Methylation sites of Histone H2B catalyzed by LaeA
Ontology highlight
ABSTRACT:
ORGANISM(S): Penicillium Oxalicum 114-2
SUBMITTER: 
Lushan Wang
PROVIDER: PXD027171 | iProX | Thu Jul 08 00:00:00 BST 2021
REPOSITORIES: iProX
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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).
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Project description:Histone lysine methylation is an important post-translational modifications (PTMs) that plays critical roles in numerous biological processes with abnormal histone lysine methylation having been associated with developmental defects and human diseases. The primary amine of all lysine residues can be mono-, di-, or trimethylated and the extent of methylation at a single site is shown to inspire unique protein function. Moreover, histone lysine methylation can activate or repress transcription depending on which sites are modified and to what degree. Therefore, a comprehensive stoichiometric analysis of histone lysine methylation is necessary to fully elucidate its function. As histones are lysine-rich and highly-hydrophilic proteins (Figure S1), trypsin digestion of histones results in small, hydrophilic peptides which often suffer from substantial losses during sample preparation, and are difficult to detect in conventional reversed phase LC-MS. Additionally, trypsin fails to cleave histone proteins when lysine residues are methylated, resulting in inaccurate estimations of site occupancy and methylation extent. Previously, lysine propionylation labeling has been developed to overcome this drawback and facilitate quantitative analysis of PTMs that frequently occur on the lysine residue of histones. However, propionylation labeling of unmodified and monomethylated lysine residues causes a mismatch in hydrophobicity and charge state between labeled and unlabeled di-/trimethylated peptides. This mismatch forces retention time and signal intensity differences that inspire inaccurate quantitation and miscalculation of site occupancy. In this work, we developed a method that facilitates blocking of free lysine groups and discrimination of native lysine methylation, enables accurate calculation of the histone lysine methylation stoichiometry.
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Project description:This SuperSeries is composed of the following subset Series: GSE22102: Histone H3 lysine 9 di-methylation as an epigenetic signature of the interferon response (sequencing) GSE24774: Histone H3 lysine 9 di-methylation as an epigenetic signature of the interferon response (WT and G9a deficient DCs) GSE24776: Histone H3 lysine 9 di-methylation as an epigenetic signature of the interferon response (WT and G9a deficient MEFs) Refer to individual Series
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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
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Project description:G9a and GLP lysine methyltransferases form a heterodimeric complex that is responsible for the bulk of cellular mono- and di-methylation on histone H3 lysine 9 (H3K9me1/me2). Widely Interspaced Zinc finger (WIZ) associates with the G9a/GLP protein complex, but its role in lysine methylation is poorly defined. Here, we show that WIZ regulates global H3K9me2 levels through a mechanism that involves retention of G9a on chromatin. We also show that WIZ-mediated chromatin loss of G9a/GLP results in altered gene expression and protein-protein interactions that are distinguishable from that of using a molecule-induced enzymatic inhibitor towards G9a/GLP – thus providing evidence that the G9a/GLP/WIZ complex has unique functions when bound to chromatin that are independent of the H3K9me2 mark DMSO, UNC0638, NS, and siWIZ treatments to HEK293T cells, assessing G9a and H3K9me2 localization, each performed in duplicate, plus one input control for each cell condition. 30 samples total.
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Project description:Polycomb Repressor Complex 2 (PRC2) is a multi-protein epigenetic regulator complex that plays critical roles in early development and tissue differentiation. The complex catalyzes the methylation of histone H3 lysine 27 (H3K27). The tri-methyl state (H3K27me3) is well studied as an agent of gene repression, but more recently distinct roles for the mono- and di-methyl states have been discovered. In order to more fully understand how PRC2-associated factors might influence H3K27 status, we screened for physical associations of PRC2 subunits. Knockdown of these factors was used to produce functional interaction maps focused on H3K27 methylation, including proteins that have positive and negative effects on the levels each histone mark.
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Project description:Post-translational modification of proteins through methylation plays important regulatory role in biological processes. Lysine methylation on histone proteins is known to play important role in chromatin structure and function. However, non-histone protein substrates of this modification remain largely unknown. Herein, we use high resolution mass spectrometry to global screening methylated substrates and lysine- methylation sites in tomato (Solanum Lycopersicum). A total of 241 sites of lysine methylation (mono-, di-, tri-methylation) in 176 proteins with diverse biological functions and subcellular localized were identified in mix tomato with different maturity. Two putative methylation motifs were detected. KEGG pathway category enrichment analysis indicated that methylated proteins are implicated in the regulation of diverse metabolic processes, including arbon fixation in photosynthetic organisms, pentose phosphate pathway, fructose and mannose metabolism, and cysteine and methionine metabolism. Three representative proteins were selected to analyze the effect of methylated modification on protein function. In addition, quantitative RT-PCR further validated the gene expression level of some key methylated proteins during fruit ripening, which are involved in oxidation reduction process, stimulus and stress, energy metabolism, signaling transduction, fruit ripening and senescence. These data represent the first report of methylation proteomic and supply abundant resources for exploring the functions of lysine methylation in tomato and other plants.
2023-03-11 | PXD023985 | Pride
Project description:Lysine methylation of histone proteins regulates chromatin dynamics and plays important roles in diverse physiological and pathological processes. However, beyond histone proteins, the proteome-wide extent of lysine methylation is largely unknown. We have developed the naturally occurring MBT domain repeats of L3MBTL1 (3xMBT) to serve as a universal affinity reagent for detecting, enriching, and identifying proteins carrying a mono- or dimethylated lysine. The domain is broadly specific for methylated lysine ("pan-specific") and can be applied to any biological system. In experiments using two different instruments (experiment 1 - Orbitrap Elite, experiment 2 - Orbitrap Velos) we have used SILAC to compare proteins captured by 3xMBT to proteins captured by the D355N mutant, which does not bind methylated lysine. Data was analyzed using MaxQuant version 1.3.0.5 using default parameters except that mono and di-methylated lysine were included as variable modifications and modification-specific false discovery rate was set to 10%. Several hundred proteins are specifically enriched by 3xMBT and we have directly detected lysine methylation on about two dozen. We have also used our approach to identify candidate in-cell substrates of G9a and the related methyltransferase GLP. Three-way SILAC was used to compare methylated proteins between cells treated with UNC0638, a specific inhibitor of G9a and GLP, and cells treated with vehicle control. We find reduced capture of the known G9a substrates WIZ and ACIN1, as well as identifying DNA ligase 1 as a potential target for G9a/GLP. Together, our results demonstrate a powerful new approach for global and quantitative analysis of methylated lysine, and they represent the first systems biology understanding of lysine methylation.
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