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Proteomics analysis of ligated and recombinant human histones H3 and H4

ABSTRACT: A library of unmodified and differentially modified human histones H3 and H4 was prepared using native chemical ligation as described previously (Bartke et al., 2010; Nakamura et al., 2019). The modification status of histone H3 and H4 products was confirmed by LC-MS/MS.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Andrey Tvardovskiy

LAB HEAD: Till Bartke

PROVIDER: PXD020773 | Pride | 2023-12-10

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	H3K27ac.raw	Raw
	H3K27me2.raw	Raw
	H3K27me3.raw	Raw
	H3K4me1.raw	Raw
	H3K4me1K18acK23acK27me3.raw	Raw

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Publications

Decoding chromatin states by proteomic profiling of nucleosome readers.

Lukauskas Saulius S Tvardovskiy Andrey A Nguyen Nhuong V NV Stadler Mara M Faull Peter P Ravnsborg Tina T Özdemir Aygenli Bihter B Dornauer Scarlett S Flynn Helen H Lindeboom Rik G H RGH Barth Teresa K TK Brockers Kevin K Hauck Stefanie M SM Vermeulen Michiel M Snijders Ambrosius P AP Müller Christian L CL DiMaggio Peter A PA Jensen Ole N ON Schneider Robert R Bartke Till T

Nature 20240306 8004

DNA and histone modifications combine into characteristic patterns that demarcate functional regions of the genome<sup>1,2</sup>. While many 'readers' of individual modifications have been described<sup>3-5</sup>, how chromatin states comprising composite modification signatures, histone variants and internucleosomal linker DNA are interpreted is a major open question. Here we use a multidimensional proteomics strategy to systematically examine the interaction of around 2,000 nuclear proteins wi ...[more]

PMID: 38448585

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Histone modifications associated with the histone chaperone DAXX

Project description:A multitude of histone chaperones is required to protect histones from their biosynthesis to DNA deposition. They cooperate through the formation of co-chaperone complexes, but the crosstalk between nucleosome assembly pathways is unclear. Using explorative interactomics approaches, we map the organization of the histone H3-H4 chaperones network and define the interplay between histone chaperones systems. We identify and validate a panel of novel histone (PTM) dependent complexes. We show DAXX acts separately from the rest of the network, recruiting heterochromatin factors and promoting lysine 9 tri-methylated new histone H3.3 prior to deposition onto DNA. With its functionality, DAXX provides a molecular mechanism for de novo heterochromatin assembly.

2023-03-06 | PXD034924 | Pride

Quantification of histone H3 and H4 modifications by mass spectrometry in wildtype and MCM2-2A mutants of mice ESCs

Project description:During DNA replication modified parental histones H3-H4 are segregated almost symmetrically to the two daughter DNA strands enabling mitotic inheritance of histone PTMs. Whether heritable histone modifications confer epigenetic regulation by maintaining chromatin states and gene expression is unclear. Using MCM2 histone-binding mutant embryonic stem cells (ESCs) and mass spectrometry, we show that asymmetric segregation of parental histones to the leading strand causes imbalanced inheritance of histone H3K27 methylations to daughter cells.

2023-08-30 | PXD020326 | Pride

Sample preparation procedure for mass spectrometric analysis of plant histones

Project description:The protocol for preparing samples of plant histones prior to LC-MS/MS analysis was established, using a filter-aided sample preparation (FASP) technique to remove natural contaminants and chemical additives incompatible with MS-based procedures. The protocol consists of nuclei isolation, histone extraction into acidic solvent, protein propionylation and on-membrane digestion, then peptide propionylation in solution. Two variants of the derivatization protocol, in-solution propionylation in the vial (PROP-in-SOL) and propionylation on the filter unit (PROP-on-FILTER), were compared by efficiency of the labeling and by abundance of identified histone peptide forms.

2018-09-20 | PXD010102 | Pride

De novo mutations of MSL3 cause a novel X-linked syndrome due to impaired histone H4 lysine 16 acetylation

Project description:Epigenetic regulation by histone acetylation plays a key role in cellular homeostasis and its misregulation is associated with human disease. Histone 4 Lysine 16 acetylation (H4K16ac) serves a unique role amongst the many histone modifications as it directly affects chromatin structure1. The Male Specific Lethal (MSL) complex associated MOF/KAT8 histone acetyl transferase is responsible for bulk H4K16ac in flies and mammals. Yet, its importance during human development and a potential involvement in human pathologies remains largely unknown. Here, we uncover that pathogenic variants in MSL3, a component of the MSL complex, are causative for a new recognizable X-linked syndrome affecting Histone 4 Lysine 16 acetylation (H4K16ac) in both male and female individuals. Common clinical features of the syndrome include global developmental delay comprising profound speech delay, delayed ability to walk and craniofacial dysmorphism. Using patient-derived primary fibroblasts, we demonstrate that de novo variants or deletions of MSL3 affect the assembly and enzymatic activity of the MSL complex, hence impacting on global H4K16ac levels. Transcriptome analysis from patient cells showed misregulation of cellular pathways involved in serotonergic signaling, morphogenesis, axon guidance and cell structure. Finally, using HDAC inhibitor treatment, we can rescue expression of downregulated target genes, offering potential therapeutic avenues for MSL3-mutated patients. Taken together, we characterize a novel syndrome, named ILyADe (Impaired Lysine 16 acetylation associated disorder), which is caused by mutations of an epigenetic regulator, allowing us for the first time to unravel the crucial role of H4K16ac during human development. ILyaDe thus constitutes a new class of syndromes associated with misregulation of a single epigenetic modification caused by a genetic alteration.

2018-09-18 | PXD009317 | Pride

Widespread hydroxylation of unstructured lysine-rich protein domains by JMJD6

Project description:The Jumonji domaining-containing protein JMJD6 is a 2-oxoglutarate dependent dioxygenase that has been implicated in a broad range of biological functions. Cellular studies have implicated the enzyme in chromatin biology, transcription, DNA repair, mRNA splicing and co-transcriptional processing. Although not all studies agree, JMJD6 has been reported to catalyse both hydroxylation of lysine residues and demethylation of arginine residues. However, despite extensive study and the indirect implication of JMJD6 catalysis in many cellular processes, direct assignment of JMJD6 catalytic substrates has been limited. Examination of a site of reported prolyl hydroxylation within a lysine-rich region of the bromodomain protein BRD4 led us to conclude that hydroxylation was in fact on lysine and catalysed by Jmjd6. This prompted a wider search for JMJD6-catalysed protein modifications deploying mass spectrometric methods designed to identify novel substrate associations and facilitate analysis of lysine-rich regions by LC-MSMS. Using derivatization of lysine with propionic anhydride to improve the analysis of tryptic peptides and a pharmacological inhibitor of JMJD6 to stabilise enzyme/substrate associations, we report over 100 sites of JMJD6-catalysed lysyl hydroxylation on 48 protein substrates including 19 sites of hydroxylation on BRD4. Most hydroxylations were within lysine-rich regions that are predicted to be unstructured; in some multiple modifications were observed on adjacent lysine residues. Almost all of the JMJD6 substrates defined in this study have been associated with membraneless organelle formation. Taken together with findings implicating lysine-rich regions in subcellular partitioning by liquid-liquid phase separation, our findings raise the possibility that JMJD6 may play a role in regulating such processes in response to stresses, including hypoxia. This deposition contains all hydroxylysine assignment data.

2022-08-02 | PXD031221 | Pride

Comprehensive Histone Epigenetics (CHEpi): A mass spectrometry based screening assay to measure epigenetic toxicity

Project description:Epigenetics have taken center stage in the study of diseases such as cancer, diabetes, and neurodegeneration. For nearly thirty years, it has been known that cancer cells exhibit abnormal DNA methylation patterns. In contrast, the large scale analysis of histone posttranslational modifications (hPTMs) has lagged behind because classically, histone modification analysis has relied on site specific antibody-based techniques. Mass spectrometry (MS) is a technique that holds the promise to picture the histone code comprehensively in a single experiment. Therefore, we developed an MS-based method that is capable of tracking all possible hPTMs in an untargeted approach. In this way, trends in single and combinatorial hPTMs can be reported and enable prediction of the epigenetic toxicity of compounds. Moreover, this method is based on the use of human embryonic stem cells (hESCs), thereby omitting the need to sacrifice laboratory animals. Improving the workflow and the user-friendliness of the assay is an ongoing effort. Still, this novel toxicoepigenetic assay and the data it generates holds great potential for, among others, pharmaceutical industry, food science, clinical diagnostics and, environmental toxicity screening.

2020-10-12 | PXD019652 | Pride

Large scale toxicoepigenetics on histones: a mass spectrometry-based screening assay applied to developmental toxicity

Project description:Toxicoepigenetics is an emerging field that studies the toxicological impact of compounds on protein expression through heritable, non-genetic mechanisms, such as histone post-translational modifications (hPTMs). Due to substantial progress in the large-scale study of hPTMs, integration into the field of toxicology is a promising addition that offers the opportunity to gain novel insights into toxicological phenomena. Moreover, there is a growing demand for high-throughput human-based in vitro assays for toxicity testing and especially for developmental toxicity. Consequently, we developed a mass spectrometry based proof-of-concept to develop an assay to screen the histone code and hence detecting multiple hPTMs changes simultaneously in human embryonic stem cells. To prove the applicability and performance, we first validated the untargeted workflow with valproic acid (VPA), a histone deacetylase inhibitor. These results demonstrate that our workflow is capable of mapping the hPTM-dynamics, with a general increase in acetylations as an internal control. To illustrate the scalability, the workflow was applied on a proof-of-concept dose-response library of a total of ten compounds with either i) a known effect on the hPTMs (BIX-01294, 3-Deazaneplanocin A, Trichostatin A, and VPA), ii) a presumed developmental toxicity, including compounds of abuse (Caffeine, Ethanol, Nicotine, Methotrexate, and All-trans retinoic acid), or iii) no proven embryotoxicity (Penicillin G). In conclusion, we show that toxicoepigenetic screening on histones is feasible and yields very rich data that holds great potential, not only for applications in the pharmaceutical industry, but also for environmental toxicity and food safety.

2022-02-17 | PXD026468 | Pride

Histone acetyltransferase 2 links H4 acetylation, H2A.Z deposition and transcription start site-choice in African trypanosomes

Project description:Both, acetylation of histones and of histone variant H2A.Z are conserved features of eukaryotic transcription start sites (TSSs) and both features appear to be critical for correct transcription initiation. However, complex patterns of transcriptional regulation have complicated the establishment of functional links between histone acetylation, H2A.Z deposition and their importance in transcription regulation. To elucidate these links, we took advantage of the unusual genome organization in Trypanosoma brucei, a highly divergent eukaryote. In T. brucei genes are organized in long polycistronic transcription units, drastically reducing the sites of transcription initiation. Employing a highly sensitive and quantitative mass-spectrometry-based approach, we quantified the genome-wide histone acetylation and methylation pattern and identified various acetyl and methyl marks exclusively enriched at TSSs In addition, we show that deletion of histone acetyltransferase 2 results in a loss of H4 acetylation at TSSs, a loss of H2A.Z deposition at TSSs and a shift in the sites of transcription initiation. Combined, our findings demonstrate an evolutionary conserved link between histone H4 acetylation, H2A.Z deposition and RNA transcription initiation.

2020-03-24 | PXD014452 | Pride

Proteomic profiling of histone acetylation pattern in Arabidopsis

Project description:The levels of acetylated and non-acetylated histone peptide forms were quantified in A. thaliana plants with loss of function of HDA6, and plants exposed to HDAs inhibitors (trichostatin and sodium butyrate)during germination .

2019-10-14 | PXD014739 | Pride

Complete loss of H3K9 methylation dissolves mouse heterochromatin organization

Project description:Histone H3 lysine 9 (H3K9) methylation is a central epigenetic modification that defines heterochromatin from unicellular to multicellular organisms. In mammalian cells, H3K9 methylation can be catalyzed by at least six distinct SET domain enzymes: Suv39h1/Suv39h2, Eset1/Eset2 and G9a/Glp. We used mouse embryonic fibroblasts (MEFs) with a conditional mutation for Eset1 and introduced progressive deletions for the other SET domain genes by CRISPR/Cas9 technology. Compound mutant MEFs for all 6 SET domain methyltransferase (KMT) genes lack all H3K9 methylation states, derepress nearly all families of repeat elements and display genomic instabilities. Strikingly, the 6KO H3K9 KMT MEFs no longer maintain heterochromatin organization and have lost electron-dense heterochromatin. This is the first analysis of H3K9 methylation deficient mammalian chromatin and reveals a crucial function for H3K9 methylation in protecting heterochromatin organization and genome integrity.

2021-05-27 | PXD018175 | Pride

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