Nuclear Histone 3 Post-Translational Modification Profiling in Whole Cells using Spectral Flow Cytometry

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.

ADVERTISEMENT RETURN TO ISSUEPREVReviewNEXTQuantitative Proteomic Analysis of Histone ModificationsHe Huang†, Shu Lin‡, Benjamin A. Garcia‡, and Yingming Zhao*†View Author Information† Ben May Department of Cancer Research, The University of Chicago, Chicago, Illinois 60637, United States‡ Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States*Tel.: (773) 834-1561. Fax: (773) 702-3701. E-mail: [email protected]Cite this: Chem. Rev. 2015, 115, 6, 2376–2418Publication Date (Web):February 17, 2015Publication History Received2 September 2014Published online17 February 2015Published inissue 25 March 2015https://pubs.acs.org/doi/10.1021/cr500491uhttps://doi.org/10.1021/cr500491ureview-articleACS PublicationsCopyright © 2015 American Chemical SocietyRequest reuse permissionsArticle Views9003Altmetric-Citations286LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose SUBJECTS:Ions,Mass spectrometry,Monomers,Peptides and proteins,Protein structure Get e-Alerts

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

Unlocking the potential of chemical probes for methyl-lysine reader proteins.

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.

The California Grunion, Leuresthes tenuis, can experience a broad range of salinities during their early life stages, with their nursery grounds measuring salinities of 21 to 42ppt under normal conditions. Due to the unique subterrestrial incubation of their eggs within the sand of California beaches, it is not unlikely for this marine fish to hatch in non-seawater conditions, whereafter the larvae may transition to relatively diluted estuaries or concentrated harbors to develop as juveniles for several months. Consequently, they are an ideal system to study the impacts of ecologically relevant salinity stress on the alteration of proteins and histone post-translational modifications (PTMs) during early development. The study of the relationship between the critical window of development (CWD), in which sexual bipotential is lost, and the longevity of protein landscape and histone PTM changes has not been widely explored. The CWD is of interest as this is when the gametes of this species become set. Therefore, we hypothesize that histone PTM changes that occur during this time may thus be a form of heritable epigenetics. Thus, hypo-and-hyperosmotic exposures of L. tenuis hatchlings during this window, and past it, attempt to identify if the CWD is relevant to the presence and persistence of protein and histone PTM changes under salinity stress. We hypothesize that exposure to osmotic stress during the CWD will result in induced compensatory mechanisms in the protein landscape of the California grunion larvae, as well as persistent alterations in the histone PTMs in the larvae. To elucidate the relationship between these factors, California grunion larvae were exposed in replicate to one of three chronic salinity treatments (16ppt, 32ppt, or 40ppt) for the duration of their CWD (68dph), with 16ppt and 40ppt inducing hypoosmotic and hyperosmotic stress respectively. Chronic post-CWD exposures and recovery times until 98dph were used to determine whether the CWD or prolonged salinity exposures were key to alterations in the histone PTM and protein landscape of the larvae. After each timepoint, L. tenuis were culled and processed for histone PTM enrichment and proteomic analysis. At the time of culling, there were no significant differences in survivorship between replicates and experimental groups, with statistically significant different deaths between groups occurring only shortly after hatching. Skyline and MSstats were used in the analysis of the statistical significance of differential regulation of proteins and histone PTMs under stress conditions versus the controls. Of special interest is the osmoregulatory mechanisms involved in the myo-inositol biosynthesis pathway, with analysis focusing on the abundance of histone acetylation, amidation, ubiquitylation, and 4-hydroxynonelation in histone PTMs, as the accumulations of these have been found to be correlated with osmotic stress in some fish tissues. Funding provided by NSF IOS-2209383 and NSF GRFP. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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

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.

Chapter 15 - Useful methods to study epigenetic marks: DNA methylation, histone modifications, chromatin structure, and noncoding RNAs

The pannexins (Panx1, -2, and -3) are a mammalian family of putative single membrane channels discovered through homology to invertebrate gap junction-forming proteins, the innexins. Because connexin gap junction proteins are known regulators of neural stem and progenitor cell proliferation, migration, and specification, we asked whether pannexins, specifically Panx2, play a similar role in the postnatal hippocampus. We show that Panx2 protein is differentially expressed by multipotential progenitor cells and mature neurons. Both in vivo and in vitro, Type I and IIa stem-like neural progenitor cells express an S-palmitoylated Panx2 species localizing to Golgi and endoplasmic reticulum membranes. Protein expression is down-regulated during neurogenesis in neuronally committed Type IIb and III progenitor cells and immature neurons. Panx2 is re-expressed by neurons following maturation. Protein expressed by mature neurons is not palmitoylated and localizes to the plasma membrane. To assess the impact of Panx2 on neuronal differentiation, we used short hairpin RNA to suppress Panx2 expression in Neuro2a cells. Knockdown significantly accelerated the rate of neuronal differentiation. Neuritic extension and the expression of antigenic markers of mature neurons occurred earlier in stable lines expressing Panx2 short hairpin RNA than in controls. Together, these findings describe an endogenous post-translational regulation of Panx2, specific to early neural progenitor cells, and demonstrate that this expression plays a role in modulating the timing of their commitment to a neuronal lineage.

Based on the analysis of literary sources of PubMed, MedLine, The Cochrane Library, EMBASE database, the authors of the article give general provisions regarding post-translational modifications of histones (small proteins with a molecular weight of 10–15 kDa, which make up the largest part of nuclear proteins), which are associated with the development of metabolic dysfunction-associated fatty liver disease. The authors emphasize that post-translational histone modifications regulate the activity of gene expression, and each of these types differently changes the structure of chromatin and, as a result, gene expression. Currently, more than 20 types of histone protein modifications have been identified (acetylation, biotinylation, butyrylation, 2-hydroxybutyrylation, ADP-ribosylation, N-formylation, hydroxylation, glycosylation, glutarylation, dopaminylation, proline isomerization and aspartic acid carbonylation, crotonylation, lactylation, malonylation, methylation, propionylation, succinylation, SUMOylation, ubiquitination, phosphorylation, citrullination). Epigenetic and epitranscriptomic changes are induced by lifestyle, especially the nature of diet and physical activity, by the influence of exogenous and endogenous factors. Prolonged epigenetic changes that determine the expression of target genes can be accompanied by the development of metabolic disorders and the progression of metabolic dysfunction-associated fatty liver disease. Histone modification is carried out by site-specific enzymes: writers, which identify a marker, and erasers, which “erase” a marker. Post-translational histone modifications change the local physicochemical environment and, based on this, directly affect the structure of the nucleosome and chromatin. Also, post-translational modifications of the N- and C-terminal tails of histone proteins act as “docking sites” that recruit specific molecular readers. Readers of histone modifications can act both in the intranucleosomal space, modifying adjacent histone sites or recruiting transcription factors, transcription activators and repressors, and in the internucleosomal space. The authors also describe the pathophysiological significance of post-translational histone modifications in the development of metabolic dysfunction-associated fatty liver disease, the diagnostic value of epigenetic biomarkers, and the potential of pharmacological management of histone modifications to achieve inhibition of the activity of the pathological process.

Gene expression is regulated by chromatin DNA methylation and other features, including histone post-translational modifications (PTMs), chromatin remodelers and transcription factor occupancy. A complete understanding of gene regulation will require the mapping of these chromatin features in small cell number samples. Here we describe a novel genome-wide chromatin profiling technology, named as Nicking Enzyme Epitope targeted DNA sequencing (NEED-seq). NEED-seq offers antibody-targeted controlled nicking by Nt.CviPII-pGL fusion to study specific protein-DNA complexes in formaldehyde fixed cells, allowing for both visual and genomic resolution of epitope bound chromatin. When applied to nuclei, NEED-seq yielded genome-wide profile of chromatin-associated proteins and histone PTMs. Additionally, NEED-seq of lamin B1 and B2 demonstrated their association with heterochromatin. Lamin B1- and B2-associated domains (LAD) segregated to three different states, and states with stronger LAD correlated with heterochromatic marks. Hi-C analysis displayed A and B compartment with equal lamin B1 and B2 distribution, although methylated DNA remained high in B compartment. LAD clustering with Hi-C resulted in subcompartments, with lamin B1 and B2 partitioning to facultative and constitutive heterochromatin, respectively, and were associated with neuronal development. Thus, lamin B1 and B2 show structural and functional partitioning in mammalian nucleus.

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.

BackgroundHistone Post-Translational Modifications (PTMs) are important epigenetic marks, whose specific pattern over the chromatin plays a critical role in turning the corresponding gene on/off. During DNA replication in mitotic cells, the histone PTMs are dislodged from the mother chromatid, ahead of the replication fork, and distributed uniformly at random among the daughter chromatids. We show that maintaining the fidelity of a primary PTM pattern, from the partial information available after replication, can be significantly improved by the presence of an additional antagonistic PTM sequence in the mother.Results Building on our previous study which proposed mechanisms for maintaining fidelity by utilizing only half the parental nucleosomes, the current work considers the effect of an additional antagonistic PTM sequence. We represent the joint PTM sequence by an appropriate Markov model and the DNA replication fork as a noisy communication channel. An optimal Bayesian sequence estimator is then employed at each of the daughter chromatids to reconstruct the primary PTM pattern. A high-fidelity reconstruction, potentially aided by the enzyme machinery, is shown to be possible in the presence of epigenetic memory. The structural properties derived for the optimal estimator are then verified through simulations, which show the improvement in fidelity of inheritance with antagonism. This is further supported empirically through observations from some recent experimental dataConclusionsOur work provides a computational model to quantify the effect of combinatorial histone PTMs in epigenetic inheritance. The fidelity of reconstruction of the primary histone PTM post-replication, is shown to be enhanced in the presence of antagonistic PTMs in the vicinity.

This study sheds new light on the timescale through which histone post-translational modifications (PTMs) respond to environmental stimuli, demonstrating that the histone PTM response does not necessarily precede the proteomic response or acclimation. After a variety of salinity treatments were administered to Mozambique tilapia (Oreochromis mossambicus) throughout their lifetimes, we quantified 343 histone PTMs in the gills of each fish. We show here that histone PTMs differ dramatically between fish exposed to distinct environmental conditions for 18 months, and that the majority of these histone PTM alterations persist for at least 4 weeks, irrespective of further salinity changes. However, histone PTMs respond minimally to 4-week-long periods of salinity acclimation during adulthood. The results of this study altogether signify that patterns of histone PTMs in individuals reflect their prolonged exposure to environmental conditions.