A mass spectrometry-based assay using metabolic labeling to rapidly monitor chromatin accessibility of modified histone proteins

Abstract Histone post-translational modifications (PTMs) contribute to chromatin accessibility due to their chemical properties and their ability to recruit enzymes responsible for DNA readout and chromatin remodeling. To date, more than 400 different histone PTMs and thousands of combinations of PTMs have been identified, the vast majority with still unknown biological function. Identification and quantification of histone PTMs has become routine in mass spectrometry (MS) but, since raising antibodies for each PTM in a study can be prohibitive, lots of potential is lost from MS datasets when uncharacterized PTMs are found to be significantly regulated. We developed an assay that uses metabolic labeling and MS to associate chromatin accessibility with histone PTMs and their combinations. The labeling is achieved by spiking in the cell media a 5x concentration of stable isotope labeled arginine and allow cells to grow for at least one cell cycle. We quantified the labeling incorporation of about 200 histone peptides with a proteomics workflow, and we confirmed that peptides carrying PTMs with extensively characterized roles in active transcription or gene silencing were in highly or poorly labeled forms, respectively. Data were further validated using next-generation sequencing to assess the transcription rate of chromatin regions modified with five selected PTMs. Furthermore, we quantified the labeling rate of peptides carrying co-existing PTMs, proving that this method is suitable for combinatorial PTMs. We focus on the abundant bivalent mark H3K27me3K36me2, showing that H3K27me3 dominantly represses histone swapping rate even in the presence of the more permissive PTM H3K36me2. Together, we envision this method will help to generate hypotheses regarding histone PTM functions and, potentially, elucidate the role of combinatorial histone codes.

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The challenge of detecting modifications on proteins

Essays in Biochemistry ◽

10.1042/ebc20190055 ◽

2020 ◽

Vol 64 (1) ◽

pp. 135-153 ◽

Cited By ~ 1

Author(s):

Lauren Elizabeth Smith ◽

Adelina Rogowska-Wrzesinska

Keyword(s):

Mass Spectrometry ◽

Protein Function ◽

Chemical Properties ◽

Lysine Acetylation ◽

Mass Determination ◽

Post Translational Modifications ◽

Site Specific ◽

The Common

Abstract Post-translational modifications (PTMs) are integral to the regulation of protein function, characterising their role in this process is vital to understanding how cells work in both healthy and diseased states. Mass spectrometry (MS) facilitates the mass determination and sequencing of peptides, and thereby also the detection of site-specific PTMs. However, numerous challenges in this field continue to persist. The diverse chemical properties, low abundance, labile nature and instability of many PTMs, in combination with the more practical issues of compatibility with MS and bioinformatics challenges, contribute to the arduous nature of their analysis. In this review, we present an overview of the established MS-based approaches for analysing PTMs and the common complications associated with their investigation, including examples of specific challenges focusing on phosphorylation, lysine acetylation and redox modifications.

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Histone modifications during the life cycle of the brown alga Ectocarpus

10.1101/2020.03.09.980763 ◽

2020 ◽

Cited By ~ 3

Author(s):

Simon Bourdareau ◽

Leila Tirichine ◽

Bérangère Lombard ◽

Damarys Loew ◽

Delphine Scornet ◽

...

Keyword(s):

Life Cycle ◽

Brown Algae ◽

Land Plant ◽

Chromatin Modifications ◽

Post Translational Modifications ◽

Transcription Start Sites ◽

Histone Ptms ◽

Genomic Regions ◽

Active Genes

AbstractBackgroundBrown algae evolved complex multicellularity independently of the animal and land plant lineages and are the third most developmentally complex phylogenetic group on the planet. An understanding of developmental processes in this group is expected to provide important insights into the evolutionary events necessary for the emergence of complex multicellularity. Here we have focused on mechanisms of epigenetic regulation involving post-translational modifications (PTMs) of histone proteins.ResultsA total of 47 histone PTMs were identified, including a novel mark H2AZR38me1, but Ectocarpus lacks both H3K27me3 and the major polycomb complexes. ChIP-seq identified PTMs associated with transcription start sites (TSSs) and gene bodies of active genes, and with transposons. H3K79me2 exhibited an unusual pattern, often marking large genomic regions spanning several genes. TSSs of closely spaced divergently transcribed gene pairs shared a common nucleosome depleted region and exhibited shared histone PTM peaks. Overall, patterns of histone PTMs were stable through the life cycle. Analysis of histone PTMs at generation-biased genes identified a correlation between the presence of specific chromatin marks and the level of gene expression.ConclusionsThe overview of histone PTMs in the brown algae presented here will provide a foundation for future studies aimed at understanding the role of chromatin modifications in the regulation of brown algal genomes.

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A genetic analysis reveals novel histone residues required for transcriptional reprogramming upon stress

Nucleic Acids Research ◽

10.1093/nar/gkaa081 ◽

2020 ◽

Vol 48 (7) ◽

pp. 3455-3475

Author(s):

Cristina Viéitez ◽

Gerard Martínez-Cebrián ◽

Carme Solé ◽

René Böttcher ◽

Clement M Potel ◽

...

Keyword(s):

Transcriptional Activation ◽

Growth Conditions ◽

Proof Of Concept ◽

Transcriptional Program ◽

Post Translational Modifications ◽

Transcriptional Reprogramming ◽

Histone Ptms ◽

Response To Stress ◽

State Growth

Abstract Cells have the ability to sense, respond and adapt to environmental fluctuations. Stress causes a massive reorganization of the transcriptional program. Many examples of histone post-translational modifications (PTMs) have been associated with transcriptional activation or repression under steady-state growth conditions. Comparatively less is known about the role of histone PTMs in the cellular adaptive response to stress. Here, we performed high-throughput genetic screenings that provide a novel global map of the histone residues required for transcriptional reprogramming in response to heat and osmotic stress. Of note, we observed that the histone residues needed depend on the type of gene and/or stress, thereby suggesting a ‘personalized’, rather than general, subset of histone requirements for each chromatin context. In addition, we identified a number of new residues that unexpectedly serve to regulate transcription. As a proof of concept, we characterized the function of the histone residues H4-S47 and H4-T30 in response to osmotic and heat stress, respectively. Our results uncover novel roles for the kinases Cla4 and Ste20, yeast homologs of the mammalian PAK2 family, and the Ste11 MAPK as regulators of H4-S47 and H4-T30, respectively. This study provides new insights into the role of histone residues in transcriptional regulation under stress conditions.

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The role of HIRA-dependent H3.3 deposition and its modifications in the somatic hypermutation of immunoglobulin variable regions

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2114743118 ◽

2021 ◽

Vol 118 (50) ◽

pp. e2114743118

Author(s):

Guojun Yu ◽

Yongwei Zhang ◽

Varun Gupta ◽

Jinghang Zhang ◽

Thomas MacCarthy ◽

...

Keyword(s):

B Cells ◽

Somatic Hypermutation ◽

Variable Region ◽

Chromatin Accessibility ◽

Variable Regions ◽

Igh Locus ◽

Human B Cells ◽

Active Transcription ◽

Activation Induced Deaminase

The H3.3 histone variant and its chaperone HIRA are involved in active transcription, but their detailed roles in regulating somatic hypermutation (SHM) of immunoglobulin variable regions in human B cells are not yet fully understood. In this study, we show that the knockout (KO) of HIRA significantly decreased SHM and changed the mutation pattern of the variable region of the immunoglobulin heavy chain (IgH) in the human Ramos B cell line without changing the levels of activation-induced deaminase and other major proteins known to be involved in SHM. Except for H3K79me2/3 and Spt5, many factors related to active transcription, including H3.3, were substantively decreased in HIRA KO cells, and this was accompanied by decreased nascent transcription in the IgH locus. The abundance of ZMYND11 that specifically binds to H3.3K36me3 on the IgH locus was also reduced in the HIRA KO. Somewhat surprisingly, HIRA loss increased the chromatin accessibility of the IgH V region locus. Furthermore, stable expression of ectopic H3.3G34V and H3.3G34R mutants that inhibit both the trimethylation of H3.3K36 and the recruitment of ZMYND11 significantly reduced SHM in Ramos cells, while the H3.3K79M did not. Consistent with the HIRA KO, the H3.3G34V mutant also decreased the occupancy of various elongation factors and of ZMYND11 on the IgH variable and downstream switching regions. Our results reveal an unrecognized role of HIRA and the H3.3K36me3 modification in SHM and extend our knowledge of how transcription-associated chromatin structure and accessibility contribute to SHM in human B cells.

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Accelerating the field of epigenetic histone modification through mass spectrometry-based approaches

Molecular & Cellular Proteomics ◽

10.1074/mcp.r120.002257 ◽

2020 ◽

pp. mcp.R120.002257

Author(s):

Congcong Lu ◽

Mariel Coradin ◽

Elizabeth G Porter ◽

Benjamin A Garcia

Keyword(s):

Mass Spectrometry ◽

Molecular Biology ◽

Histone Modification ◽

High Throughput ◽

Epigenetic Regulation ◽

Associated Factors ◽

Chemical Biology ◽

Post Translational Modifications ◽

Histone Ptms ◽

Chromatin Biology

Histone post-translational modifications (PTMs) are one of the main mechanisms of epigenetic regulation. Dysregulation of histone PTMs leads to many human diseases, such as cancer. Due to its high-throughput, accuracy, and flexibility, mass spectrometry (MS) has emerged as a powerful tool in the epigenetic histone modification field, allowing the comprehensive and unbiased analysis of histone PTMs and chromatin-associated factors. Coupled with various techniques from molecular biology, biochemistry, chemical biology and biophysics, MS has been employed to characterize distinct aspects of histone PTMs in the epigenetic regulation of chromatin functions. In this review we will describe advancements in the field of MS that have facilitated the analysis of histone PTMs and chromatin biology.

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Role of mass spectrometry in mapping strain variation and post-translational modifications of viral proteins

Biological Mass Spectrometry ◽

10.1002/bms.1200191104 ◽

1990 ◽

Vol 19 (11) ◽

pp. 646-654 ◽

Cited By ~ 13

Author(s):

Jeffrey J. Gorman ◽

Gary L. Corino ◽

Brian J. Shiell

Keyword(s):

Mass Spectrometry ◽

Viral Proteins ◽

Strain Variation ◽

Post Translational Modifications

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PROTEIN DYNAMICS IN PHOSPHORYLATION-MEDIATED ALLOSTERY PROBED BY AMIDE EXCHANGE MASS SPECTROMETRY

COSMOS ◽

10.1142/s0219607713300014 ◽

2013 ◽

Vol 09 (01) ◽

pp. 19-27

Author(s):

MADHUBRATA GHOSH ◽

GANESH S. ANAND

Keyword(s):

Mass Spectrometry ◽

Protein Dynamics ◽

Protein Function ◽

Post Translational Modifications ◽

Exchange Mass Spectrometry ◽

Amide Exchange ◽

Hydrogen Deuterium ◽

Deuterium Exchange Mass Spectrometry ◽

And Function

A major goal of molecular biology is to correlate molecular structure with function. Since most enzymes and biological catalysts are proteins, the focus for correlating 'form' with 'function' has been entirely on protein macromolecular structure. It is obvious that any understanding of protein function must come through an understanding protein dynamics. Furthermore, all of the regulatory reactions are through changes in dynamics brought about by post-translational modifications, the most important of which is phosphorylation. This review highlights the important role of covalent phosphorylation and noncovalent phosphates in regulating allosteric effects and function through a study of protein dynamics. Mass spectrometry is a relatively new and increasingly important tool for describing protein dynamics. All examples described in this review have been studied by amide hydrogen/deuterium exchange mass spectrometry.

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PTMViz: a tool for analyzing and visualizing histone post translational modification data

BMC Bioinformatics ◽

10.1186/s12859-021-04166-9 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Kevin Chappell ◽

Stefan Graw ◽

Charity L. Washam ◽

Aaron J. Storey ◽

Chris Bolden ◽

...

Keyword(s):

Mass Spectrometry ◽

Histone Modifications ◽

Drug Exposure ◽

Regulation Of Gene Expression ◽

Mass Spectrometry Data ◽

Saline Control ◽

Post Translational Modifications ◽

Histone Ptms ◽

Gene And Protein Expression ◽

Data Tables

Abstract Background Histone post-translational modifications (PTMs) play an important role in our system by regulating the structure of chromatin and therefore contribute to the regulation of gene and protein expression. Irregularities in histone PTMs can lead to a variety of different diseases including various forms of cancer. Histone modifications are analyzed using high resolution mass spectrometry, which generate large amounts of data that requires sophisticated bioinformatics tools for analysis and visualization. PTMViz is designed for downstream differential abundance analysis and visualization of both protein and/or histone modifications. Results PTMViz provides users with data tables and visualization plots of significantly differentiated proteins and histone PTMs between two sample groups. All the data is packaged into interactive data tables and graphs using the Shiny platform to help the user explore the results in a fast and efficient manner to assess if changes in the system are due to protein abundance changes or epigenetic changes. In the example data provided, we identified several proteins differentially regulated in the dopaminergic pathway between mice treated with methamphetamine compared to a saline control. We also identified histone post-translational modifications including histone H3K9me, H3K27me3, H4K16ac, and that were regulated due to drug exposure. Conclusions Histone modifications play an integral role in the regulation of gene expression. PTMViz provides an interactive platform for analyzing proteins and histone post-translational modifications from mass spectrometry data in order to quickly identify differentially expressed proteins and PTMs.

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Epigenetic dysregulation from chromosomal transit in micronuclei

10.1101/2022.01.12.475944 ◽

2022 ◽

Author(s):

Albert Agustinus ◽

Ramya Raviram ◽

Bhargavi Dameracharla ◽

Jens Luebeck ◽

Stephanie Stransky ◽

...

Keyword(s):

Chromosomal Instability ◽

Chromatin Accessibility ◽

Epigenetic Alterations ◽

Copy Number Alterations ◽

Mitotic Chromosomes ◽

Post Translational Modifications ◽

Positional Bias ◽

Histone Ptms ◽

Mitotic Abnormalities ◽

Intergenic Regions

Chromosomal instability (CIN) and epigenetic alterations are characteristics of advanced and metastatic cancers [1-4], yet whether they are mechanistically linked is unknown. Here we show that missegregation of mitotic chromosomes, their sequestration in micronuclei [5, 6], and subsequent micronuclear envelope rupture [7] profoundly disrupt normal histone post-translational modifications (PTMs), a phenomenon conserved across humans and mice as well as cancer and non-transformed cells. Some of the changes to histone PTMs occur due to micronuclear envelope rupture whereas others are inherited from mitotic abnormalities prior to micronucleus formation. Using orthogonal techniques, we show that micronuclei exhibit extensive differences in chromatin accessibility with a strong positional bias between promoters and distal or intergenic regions. Finally, we show that inducing CIN engenders widespread epigenetic dysregulation and that chromosomes which transit in micronuclei experience durable abnormalities in their accessibility long after they have been reincorporated into the primary nucleus. Thus, in addition to genomic copy number alterations, CIN can serve as a vehicle for epigenetic reprogramming and heterogeneity in cancer.

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