Analysis Of Histone Post-translational Modifications Research Articles

Abstract 6413: Biomarkers of response and progression to Pembrolizumab and Vorinostat combination in late-stage squamous cell carcinoma patients of different locations included in the PEVOsq basket trial

Abstract Background: Epigenetic modulation plays a major role in escaping tumor immunosurveillance. PEVOsq (NCT04357873) was an open-label, non-randomized, multi-center, basket phase II trial evaluating the efficacy of pembrolizumab (P) and the vorinostat (V) HDAC inhibitor in 112 patients (pts) with recurrent and/or metastatic squamous cell carcinoma (SCC) of the cervix, head and neck, anus, vulva/vagina, penis, and lung. Results showed an objective response rate (ORR) of 25%, the highest being in anal (31%) and cervical (39%) (ASCO #397612). We report here early biomarkers of response and progression to P+V. Patients and Methods: Pts had to undergo a tumor biopsy at baseline, and optional biopsies on-treatment (after cycle 2), and at progression for translational analyses. PD-L1 expression (n=102), tumor-infiltrating lymphocytes quantification (n=39), HPV typing (n=111), WES (n=83), RNAseq (n=82) and liquid chromatography-mass spectrometry (LC-MS) quantitative analysis of histone post-translational modifications (PTMs) (n=44) were performed on baseline samples.SNVs, CNVs, TMB, MSI status (by MSI sensor), aneuploidy score (AS), mutational signatures, immune signatures, and histone modifications were correlated with response and progression. Results: Sixty-six pts were HPV+ (59%). Most frequently altered genes were PIK3CA (30%), TP53 (30%), KMT2D/C (18 and 17%), and FGF3/4 (17%). MSI was detected in 4 pts (5%), and TMB-high (>10mut/Mb) in 13 pts (16%). High AS (>10) was detected in 34 pts (40%), and was significantly associated with HPV- status (p=0.03). TMB high and MSI were associated with MMR mutational signatures (p<0.01). TMB high was also associated with APOBEC (p=0.02) and DNA polymerase (POL) signatures (p <0.01). Biomarkers associated with improved overall survival (OS) included HPV positivity (7.8 months versus 17.4 months, p<0.01), high TMB (not reached versus 10.3 months, p=0.01), and high PD-L1 CPS score (13.2 months versus 7.0 months, p=0.06).Deconvolution of immune cell infiltration showed an expected trend for increased CD8 and decreased neutrophil infiltration in non progressor pts.Interestingly, LC-MS histone PTMs profiling showed a significant decrease in H4K5K8K12K16-4ac (p=0.05) and increase in H3K27me2K36me1 in non progressor pts (p=0.03). At the transcriptomic level, although not statistically significant, we observed modulation in the expression of genes associated with H3K36me1 (KDM2A, SETD3, SMYD2), and H3K27me3 (KDM6A) regulation, as well as HDAC genes. Conclusions: HPV+ status was associated with improved OS, together with a high TMB and PD-L1 expression. CD8, macrophages and neutrophil signatures at baseline were associated with response. Promising epigenetic biomarkers were identified using LC/MS and will be correlated to transcriptomic and genomic data. Citation Format: Christophe Le Tourneau, Luca Mazzarella, Bastien Cabarrou, Miguel Francisco, Célia Dupain, Maral Halladjian, Gianmaria Frige, Bruno Duso, Yinxiu Zhan, Elena Guerini-Rocco, Roberta Noberini, Tiziana Bonaldi, Maria Manuela Tonini, Grégoire Marret, Clélia Coutzac, Elodie Coquan, Mathilde Saint-Ghislain, Olivia Le Saux, Laetitia Chanas, François Legrand, Daria Maria Filippini, Emmanuelle Jeannot, Stephan Bernhart, Gabor Balogh, Marta Jimenez, Thomas Filleron, Nicolas Servant, Maud Kamal. Biomarkers of response and progression to Pembrolizumab and Vorinostat combination in late-stage squamous cell carcinoma patients of different locations included in the PEVOsq basket trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6413.

A Strategy to Characterize the Global Landscape of Histone Post-Translational Modifications Within Tissues of Nonmodel Organisms.

Histone post-translational modifications (PTMs) are epigenetic marks that play a critical role in the expression and maintenance of DNA, but they remain largely uninvestigated in nonmodel organisms due to technical challenges. To begin alleviating this issue, we developed a workflow for histone PTM analysis in Mozambique tilapia (Oreochromis mossambicus), being a widespread and environmentally hardy fish, using mass spectrometry methods. By incorporating multiple protein digestion methods into the preparation of each sample, we reliably quantified 214 biologically relevant histone PTMs. All of these histone PTMs, collectively referred to as the global histone PTM landscape, were characterized in the gills, kidney, and testes of this fish. By comparing the global histone PTM landscape between the three tissues, we found that 91.59% of histone PTMs were tissue-dependent. The workflow and tools for histone PTM analysis described in this study are now publicly available and enable comprehensive investigation into the influence of environmental stress on histone PTMs in nonmodel organisms. Given the functionality and flexibility of histone PTMs, we anticipate that the study of histone PTMs in ecologically relevant contexts will provide ground-breaking insights into comparative physiology and evolution.

Mass Spectrometry-Based Analysis of Histone Posttranslational Modifications from Laser Microdissected Samples.

The analysis of histone posttranslational modifications (PTMs) in clinical samples has gained considerable interest due to the increasing knowledge about the implication of epigenetics in a multitude of physiological and pathological processes. Mass spectrometry (MS) has emerged as the most accurate and versatile tool to detect and quantify histone PTMs and has also been applied to clinical specimens, thanks to protocols developed during the past years. However, the requirement for relatively large amounts of material has so far impaired the application of these approaches to samples available in limited amounts. To address this issue, we have recently streamlined the protein extraction procedure from low-amount clinical samples and optimized the digestion step, obtaining a protocol suitable for the analysis of the most common histone PTMs from laser microdissected tissue areas containing down to 1000 cells, which we will describe in this chapter.

A Super-SILAC Approach for Profiling Histone Posttranslational Modifications.

Histone posttranslational modifications (PTMs) play an important role in the regulation of gene expression and have been implicated in a multitude of physiological and pathological processes. During the last decade, mass spectrometry (MS) has emerged as the most accurate and versatile tool to quantitate histone PTMs. Stable-isotope labeling by amino acids in cell culture (SILAC) is an MS-based quantitation strategy involving metabolic labeling of cells, which has been applied to global protein profiling as well as histone PTM analysis. The classical SILAC approach is associated with reduced experimental variability and high quantitation accuracy, but provides limited multiplexing capabilities and can be applied only to actively dividing cells, thus excluding clinical samples. Both limitations are overcome by an evolution of classical SILAC involving the use of a mix of heavy-labeled cell lines as a spike-in standard, known as "super-SILAC". In this chapter, we will provide a detailed description of the optimized protocol used in our laboratory to generate a histone-focused super-SILAC mix and employ it as an internal standard for histone PTM quantitation.

The histone code of the fungal genus Aspergillus uncovered by evolutionary and proteomic analyses.

Chemical modifications of DNA and histone proteins impact the organization of chromatin within the nucleus. Changes in these modifications, catalysed by different chromatin-modifying enzymes, influence chromatin organization, which in turn is thought to impact the spatial and temporal regulation of gene expression. While combinations of different histone modifications, the histone code, have been studied in several model species, we know very little about histone modifications in the fungal genus Aspergillus, whose members are generally well studied due to their importance as models in cell and molecular biology as well as their medical and biotechnological relevance. Here, we used phylogenetic analyses in 94 Aspergilli as well as other fungi to uncover the occurrence and evolutionary trajectories of enzymes and protein complexes with roles in chromatin modifications or regulation. We found that these enzymes and complexes are highly conserved in Aspergilli, pointing towards a complex repertoire of chromatin modifications. Nevertheless, we also observed few recent gene duplications or losses, highlighting Aspergillus species to further study the roles of specific chromatin modifications. SET7 (KMT6) and other components of PRC2 (Polycomb Repressive Complex 2), which is responsible for methylation on histone H3 at lysine 27 in many eukaryotes including fungi, are absent in Aspergilli as well as in closely related Penicillium species, suggesting that these lost the capacity for this histone modification. We corroborated our computational predictions by performing untargeted MS analysis of histone post-translational modifications in Aspergillus nidulans. This systematic analysis will pave the way for future research into the complexity of the histone code and its functional implications on genome architecture and gene regulation in fungi.

The impact of cholesterol lowering drugs on metabolism and epigenetics

Background and Aims : Cardiovascular diseases (CVDs) are the leading cause of death globally. In patients with CVDs, treatment with cholesterol lowering drugs has been shown to reduce cardiovascular events. Bempedoic acid is a novel drug for lowering low-density lipoprotein cholesterol that has recently been approved for clinical use. Bempedoic acid specifically inhibits ATP citrate lyase (ACLY) in the liver, an enzyme that converts citrate to acetyl-CoA and acts at the interface of carbohydrate and lipid metabolism. ACLY is not only cytoplasmic, but is also found in the nucleus, where it regulates histone acetylation levels. Yet, it is unknown if and how bempedoic acid impacts ACLYs regulatory function in the nucleus.Methods: By combining chromatin immunoprecipitation sequencing, transcriptomics, tracer metabolomics and mass-spectrometry based histone post-translational modification analysis in primary human hepatocytes, we systematically investigate the impact of bempedoic acid on ACLY at different regulatory levels.Results: Our data from mouse liver tissue revealed binding of ACLY to promoter regions of metabolic genes. Consistently, gene expression analyses in primary human hepatocytes showed reduced expression of ACLY bound metabolic genes in response to bempedoic acid. Furthermore, histone modification analysis via mass spectrometry and ChIP-seq profiles displayed a highly specific loss of H3K9ac, a promoter-based histone modification known to stimulate gene transcription, upon bempedoic acid treatment. Metabolite profiling revealed reduction in lipid metabolism following bempedoic acid administration.Conclusions: Taken together, these data suggest that bempedoic acid's mechanism of action involves reprogramming of the epigenetic and transcriptional landscape of ACLY associated genes, together with a reduction in lipid production in hepatocytes. Background and Aims : Cardiovascular diseases (CVDs) are the leading cause of death globally. In patients with CVDs, treatment with cholesterol lowering drugs has been shown to reduce cardiovascular events. Bempedoic acid is a novel drug for lowering low-density lipoprotein cholesterol that has recently been approved for clinical use. Bempedoic acid specifically inhibits ATP citrate lyase (ACLY) in the liver, an enzyme that converts citrate to acetyl-CoA and acts at the interface of carbohydrate and lipid metabolism. ACLY is not only cytoplasmic, but is also found in the nucleus, where it regulates histone acetylation levels. Yet, it is unknown if and how bempedoic acid impacts ACLYs regulatory function in the nucleus. Methods: By combining chromatin immunoprecipitation sequencing, transcriptomics, tracer metabolomics and mass-spectrometry based histone post-translational modification analysis in primary human hepatocytes, we systematically investigate the impact of bempedoic acid on ACLY at different regulatory levels. Results: Our data from mouse liver tissue revealed binding of ACLY to promoter regions of metabolic genes. Consistently, gene expression analyses in primary human hepatocytes showed reduced expression of ACLY bound metabolic genes in response to bempedoic acid. Furthermore, histone modification analysis via mass spectrometry and ChIP-seq profiles displayed a highly specific loss of H3K9ac, a promoter-based histone modification known to stimulate gene transcription, upon bempedoic acid treatment. Metabolite profiling revealed reduction in lipid metabolism following bempedoic acid administration. Conclusions: Taken together, these data suggest that bempedoic acid's mechanism of action involves reprogramming of the epigenetic and transcriptional landscape of ACLY associated genes, together with a reduction in lipid production in hepatocytes.

An Efficient Chromatin Immunoprecipitation Protocol for the Analysis of Histone Modification Distributions in the Brown Alga Ectocarpus.

The brown algae are an important but understudied group of multicellular marine organisms. A number of genetic and genomic tools have been developed for the model brown alga Ectocarpus; this includes, most recently, chromatin immunoprecipitation methodology, which allows genome-wide detection and analysis of histone post-translational modifications. Post-translational modifications of histone molecules have been shown to play an important role in gene regulation in organisms from other major eukaryotic lineages, and this methodology will therefore be a very useful tool to investigate genome function in the brown algae. This article provides a detailed, step-by-step description of the Ectocarpus ChIP protocol, which effectively addresses the difficult problem of efficiently extracting chromatin from cells protected by a highly resistant cell wall. The protocol described here will be an essential tool for the future application of chromatin analysis methodologies in brown algal research.

The Current State of Chromatin Immunoprecipitation (ChIP) from FFPE Tissues.

Cancer cells accumulate epigenomic aberrations that contribute to cancer initiation and progression by altering both the genomic stability and the expression of genes. The awareness of such alterations could improve our understanding of cancer dynamics and the identification of new therapeutic strategies and biomarkers to refine tumor classification and treatment. Formalin fixation and paraffin embedding (FFPE) is the gold standard to preserve both tissue integrity and organization, and, in the last decades, a huge number of biological samples have been archived all over the world following this procedure. Recently, new chromatin immunoprecipitation (ChIP) techniques have been developed to allow the analysis of histone post-translational modifications (PTMs) and transcription factor (TF) distribution in FFPE tissues. The application of ChIP to genome-wide chromatin studies using real archival samples represents an unprecedented opportunity to conduct retrospective clinical studies thanks to the possibility of accessing large cohorts of samples and their associated diagnostic records. However, although recent attempts to standardize have been made, fixation and storage conditions of clinical specimens are still extremely variable and can affect the success of chromatin studies. The procedures introduced in the last few years dealt with this problem proponing successful strategies to obtain high-resolution ChIP profiles from FFPE archival samples. In this review, we compare the different FFPE-ChIP techniques, highlighting their strengths, limitations, common features, and peculiarities, as well as pitfalls and caveats related to ChIP studies in FFPE samples, in order to facilitate their application.

Spatial epi-proteomics enabled by histone post-translational modification analysis from low-abundance clinical samples

BackgroundIncreasing evidence linking epigenetic mechanisms and different diseases, including cancer, has prompted in the last 15 years the investigation of histone post-translational modifications (PTMs) in clinical samples. Methods allowing the isolation of histones from patient samples followed by the accurate and comprehensive quantification of their PTMs by mass spectrometry (MS) have been developed. However, the applicability of these methods is limited by the requirement for substantial amounts of material.ResultsTo address this issue, in this study we streamlined the protein extraction procedure from low-amount clinical samples and tested and implemented different in-gel digestion strategies, obtaining a protocol that allows the MS-based analysis of the most common histone PTMs from laser microdissected tissue areas containing as low as 1000 cells, an amount approximately 500 times lower than what is required by available methods. We then applied this protocol to breast cancer patient laser microdissected tissues in two proof-of-concept experiments, identifying differences in histone marks in heterogeneous regions selected by either morphological evaluation or MALDI MS imaging.ConclusionsThese results demonstrate that analyzing histone PTMs from very small tissue areas and detecting differences from adjacent tumor regions is technically feasible. Our method opens the way for spatial epi-proteomics, namely the investigation of epigenetic features in the context of tissue and tumor heterogeneity, which will be instrumental for the identification of novel epigenetic biomarkers and aberrant epigenetic mechanisms.

A Chemical Acetylation-Based Mass Spectrometry Platform for Histone Methylation Profiling

Histones are highly posttranslationally modified proteins that regulate gene expression by modulating chromatin structure and function. Acetylation and methylation are the most abundant histone modifications, with methylation occurring on lysine (mono-, di-, and trimethylation) and arginine (mono- and dimethylation) predominately on histones H3 and H4. In addition, arginine dimethylation can occur either symmetrically (SDMA) or asymmetrically (ADMA) conferring different biological functions. Despite the importance of histone methylation on gene regulation, characterization and quantitation of this modification have proven to be quite challenging. Great advances have been made in the analysis of histone modification using both bottom-up and top-down mass spectrometry (MS). However, MS-based analysis of histone posttranslational modifications (PTMs) is still problematic, due both to the basic nature of the histone N-terminal tails and to the combinatorial complexity of the histone PTMs. In this report, we describe a simplified MS-based platform for histone methylation analysis. The strategy uses chemical acetylation with d0-acetic anhydride to collapse all the differently acetylated histone forms into one form, greatly reducing the complexity of the peptide mixture and improving sensitivity for the detection of methylation via summation of all the differently acetylated forms. We have used this strategy for the robust identification and relative quantitation of H4R3 methylation, for which stoichiometry and symmetry status were determined, providing an antibody-independent evidence that H4R3 is a substrate for both Type I and Type II PRMTs. Additionally, this approach permitted the robust detection of H4K5 monomethylation, a very low stoichiometry methylation event (0.02% methylation). In an independent example, we developed an in vitro assay to profile H3K27 methylation and applied it to an EZH2 mutant xenograft model following small-molecule inhibition of the EZH2 methyltransferase. These specific examples highlight the utility of this simplified MS-based approach to quantify histone methylation profiles.

Histone Purification Combined with High-Resolution Mass Spectrometry to Examine Histone Post-Translational Modifications and Histone Variants in Caenorhabditis elegans.

Histones are the major proteinaceous component of chromatin in eukaryotic cells and an important part of the epigenome, affecting most DNA‐related events, including transcription, DNA replication, and chromosome segregation. The properties of histones are greatly influenced by their post‐translational modifications (PTMs), over 200 of which are known today. Given this large number, researchers need sophisticated methods to study histone PTMs comprehensively. In particular, mass spectrometry (MS)−based approaches have gained popularity, allowing for the quantification of dozens of histone PTMs at once. Using these approaches, even the study of co‐occurring PTMs and the discovery of novel PTMs become feasible. The success of MS‐based approaches relies substantially on obtaining pure and well‐preserved histones for analysis, which can be difficult depending on the source material. Caenorhabditis elegans has been a popular model organism to study the epigenome, but isolation of pure histones from these animals has been challenging. Here, we address this issue, presenting a method for efficient isolation of pure histone proteins from C. elegans at good yield. Further, we describe an MS pipeline optimized for accurate relative quantification of histone PTMs from C. elegans. We alkylate and tryptically digest the histones, analyze them by bottom‐up MS, and then evaluate the resulting data by a C. elegans−adapted version of the software EpiProfile 2.0. Finally, we show the utility of this pipeline by determining differences in histone PTMs between C. elegans strains that age at different rates and thereby achieve very different lifespans. © 2020 The Authors. Basic Protocol 1: Large‐scale growth and harvesting of synchronized C. elegans Basic Protocol 2: Nuclear preparation, histone extraction, and histone purification Basic Protocol 3: Bottom‐up mass spectrometry analysis of histone PTMs and histone variants

Improvements on the quantitative analysis of Trypanosoma cruzi histone post translational modifications: Study of changes in epigenetic marks through the parasite's metacyclogenesis and life cycle

Trypanosome histone N-terminal sequences are very divergent from the other eukaryotes, although they are still decorated by post-translational modifications (PTMs). Here, we used a highly robust workflow to analyze histone PTMs in the parasite Trypanosoma cruzi using mass spectrometry-based (MS-based) data-independent acquisition (DIA). We adapted the workflow for the analysis of the parasite's histone sequences by modifying the software EpiProfile 2.0, improving peptide and PTM quantification accuracy. This workflow could now be applied to the study of 141 T. cruzi modified histone peptides, which we used to investigate the dynamics of histone PTMs along the metacyclogenesis and the life cycle of T. cruzi. Global levels of histone acetylation and methylation fluctuates along metacyclogenesis, however most critical differences were observed between parasite life forms. More than 66 histone PTM changes were detected. Strikingly, the histone PTM pattern of metacyclic trypomastigotes is more similar to epimastigotes than to cellular trypomastigotes. Finally, we highlighted changes at the H4 N-terminus and at H3K76 discussing their impact on the trypanosome biology. Altogether, we have optimized a workflow easily applicable to the analysis of histone PTMs in T. cruzi and generated a dataset that may shed lights on the role of chromatin modifications in this parasite. SignificanceTrypanosomes are unicellular parasites that have divergent histone sequences, no chromosome condensation and a peculiar genome/gene regulation. Genes are transcribed from divergent polycistronic regions and post-transcriptional gene regulation play major role on the establishment of transcripts and protein levels. In this regard, the fact that their histones are decorated with multiple PTMs raises interesting questions about their role. Besides, this digenetic organism must adapt to different environments changing its metabolism accordingly. As metabolism and epigenetics are closely related, the study of histone PTMs in trypanosomes may enlighten this strikingly, and not yet fully understood, interplay. From a biomedical perspective, the comprehensive study of molecular mechanisms associated to the metacyclogenesis process is essential to create better strategies for controlling Chagas disease.

A Novel Data Analysis Pipeline for Bottoms‐Up Mass Spectrometry Proteomic Analysis of Primary Tissues

Modern mass spectrometry‐based proteomic studies require reliable, and robust data analysis for a comprehensive and quantitative profile of histone post‐translational modifications (PTM). Studies involving primary tissue samples face additional challenges in comparative sample analysis, due to various protein concentrations, multiple modification sites and modification states. A novel data analysis pipeline was developed in order to streamline the analysis process and normalize samples of varying concentration. This is relevant in the proteomic analysis of addiction, where various modification isoforms can correlate with different addiction phenotypes. A self‐administration model addiction study of methamphetamine (METH) addiction was used to develop this pipeline.Following nuclear protein isolation, proteins underwent tryptic digestion and histone H3 samples were derivatized with propionic anhydride. Identification, validation, and label‐free quantification of histone modifications was performed. Protein loading of different samples was normalized, using high abundance, proteotypic unmodified peptides. To localize changes in specific site and state, peptide isoforms for each site‐ and state‐specific modification were binned. Using this approach, we were able to identify histone modifications that underwent statistically significant changes in METH self‐administrated rat brain samples. In addition, we were able to identify histone modifications that are indicative of METH controllability in tested individual subjects. This data analysis pipeline has led to robust analysis of histone PTMs. Information on changes in site‐ and state‐specific histone modifications can be integrated with RNA‐seq data to elucidate driving chromatin pathways in the formation and maintenance of pathophysiological epigenetic states.Support or Funding InformationThis project was supported in part by the University of New Hampshire Hamel Center for Undergraduate Research.

Comprehensive histone epigenetics: A mass spectrometry based screening assay to measure epigenetic toxicity

Evidence of the involvement of epigenetics in pathologies such as cancer, diabetes, and neurodegeneration has increased global interest in epigenetic modifications. For nearly thirty years, it has been known that cancer cells exhibit abnormal DNA methylation patterns. In contrast, the large-scale analysis of histone post-translational 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 cells to provide preliminary data, thereby omitting the need to sacrifice laboratory animals. Improving the workflow and the user-friendliness in order to become a high throughput, easily applicable, toxicological screening 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. •There is a growing interest in epigenetic modifications, and more specifically in histone post-translational modifications (hPTMs).•We describe an MS-based workflow that is capable of tracking all possible hPTMs in an untargeted approach that makes use of human cells.•Improving the workflow and the user-friendliness in order to become a high throughput, easily applicable, toxicological screening assay is an ongoing effort.

A Workflow for Ultra-rapid Analysis of Histone Post-translational Modifications with Direct-injection Mass Spectrometry.

Chromatin modifications, like histone post translational modifications (PTMs), are critical for tuning gene expression and many other aspects of cell phenotype. Liquid chromatography coupled to mass spectrometry (LC-MS) has become the most suitable method to analyze histones and histone PTMs in a large-scale manner. Selected histone PTMs have known functions, and their aberrant regulation is linked to a wide variety of diseases, including cancer. However, histone analysis is scarcely used in diagnostics, partially due to the limited throughput and not ideal reproducibility of LC-MS based analysis. We describe a workflow that allows for high-throughput sample preparation is less than a day using 96-well plates. Following preparation, samples are sprayed into MS without LC, using an automated direct injection (DI-MS) method. Each analysis provides accurate quantification for 29 peptide sequences with 45 PTMs (methylations, acetylations and phosphorylations) for a total of 151 histone marks plus 16 unmodified histone peptides for relative quantification of histone variants. This workflow allows for < 1 min MS runs and higher reproducibility and robustness due to the absence of carryover or LC-based batch effects. Finally, we describe an engineered peptide sequence used to accurately monitor the efficiency of sample preparation, which can be detected during the DI-MS run.

A practical guide for analysis of histone post-translational modifications by mass spectrometry: Best practices and pitfalls.

Advances in mass spectrometry (MS) have revolutionized the ability to measure global changes in histone post-translational modifications (PTMs). The method routinely quantifies over 60 modification states in a single sample, far exceeding the capabilities of traditional western blotting. Thus, MS-based histone analysis has become an increasingly popular tool for understanding how genetic and environmental factors influence epigenetic states. However, histone proteomics experiments exhibit unique challenges, such as batch-to-batch reproducibility, accurate peak integration, and noisy data. Here, we discuss the steps of histone PTM analysis, from sample preparation and peak integration to data analysis and validation. We outline a set of best practices for ensuring data quality, accurate normalization, and robust statistics. Using these practices, we quantify histone modifications in 5 human cell lines, revealing that each cell line exhibits a unique epigenetic signature. We also provide a reproducible workflow for histone PTM analysis in the form of an R script, which is freely available at https://github.com/DenuLab/HistoneAnalysisWorkflow.

Outer Membrane Protease OmpT-Based Strategy for Simplified Analysis of Histone Post-Translational Modifications by Mass Spectrometry.

Histone modifications play an important role in regulating transcriptional gene expression and chromatin processes in eukaryotes. Increasing researches proved that aberrant post-translational modifications (PTMs) of histones is associated with many diseases. However, MS-based identification and quantification of histone PTMs are still challenging. Although classic chemical derivatization in conjunction with trypsin digestion is widely used for histone PTMs analysis in a bottom-up strategy, several side reactions have been observed in practice. In this work, outer membrane protease T (OmpT) was utilized as a protease for direct histone proteolysis and generated appropriate lengths of histone peptides for retention on reversed-phase chromatography. The powerful and unique tolerance of OmpT for modified lysines and arginines was demonstrated and can be quantitatively described for the first time, making it useful for detecting natural modifications. Using the optimized digestion conditions, we succeeded in identifying 121 histone marks from HEK293T cells, 42 of which were previously unreported. Additionally, histone H3 PTMs were quantitatively profiled in the KMS11 multiple myeloma cells and NSD2 selective knockout KMS11cells, revealing that NSD2 was of high specificity on H3K36 dimethylation. Histone chemical derivatizations are not required in our strategy, showing a remarkable strength over the conventional trypsin-based workflow.

Quantitation of Single and Combinatorial Histone Modifications by Integrated Chromatography of Bottom-up Peptides and Middle-down Polypeptide Tails.

The analysis of histone post-translational modifications (PTMs) by mass spectrometry (MS) has been critical to the advancement of the field of epigenetics. The most sensitive and accurate workflow is similar to the canonical proteomics analysis workflow (bottom-up MS), where histones are digested into short peptides (4-20 aa) and quantitated in extracted ion chromatograms. However, this limits the ability to detect even very common co-occurrences of modifications on histone proteins, preventing biological interpretation of PTM crosstalk. By digesting with GluC rather than trypsin, it is possible to produce long polypeptides corresponding to intact histone N-terminal tails (50-60 aa), where most modifications reside. This middle-down MS approach is used to study distant PTM co-existence. However, the most sensitive middle-down workflow uses weak cation exchange-hydrophilic interaction chromatography (WCX-HILIC), which is less robust than conventional reversed-phase chromatography. Additionally, since the buffer systems for middle-down and bottom-up proteomics differ substantially, it is cumbersome to toggle back and forth between both experimental setups on the same LC system. Here, we present a new workflow using porous graphitic carbon (PGC) as a stationary phase for histone analysis where bottom-up and middle-down sized histone peptides can be analyzed simultaneously using the same reversed-phase buffer setup. By using this protocol for middle-down sized peptides, we identified 406 uniquely modified intact histone tails and achieved a correlation of 0.85 between PGC and WCX-HILICLC methods. Together, our method facilitates the analysis of single and combinatorial histone PTMs with much simpler applicability for conventional proteomics labs than the state-of-the-art middle-down MS.

HSWATH: Unlocking SWATH's Full Potential for an Untargeted Histone Perspective.

Mass spectrometry (MS) has become the technique of choice for large-scale analysis of histone post-translational modifications (hPTMs) and their combinatorial patterns, especially in untargeted settings where novel discovery-driven hypotheses are being generated. However, MS-based histone analysis requires a distinct sample preparation, acquisition, and data analysis workflow when compared to traditional MS-based approaches. To this end, sequential window acquisition of all theoretical fragment ion spectra (SWATH) has great potential, as it allows for untargeted accurate identification and quantification of hPTMs. Here, we present a complete SWATH workflow specifically adapted for the untargeted study of histones (hSWATH). We assess its validity on a technical dataset of time-lapse deacetylation of a commercial histone extract using HDAC1, which contains a ground truth, i.e., acetylated substrate peptides reduce in intensity. We successfully apply this workflow in a biological setting and subsequently investigate the differential response to HDAC inhibition in different breast cancer cell lines.

The Chromatin Environment Around Interneuron Genes in Oligodendrocyte Precursor Cells and Their Potential for Interneuron Reprograming.

Oligodendrocyte precursor cells (OPCs), also known as NG2 glia, arise from neural progenitor cells in the embryonic ganglionic eminences that also generate inhibitory neurons. They are ubiquitously distributed in the central nervous system, remain proliferative through life, and generate oligodendrocytes in both gray and white matter. OPCs exhibit some lineage plasticity, and attempts have been made to reprogram them into neurons, with varying degrees of success. However, little is known about how epigenetic mechanisms affect the ability of OPCs to undergo fate switch and whether OPCs have a unique chromatin environment around neuronal genes that might contribute to their lineage plasticity. Our bioinformatic analysis of histone posttranslational modifications at interneuron genes in OPCs revealed that OPCs had significantly fewer bivalent and repressive histone marks at interneuron genes compared to astrocytes or fibroblasts. Conversely, OPCs had a greater degree of deposition of active histone modifications at bivalently marked interneuron genes than other cell types, and this was correlated with higher expression levels of these genes in OPCs. Furthermore, a significantly higher proportion of interneuron genes in OPCs than in other cell types lacked the histone posttranslational modifications examined. These genes had a moderately high level of expression, suggesting that the “no mark” interneuron genes could be in a transcriptionally “poised” or “transitional” state. Thus, our findings suggest that OPCs have a unique histone code at their interneuron genes that may obviate the need for erasure of repressive marks during their fate switch to inhibitory neurons.