Quantitative proteomic analysis of histone modifications.

Post-translational modifications (PTMs) of histones play an important role in many cellular processes, notably gene regulation. Using a combination of mass spectrometric and immunobiochemical approaches, we show that the PTM profile of histone H3 differs significantly among the various model organisms examined. Unicellular eukaryotes, such as Saccharomyces cerevisiae (yeast) and Tetrahymena thermophila (Tet), for example, contain more activation than silencing marks as compared with mammalian cells (mouse and human), which are generally enriched in PTMs more often associated with gene silencing. Close examination reveals that many of the better-known modified lysines (Lys) can be either methylated or acetylated and that the overall modification patterns become more complex from unicellular eukaryotes to mammals. Additionally, novel species-specific H3 PTMs from wild-type asynchronously grown cells are also detected by mass spectrometry. Our results suggest that some PTMs are more conserved than previously thought, including H3K9me1 and H4K20me2 in yeast and H3K27me1, -me2, and -me3 in Tet. On histone H4, methylation at Lys-20 showed a similar pattern as H3 methylation at Lys-9, with mammals containing more methylation than the unicellular organisms. Additionally, modification profiles of H4 acetylation were very similar among the organisms examined.

Histones variant composition and post-translational modifications (PTMs) of core and linker histones jointly orchestrate chromatin architecture and tissue-specific gene regulation. However, capturing the full complexity of the "histone code" in native human tissues remains challenging. Here we present a human cardiac histone proteoform atlas-comprising intact histone variants with combinatorial PTMs-enabled by a streamlined top-down proteomics workflow. In a single one-dimensional reversed-phase liquid chromatography (LC)-mass spectrometry (MS) run, we achieve baseline separation of all four core histone families (H2A, H2B, H3, and H4) together with the linker histone H1 directly from human myocardium. Notably, this intact-protein analysis preserves the connectivity of co-occurring PTMs to individual histone molecules to reveal the combinatorial histone code, enabling proteoform-level quantification of histone-variant composition and PTM stoichiometry at the chromatographic scale. Targeted MS/MS directly resolves co-occurring PTM combinations and distinguishes highly homologous histone isoforms. Using this approach, we assemble the first comprehensive cardiac histone proteoform atlas and uncover previously unobserved histone proteoforms. More broadly, this top-down proteomics workflow provides a robust framework for proteoform-resolved analysis of histone variants and PTMs in complex biological systems.

Chapter One - Examining Histone Posttranslational Modification Patterns by High-Resolution Mass Spectrometry

Nuclear events such as chromatin condensation, DNA cleavage at internucleosomal sites, and histone release from chromatin are recognized as hallmarks of apoptosis. However, there is no complete understanding of the molecular events underlying these changes. It is likely that epigenetic changes such as DNA methylation and histone modifications that are involved in chromatin dynamics and structure are also involved in the nuclear events described. In this report we have shown that apoptosis is associated with global DNA hypomethylation and histone deacetylation events in leukemia cells. Most importantly, we have observed a particular epigenetic signature for early apoptosis defined by a release of hypoacetylated and trimethylated histone H4 and internucleosomal fragmented DNA that is hypermethylated and originates from perinuclear heterochromatin. These findings provide one of the first links between apoptotic nuclear events and epigenetic markers.

Chromatin modifications, epigenetics, and how protozoan parasites regulate their lives

Nothing Is Yet Set in (Hi)stone: Novel Post-Translational Modifications Regulating Chromatin Function

Post-translational modifications of histone and non-histone proteins in epigenetic regulation and translational applications in alcohol-associated liver disease: Challenges and research opportunities

Background: Epigenetic alterations can impact gene expression and have been implicated in tumorigenesis. One major process is the post-translational modification (PTM) of histone proteins governed by histone-modifying enzyme (HME) activity. There is growing evidence that the dysregulation of HMEs plays an essential role in cancer initiation and progression. In this study, we hypothesized that alterations in histone PTMs and their associated enzymes drive the development of castration-resistant prostate cancer (CRPC) from hormone-sensitive prostate cancer (HSPC). To address this, we performed an unbiased screen of histone PTMs in a series of prostate cancer (PC) PDXs before and after the transition to CRPC. Methods: Histone PTM levels were analyzed in 10 paired HSPC and CRPC PDXs using mass spectrometry. Histone PTM patterns were subcategorized based on common genetic alterations found in PC. RNA-sequencing data for the PDXs and publicly available human PC datasets were used to further characterize HMEs. Co-expression of CHD1, NSD2, and other HMEs was examined in human PC tissue microarrays (TMAs). CHD1 KO clones were generated using CRISPR and levels of histone PTMs and HMEs were assessed and quantified. Gene set enrichment analysis (GSEA) was performed to determine the pathways altered with CHD1 in CRPC PDXs. Results: A comparison of all PDX tumors revealed increased H3K14 and H4 acetylation in CRPC tumors relative to paired HSPC derivatives. We also observed distinct histone PTM patterns associated with common genetic alterations. When compared to tumors with CHD1 WT, tumors with CHD1 loss develop reduced histone PTM H3.3K36me2. CHD1 is a chromatin remodeler commonly deleted in ~ 15% of primary PCs. Screening the differential expression and activity of HMEs in these PDXs reveals that HME NSD2 correlates with CHD1. The human CRPC SU2C/PCF dataset shows a strong positive correlation between CHD1 and NSD2 mRNA (r=0.44). Human CRPC TMAs show decreased protein expression of NSD2 and its histone mark H3K36me2 is associated with reduced CHD1 levels. CHD1 KO in DU145 results in a loss of H3.3K36me2 and a 0.5-fold decrease in NSD2 expression. GSEA revealed that a reduction in the DNA repair pathway is associated with CHD1 loss. The CRPC PDXs (n=24) and human dataset SU2C/PCF 2019 showed a strong positive correlation between CHD1, NSD2, and DNA damage repair genes. Conclusions: We have identified for the first time that genetic alterations alter histone marks during the transition to castration-resistant cancer. Reduced histone H3.3K36 methylation is associated with the genetic loss of CHD1 during the transition to CRPC. The expression of NSD2 is altered in the CRPC state in a CHD1 status-dependent manner. The CHD1-NSD2 phenotype may be the target of novel therapeutic drug combinations – the subject of ongoing research. Citation Format: Tanaya A. Purohit, Joseph Gawdzik, Bing Yang, Eric A. Armstrong, Peter W. Lewis, John M. Denu, David F. Jarrard. Genetic alterations induce distinct histone post-translational modifications during the transition to castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A060.

Histones are the fundamental structural proteins intimately associated with eukaryotic DNA to form a highly ordered and condensed nucleoproteic complex termed chromatin. They are the targets of various posttranslational modifications including acetylation, methylation, phosphorylation and ubiquitination that modulate the structure/function of chromatin. The combinatorial nature of histone modifications is hypothesized to define a "histone code" that considerably extends the information potential of the genetic code, giving rise to epigenetic information. Moreover, most core histones consist of several nonallelic variants that can mark specific loci and could play an important role in establishment and maintenance of epigenetic memory. Here we will briefly present our current knowledge about histone posttranslational modifications and their implications in the regulation of epigenetic information. We will next describe core histone variants, insisting on their mode of incorporation into chromatin to discuss their epigenetic function and inheritance.

e16030 Background: Heat shock protein 90 (HSP90) is a chaperone for many proteins integral development of cancer, making inhibition of HSP90 an attractive strategy for cancer treatment. However, little is known about HSP90 inhibition-mediated bladder cancer therapy. Here, we present a quantitative proteomic study that evaluates alterations in protein expression and histone post-translational modifications (PTMs) in bladder cancer in response to HSP90 inhibition. Methods: We assessed the antitumor activity of five HSP90 inhibitors in 5637 bladder cancer cell line using MTS, cell viability, and caspase 3/7 assays. We then performed a proteomic analysis of cells before and after treatment with the HSP90 inhibitors and assessed for changes. Results: We show that the five HSP90 inhibitors (AUY922, ganetespib, SNX2112, AT13387, and CUDC305) potently inhibited the proliferation of bladder cancer 5637 cells. HSP90 inhibitors also had differential effects on cell survival between bladder cancer cells and uroepithelial cells. Our proteomic study quantified 518 twofold up-regulated and 811 twofold downregulated proteins common to both AUY922 and ganetespib treatment (P < 0.05). The subsequent bioinformatic analysis revealed that those differentially expressed proteins were involved in chromatin modifications and cell death-associated pathways. Further, quantitative proteome studies identified 14 different types of PTMs with 93 marks on the core histones, including 34 novel histone marks of butyrylation, citrullination, 2-hydroxyisobutyrylation, propionylation, and succinylation in AUY922- and ganetespib-treated 5637 cells, suggesting that HSP90 inhibitors may exert multiple cytotoxic actions in bladder cancer cells by inhibiting HSP90 activity or altering the structure of chromatin. Conclusions: This study outlines the association between proteomic changes and histone PTMs in response to HSP90 inhibitor treatment in bladder cancer cells, and supports the notion that HSP90 inhibitors both as single agents and in combination might provide new therapeutic options for bladder cancer treatment and thus warrant further preclinical exploration.

While acting upon chromatin compaction, histone post-translational modifications (PTMs) are involved in modulating gene expression through histone-DNA affinity and protein-protein interactions. These dynamic and environment-sensitive modifications are constitutive of the histone code that reflects the transient transcriptional state of the chromatin. Here we describe a global screening approach for revealing epigenetic disruption at the histone level. This original approach enables fast and reliable relative abundance comparison of histone PTMs and variants in human cells within a single LC-MS experiment. As a proof of concept, we exposed BeWo human choriocarcinoma cells to sodium butyrate (SB), a universal histone deacetylase (HDAC) inhibitor. Histone acid-extracts (n = 45) equally representing 3 distinct classes, Control, 1 mM and 2.5 mM SB, were analysed using ultra-performance liquid chromatography coupled with a hybrid quadrupole time-of-flight mass spectrometer (UPLC-QTOF-MS). Multivariate statistics allowed us to discriminate control from treated samples based on differences in their mass spectral profiles. Several acetylated and methylated forms of core histones emerged as markers of sodium butyrate treatment. Indeed, this untargeted histonomic approach could be a useful exploratory tool in many cases of xenobiotic exposure when histone code disruption is suspected.

Histone posttranslational modifications (PTMs) regulate chromatin dynamics, DNA accessibility, and transcription to expand the genetic code. Many of these PTMs are produced through cellular metabolism to offer both feedback and feedforward regulation. Herein we describe the existence of Lys and Arg modifications on histones by a glycolytic by-product, methylglyoxal (MGO). Our data demonstrate that adduction of histones by MGO is an abundant modification, present at the same order of magnitude as Arg methylation. These modifications were detected on all four core histones at critical residues involved in both nucleosome stability and reader domain binding. In addition, MGO treatment of cells lacking the major detoxifying enzyme, glyoxalase 1, results in marked disruption of H2B acetylation and ubiquitylation without affecting H2A, H3, and H4 modifications. Using RNA sequencing, we show that MGO is capable of altering gene transcription, most notably in cells lacking GLO1. Finally, we show that the deglycase DJ-1 protects histones from adduction by MGO. Collectively, our findings demonstrate the existence of a previously undetected histone modification derived from glycolysis, which may have far-reaching implications for the control of gene expression and protein transcription linked to metabolism.

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.

Besides chemical modification of histone proteins, chromatin dynamics can be modulated by histone variants. Most organisms possess multiple genes encoding for core histone proteins, which are highly similar in amino acid sequence. The Arabidopsis thaliana genome contains 11 genes encoding for histone H2B (HTBs), 13 for H2A (HTAs), 15 for H3 (HTRs), and 8 genes encoding for histone H4 (HFOs). The finding that histone variants may be expressed in specific tissues and/or during specific developmental stages, often displaying specific nuclear localization and involvement in specific nuclear processes suggests that histone variants have evolved to carry out specific functions in regulating chromatin structure and function and might be important for better understanding of growth and development and particularly the response to stress. In this review, we will elaborate on a group of core histone proteins in Arabidopsis, namely histone H2B, summarize existing data, and illuminate the potential function of H2B variants in regulating chromatin structure and function in Arabidopsis thaliana.

Histone acetylation has been shown to be required for the proper regulation of many cellular processes including transcription, DNA repair, and chromatin assembly. Acetylation of histone H3 on lysine 56 (H3K56) occurs both during the premeiotic and mitotic S phase and persists throughout DNA damage repair. To learn more about the molecular mechanism of H3K56 acetylation and factors required for this process, we surveyed the genome of the yeast Saccharomyces cerevisiae to identify genes necessary for this process. A comparative global proteomic screen identified several factors required for global H3K56 acetylation, which included histone chaperone Asf1 and a protein of an unknown function Rtt109 but not Spt10. Our results indicate that the loss of Rtt109 results in the loss of H3K56 acetylation, both on bulk histone and on chromatin, similar to that of asf1Delta or the K56Q mutation. RTT109 deletion exhibits sensitivity to DNA damaging agents similar to that of asf1Delta and H3K56Q mutants. Furthermore, Rtt109 and H3K56 acetylation appear to correlate with actively transcribed genes and associate with the elongating form of polymerase II in yeast. This histone modification is also associated with some of the transcriptionally active puff sites in Drosophila. Our results indicate a new role for the Rtt109 protein in the proper regulation of H3K56 acetylation.