2 (Ring1A/b-/-) and DNA methyltransferases (Dnmt1/3a/3b-/-) we performed comprehensive PTM analysis of histone H3 tails. We identified unique histone H3 PTM features of each of the four cell lines and we detected common combinatorial PTM features across cell lines. Using quantitative middle-down proteomics combined with probabilistic and statistical data analysis we extracted histone H3 PTM profiles for all four mESC systems. PTM crosstalk emerged as mutually exclusive histone PTMs or coordinately regulated PTMs independent of histone peptide abundance in the four model systems. We detected positive crosstalk between adjacent mono-methylated marks but strong negative crosstalk among most of the seven characterized di- and tri-methylations on lysines. We report novel features of PTM interplay involving hitherto poorly characterized arginine methylation and lysine methylation sites in histone H3, including H3R2me, H3R8me and H3K37me, which exhibited specific PTM codes suggesting a particular role in chromatin. All histone H3 PTM data is available in our publicly available CrossTalkDB repository at http://crosstalkdb.bmb.sdu.dk
Project description:Specific alterations in protein post-translational modification (PTMs) are recognized hallmarks of diseases. These modifications potentially provide a unique disease-related source of Human Leukocyte Antigen (HLA) class I-presented peptide antigens that can elicit specific immune responses. Although, phosphorylated HLA peptides have received already some attention, the frequency and characteristics of arginine methylated HLA class I peptide presentation have not been explored in detail. In a model human B-cell line we detected by mass spectrometry (MS) 149 HLA class I peptides harboring mono- and/or di-methylated arginine residues. The source proteins of these antigens play important roles in signal transduction, gene transcription and DNA repair. A striking preference was observed in presentation of arginine (di)methylated peptides predicted to bind HLA-B*07 molecules, most likely because the binding motifs of this allele resemble the substrates for arginine methyl-transferases. The HLA-B*07 peptides were preferentially di-methylated at the P3 position in the sequence, thus consecutively to the proline anchor residue at position P2. Such a proline-arginine sequnce has been associated with the arginine methyl-transferases CARM1 and PRMT5. Making use of the specific neutral losses in the MS/MS spectra we could further assign most of the peptides to be asymmetrically di-methylated, most likely by CARM1. The here presented data expand our knowledge of processing and presentation of arginine (di)methylated HLA class I peptides, indicating that this type of modification is frequently presented for recognition by T-cells and might thus present a potential target for immunotherapy.
Project description:Specific histone modifications play important roles in chromatin functions such as activation or repression of gene transcription. These participation must occur as a dynamic process, however, most of histone modification state maps reported to date only provide static pictures linking certain modification with active or silenced states. This study focused on the global histone modification variation that occurs in response to transcriptional reprogramming produced by a physiological perturbation in yeast. We have performed genome-wide chromatin immunoprecipitation analysis for eight specific histone modifications before and after of a saline stress. The most striking change is a quick deacetylation of lysines 9 and 14 of H3 and lysine 8 of H4 associated to repression of genes. Genes that are activated increase the acetylation levels at these same sites, but this acetylation process of activated genes seems minor quantitatively to that of the deacetylation of repressed genes. The observed changes in tri-methylation of lysines 4, 36 and 79 of H3 and also di-methylation of lysine 79 of H3 are much more moderate than those of acetylation. Additionally, we have produced new genome-wide maps for six histone modifications at more than five times higher resolution of previous available data and analyzed for the first time in S. cerevisiae genome wide profiles of two more, acetylation of lysine 8 of H4 and di-methylation of lysine 79 of H3. In this research we have shown that dynamic of acetylation state of histones during activation or repression of transcription is a process much quicker than methylation and therefore the changes produced in the acetylation may affect methylation but the reverse path is not possible.
Project description:Specific histone modifications play important roles in chromatin functions such as activation or repression of gene transcription. These participation must occur as a dynamic process, however, most of histone modification state maps reported to date only provide static pictures linking certain modification with active or silenced states. This study focused on the global histone modification variation that occurs in response to transcriptional reprogramming produced by a physiological perturbation in yeast. We have performed genome-wide chromatin immunoprecipitation analysis for eight specific histone modifications before and after of a saline stress. The most striking change is a quick deacetylation of lysines 9 and 14 of H3 and lysine 8 of H4 associated to repression of genes. Genes that are activated increase the acetylation levels at these same sites, but this acetylation process of activated genes seems minor quantitatively to that of the deacetylation of repressed genes. The observed changes in tri-methylation of lysines 4, 36 and 79 of H3 and also di-methylation of lysine 79 of H3 are much more moderate than those of acetylation. Additionally, we have produced new genome-wide maps for six histone modifications at more than five times higher resolution of previous available data and analyzed for the first time in S. cerevisiae genome wide profiles of two more, acetylation of lysine 8 of H4 and di-methylation of lysine 79 of H3. In this research we have shown that dynamic of acetylation state of histones during activation or repression of transcription is a process much quicker than methylation and therefore the changes produced in the acetylation may affect methylation but the reverse path is not possible. The experiments described in this study compare ChIP with a histone modification antibody to a control ChIP with a core histone antibody. Budding yeast samples were analyzed in exponential growing conditions (YPD) or after 10 minutes of 0.4M NaCl stress. For each experiment 1 or 2 biological replicates were performed.
Project description: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).