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Linking chromatin acylation mark-defined proteome and genome in living cells

ABSTRACT: A generalizable strategy with programmable site-specificity for in situ profiling of histone modifications on unperturbed chromatin remains highly desirable but challenging. We herein developed a Single-site-resolved multi-omics (SiTomics) strategy for systematic mapping of dynamic modifications, and subsequent profiling of chromatinized proteome and genome defined by specific chromatin acylations in living cells. By leveraging the genetic code expansion strategy, our SiTomics toolkit revealed distinct crotonylation (e.g., H3K56cr) and β-hydroxybutyrylation (e.g., H3K56bhb) upon short chain fatty acids stimulation, and established linkages for chromatin acylation mark-defined proteome, genome and functions. This led to the identification of GLYR1 as a distinct interacting protein in modulating H3K56cr’s gene body localization as well as the discovery of an elevated super-enhancer repertoire underlying bhb-mediated chromatin modulations. SiTomics offers a platform technology for elucidating the “metabolites-modification-regulation” axis, which is widely applicable for multi-omics profiling and functional dissection of modifications beyond acylations and proteins beyond histones.

ORGANISM(S): Homo sapiens

PROVIDER: GSE204854 | GEO | 2023/01/27

REPOSITORIES: GEO

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Linking chromatin acylation mark-defined proteome and genome in living cells

Project description:A generalizable strategy with programmable site-specificity for in situ profiling of histone modifications on unperturbed chromatin remains highly desirable but challenging. We herein developed a Single-site-resolved multi-omics (SiTomics) strategy for systematic mapping of dynamic modifications, and subsequent profiling of chromatinized proteome and genome defined by specific chromatin acylations in living cells. By leveraging the genetic code expansion strategy, our SiTomics toolkit revealed distinct crotonylation (e.g., H3K56cr) and β-hydroxybutyrylation (e.g., H3K56bhb) upon short chain fatty acids stimulation, and established linkages for chromatin acylation mark-defined proteome, genome and functions. This led to the identification of GLYR1 as a distinct interacting protein in modulating H3K56cr’s gene body localization as well as the discovery of an elevated super-enhancer repertoire underlying bhb-mediated chromatin modulations. SiTomics offers a platform technology for elucidating the “metabolites-modification-regulation” axis, which is widely applicable for multi-omics profiling and functional dissection of modifications beyond acylations and proteins beyond histones.

2023-01-27 | GSE204853 | GEO

Linking chromatin acylation mark-defined proteome and genome in living cells

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Linking chromatin acylation mark-defined proteome and genome in living cells

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Linking chromatin acylation mark-defined proteome and genome in living cells

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Prediction of gene activity based on an integrated multi-omics approach.

Project description:An increasingly common method for predicting gene activity is genome-wide chromatin immunoprecipitation of M-bM-^@M-^XactiveM-bM-^@M-^Y chromatin modifications followed by massively parallel sequencing (ChIP-seq). Using a novel ChIP-seq quantification method (cRPKM), we tested the power of such ChIP-seq strategies to predict relative protein and RNA levels at the pre-pro-B and pro-B differentiation stages in early B cell lymphopoiesis. Using a multi-omics approach that compares promoter chromatin status (ChIP-seq; published in GSE:21978) with ongoing active transcription (GRO-seq; published in GSE:40173), steady state mRNA (RNA-seq), inferred mRNA stability, and relative proteome abundance measurements (iTRAQ), we demonstrate that active chromatin modifications at promoters are a good indicator of transcription and steady state mRNA levels. Moreover, we found that promoters with active chromatin modifications exclusively in one of these cell states frequently predicted differentially expressed proteins. However, we found that many genes whose promoters have non-differential but active chromatin modifications also displayed changes in expression of their cognate proteins. This large class of developmentally and differentially regulated proteins that was uncoupled from chromatin status used mostly post-transcriptional mechanisms. Interestingly, the most differentially expressed protein in our B-cell development system, 2410004B18Rik, was regulated by a post-transcriptional mechanism, which further analyses indicated was mediated by an identified miRNA. These data provide a striking example of how our integrated multi-omics data set can be useful in uncovering regulatory mechanisms. Total RNA from mouse pre-pro-B and pro-B cells, depleted of rRNA and small RNAs, was sequenced using a strand specific, single end sequencing strategy.

2013-11-19 | E-GEOD-52450 | biostudies-arrayexpress

Optimal settings of mass spectrometry open search strategy for higher confidence

Project description:More than half of the mass spectra could not be identified in most proteome mass spectrometry experiments. Various modifications have been considered as a major reason. Open search strategy was then introduced to solve this problem, however, lacking thorough quality assessment using independent information. Here, we used the “Suspicious Discovery Rate (SDR)” based on the translatome sequencing (RNC-seq) as an independent source to assess the proteome open search strategy. We found that the open search strategy increased the spectra utilization with the cost of increasing suspicious identifications that lacks translation evidence. We further suggested that restricting the peptide FDR below 0.1% would efficiently control the suspicious identifications of open search methods and enhanced the confidence of the peptide identification with modifications than the narrow window search. These results facilitated the proper use of open search methods for higher quality of proteome identifications with the information of post-translational modifications and single amino acid polymorphisms

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Site-Link for identification of chromatinized proteome defined by site-specific histone acylations in living cells

Project description:By coupling the mass spectrometry, we developed the Site-Link strategy based on the GCE method for analyzing the interacting proteome of histone lysine acylations in living cells. As K* could mimic the natural lysine to be recognized by natural lysine synthase, histones bearing the site-specifically incorporated K*acyl, should also be also integrated into nucleosomes and further compacted into chromatin similar as Kacyl with cell passaging. The subsequent 365-nm light irradiation could activate the diazirine and covalently capture potential effector proteins on native chromatin in living cells. The effector proteins could be identified after gel-based proteomics and MS/MS analysis of the crosslinked histone-effector complexes.

2023-03-06 | PXD033959 | Pride

Site-Link for identification of chromatinized proteome defined by site-specific histone acylations in living cells

2023-03-06 | PXD029505 | Pride

Prediction of gene activity based on an integrated multi-omics approach.

Project description:An increasingly common method for predicting gene activity is genome-wide chromatin immunoprecipitation of ‘active’ chromatin modifications followed by massively parallel sequencing (ChIP-seq). Using a novel ChIP-seq quantification method (cRPKM), we tested the power of such ChIP-seq strategies to predict relative protein and RNA levels at the pre-pro-B and pro-B differentiation stages in early B cell lymphopoiesis. Using a multi-omics approach that compares promoter chromatin status (ChIP-seq; published in GSE:21978) with ongoing active transcription (GRO-seq; published in GSE:40173), steady state mRNA (RNA-seq), inferred mRNA stability, and relative proteome abundance measurements (iTRAQ), we demonstrate that active chromatin modifications at promoters are a good indicator of transcription and steady state mRNA levels. Moreover, we found that promoters with active chromatin modifications exclusively in one of these cell states frequently predicted differentially expressed proteins. However, we found that many genes whose promoters have non-differential but active chromatin modifications also displayed changes in expression of their cognate proteins. This large class of developmentally and differentially regulated proteins that was uncoupled from chromatin status used mostly post-transcriptional mechanisms. Interestingly, the most differentially expressed protein in our B-cell development system, 2410004B18Rik, was regulated by a post-transcriptional mechanism, which further analyses indicated was mediated by an identified miRNA. These data provide a striking example of how our integrated multi-omics data set can be useful in uncovering regulatory mechanisms.

2013-11-19 | GSE52450 | GEO

Multi-omic analysis of gametogenesis reveals a novel signature at the promoters and distal enhancers of active genes

Project description:Epigenetic regulation of gene expression is tightly controlled by the dynamic modification of histones by chemical groups, the diversity of which has largely expanded over the past decade with the discovery of lysine acylations, catalyzed from acyl-coenzymes A. Here, we investigated the dynamics of lysine acetylation and crotonylation on histones H3 and H4 during mouse spermatogenesis. Lysine crotonylation appeared to be of significant abundance compared to acetylation, particularly on Lys27 of histone H3 (H3K27cr) that accumulates in sperm in a cleaved form of H3. We identified the genomic localization of H3K27cr and studied its effects on transcription compared to the classical active mark H3K27ac at promoters and distal enhancers. The presence of both marks was strongly associated with highest gene expression. Assessment of their co-localization with transcription regulators (SLY, SOX30) and chromatin-binding proteins (BRD4, BORIS and CTCF) indicated systematic highest binding when both active marks were present and different selective binding when present alone at chromatin. H3K27cr and H3K27ac finally mark the building of some sperm super-enhancers. This integrated analysis of omics data provides an unprecedented level of understanding of gene expression regulation by H3K27cr in comparison to H3K27ac, and reveals both synergistic and specific actions of each histone modification.

2020-03-11 | E-MTAB-8328 | biostudies-arrayexpress

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