Combinatorial Histone Research Articles

Mass Spectrometry-based Profiling of Single-cell Histone Post-translational Modifications to Dissect Chromatin Heterogeneity.

Single-cell proteomics confidently quantifies cellular heterogeneity, yet precise quantification of post-translational modifications, such as those deposited on histone proteins, has remained elusive. Here, we developed a robust mass spectrometry-based method for the unbiased analysis of single-cell histone post-translational modifications (schPTM). schPTM identifies both single and combinatorial histone post-translational modifications (68 peptidoforms in total), which includes nearly all frequently studied histone post-translational modifications with comparable reproducibility to traditional bulk experiments. As a proof of concept, we treated cells with sodium butyrate, a histone deacetylase inhibitor, and demonstrated that our method can i) distinguish between treated and non-treated cells, ii) identify sub-populations of cells with heterogeneous response to the treatment, and iii) reveal differential co-regulation of histone post-translational modifications in the context of drug treatment. The schPTM method enables comprehensive investigation of chromatin heterogeneity at single-cell resolution and provides further understanding of the histone code.

Impact of Combinatorial Histone Modifications on Acetyllysine Recognition by the ATAD2 and ATAD2B Bromodomains.

The ATPase family AAA+ domain containing 2 (ATAD2) protein and its paralog ATAD2B have a C-terminal bromodomain (BRD) that functions as a reader of acetylated lysine residues on histone proteins. Using a structure-function approach, we investigated the ability of the ATAD2/B BRDs to select acetylated lysine among multiple histone post-translational modifications. The ATAD2B BRD can bind acetylated histone ligands that also contain adjacent methylation or phosphorylation marks, while the presence of these modifications significantly weakened the acetyllysine binding activity of the ATAD2 BRD. Our structural studies provide mechanistic insights into how ATAD2/B BRD-binding pocket residues coordinate the acetyllysine group in the context of adjacent post-translational modifications. Furthermore, we investigated how sequence changes in amino acids of the histone ligands impact the recognition of an adjacent acetyllysine residue. Our study highlights how the interplay between multiple combinations of histone modifications influences the reader activity of the ATAD2/B BRDs, resulting in distinct binding modes.

Identification of epigenetic modulators as determinants of nuclear size and shape.

The shape and size of the human cell nucleus is highly variable among cell types and tissues. Changes in nuclear morphology are associated with disease, including cancer, as well as with premature and normal aging. Despite the very fundamental nature of nuclear morphology, the cellular factors that determine nuclear shape and size are not well understood. To identify regulators of nuclear architecture in a systematic and unbiased fashion, we performed a high-throughput imaging-based siRNA screen targeting 867 nuclear proteins including chromatin-associated proteins, epigenetic regulators, and nuclear envelope components. Using multiple morphometric parameters, and eliminating cell cycle effectors, we identified a set of novel determinants of nuclear size and shape. Interestingly, most identified factors altered nuclear morphology without affecting the levels of lamin proteins, which are known prominent regulators of nuclear shape. In contrast, a major group of nuclear shape regulators were modifiers of repressive heterochromatin. Biochemical and molecular analysis uncovered a direct physical interaction of histone H3 with lamin A mediated via combinatorial histone modifications. Furthermore, disease-causing lamin A mutations that result in disruption of nuclear shape inhibited lamin A-histone H3 interactions. Oncogenic histone H3.3 mutants defective for H3K27 methylation resulted in nuclear morphology abnormalities. Altogether, our results represent a systematic exploration of cellular factors involved in determining nuclear morphology and they identify the interaction of lamin A with histone H3 as an important contributor to nuclear morphology in human cells.

Novel insights into the recognition of acetylated histone H4 tail by the TRIM24 PHD-Bromo module.

TRIM24 is a multi-functional chromatin reader, and it binds to the estrogen receptor to activate estrogen-dependent target genes associated with tumor development. TRIM24 is known to ubiquitinate p53 via an N-terminal RING domain and binds a specific combinatorial histone signature of H3K4me0/H3K23ac via its C-terminal plant homeodomain (PHD) and bromodomain (Bromo). Aberrant expression of TRIM24 positively correlates with H3K23ac levels, and high levels of both TRIM24 and H3K23ac predict poor survival of breast cancer patients. Little has been explored about the acetylated histone H4 (H4ac) signatures of TRIM24 and their biological functions. Herein, we report novel H4ac binding partners of TRIM24 and their localization in the genome. Isothermal titration calorimetry binding assay on the histone peptides revealed that the TRIM24 PHD-Bromo preferably binds to H4K5ac, H4K8ac, and H4K5acK8ac compared with other acetylated histone H4 ligands. Co-immunoprecipitation on the endogenous histones suggests that the recognition of H4ac by Bromo does not interfere with the recognition of H3K4me0 mark by the PHD domain of TRIM24. Consistent with this, TRIM24 PHD-Bromo exhibits minimal discrimination among H4ac binding partners at endogenous histone and nucleosome levels. Moreover, ChIP-seq analysis revealed that the H4K5ac and H4K8ac histone signatures strongly co-localize near the transcription start sites of different hub genes or TRIM24-targeted genes in breast cancer. In addition, the KEGG pathway analysis demonstrates that the TRIM24 and its H4ac targets are associated with several important biological pathways. Our findings describe that the H4ac recognition by TRIM24 PHD-Bromo enables access to the chromatin for specific transcriptional regulation.

CHROMATIN STATE AND MODIFICATIONS IN AGING

Abstract Program Overview: In this symposium, the speakers will discuss how genomic stability and chromatin state are affected during aging using complementary perspectives, model systems and technology. Methods such as structural genomics, genome-wide mapping, super-resolution microscopy, mass spectrometry, and enzymology are utilized to describe hallmarks of aging in models such as C. elegans, mice, and 2D vs 3D human cell culture. Dr. Lakadamyali applies innovative super-resolution microscopy to visualize and quantify the spatial organization of chromatin with nanoscale spatial resolution in single cells, revealing how disordered patches at the nucleosomal level correlate with cell phenotype. Dr. Sen will present a multi-omics approach on murine models to reveal that aged cells organize a specific type of heterochromatin unable to self-interact over long distances. These findings expose that global chromatin alterations pose a barrier and promote a cellular state that is refractory to injury sensing and repair. Finally, Dr. Sidoli will describe how mass spectrometry is applied to investigate combinatorial histone modifications that benchmark regions of decondensed heterochromatin, and show the enrichment of unusual histone modifications in a cohort of centenarian patients.

Abstract PR002: Insights into the histone code recognition by the ATAD2B bromodomain

Abstract The ATPase family, AAA domain-containing protein 2 (ATAD2/ANCCA), is an AAA nuclear co-regulator protein containing two ATPase domains and a bromodomain. Its closely related paralog, ATAD2B (KIAA1240), also contains two ATPase domains and a bromodomain. The AAA ATPase domains are broadly associated with ATP-driven molecular remodeling reactions while the bromodomains are evolutionarily conserved, chromatin ‘reader’ domains known to regulate gene expression by binding to acetyllysine on histones. Extensive studies on ATAD2 discuss its upregulation and correlation with poor prognosis in various cancers. Despite sharing high structural and sequence similarity with ATAD2, ATAD2B remains a poorly understood paralog. While previous immunostaining studies detected high expression levels of ATAD2B in breast carcinoma, there is minimal information on its role in oncogenesis. Previously, we have shown that the ATAD2B BRD can recognize mono- and di-acetyllysine modifications on N-terminal tails of histones H4 and H2A. However, since initiation and progression of cancer are characterized by multivalent histone marks, our goal is to understand how crosstalk between combinatorial histone modifications affects the acetyllysine recognition activity of the ATAD2B BRD. We hypothesize that the bromodomain recognizes the acetyllysine mark on multivalent histones and interacts with the chromatin and the ATPase domain. We expect that our results will explain the function of the ATAD2B bromodomain in the context of the overall role of ATAD2B in normal biological and cellular processes. Since bromodomains are excellent drug targets, blocking their activity will also suggest how to disrupt their possible role in oncogenesis. In this study, we have combined various innovative and interdisciplinary approaches ranging from biochemistry and structural biology to functional genomics and cellular biology to gain insights into how crosstalk between different histone H4 PTMs can modulate the chromatin ‘reader’ activity of the ATAD2B BRD. Binding affinities obtained from ITC indicate that the ATAD2B BRD can recognize the acetyllysine mark amongst multiple histone H4 PTMs. Most of these adjacent PTMs are permissible and show high affinities, while some combinations weaken the acetyllysine interaction. Our novel X-ray structures demonstrate a unique binding mode of ATAD2B BRD compared to ATAD2. While the difference in ligand selectivity could suggest that ATAD2B may regulate different biological functions, our cellular assays, including western blot and functional genomic studies, indicate that ATAD2B is conserved in its ability to bind the chromatin. Finally, we discuss how known ‘onco’ mutations within the histone H4 tail region, occurring at or around sites of PTMs, can affect the ATAD2B BRD - acetyllysine coordination. Overall, our study highlights how crosstalk between various histone PTMs regulates the ‘reader’ domain of ATAD2B and affects its recruitment to the chromatin, thus providing new insights for developing therapeutic interventions for treating various diseases. Citation Format: Margaret Phillips, Kathleen Quinn, Cameron Montgomery, Samuel P. Boyson, Sunsik Chang, Jay C. Nix, Seth E. Frietze, Karen C. Glass. Insights into the histone code recognition by the ATAD2B bromodomain. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr PR002.

Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue.

Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid tissues due to unnaturally rapid cell replication. This is particularly challenging when modeling mature chromatin, as fast replicating cells are frequently involved in DNA replication and have a heterogeneous polyploid population. Presented below is a workflow for modeling, treating, and analyzing quiescent chromatin modifications using a three-dimensional (3D) cell culture system. Using this protocol, hepatocellular carcinoma cell lines are grown as reproducible 3D spheroids in an incubator providing active nutrient diffusion and low shearing forces. Treatment with sodium butyrate and sodium succinate induced an increase in histone acetylation and succinylation, respectively. Increases in levels of histone acetylation and succinylation are associated with a more open chromatin state. Spheroids are then collected for isolation of cell nuclei, from which histone proteins are extracted for the analysis of their post-translational modifications. Histone analysis is performed via liquid chromatography coupled online with tandem mass spectrometry, followed by an in-house computational pipeline. Finally, examples of data representation to investigate the frequency and occurrence of combinatorial histone marks are shown.

Chromatin profiling in human neurons reveals aberrant roles for histone acetylation and BET family proteins in schizophrenia

Schizophrenia (SZ) is a psychiatric disorder with complex genetic risk dictated by interactions between hundreds of risk variants. Epigenetic factors, such as histone posttranslational modifications (PTMs), have been shown to play critical roles in many neurodevelopmental processes, and when perturbed may also contribute to the precipitation of disease. Here, we apply an unbiased proteomics approach to evaluate combinatorial histone PTMs in human induced pluripotent stem cell (hiPSC)-derived forebrain neurons from individuals with SZ. We observe hyperacetylation of H2A.Z and H4 in neurons derived from SZ cases, results that were confirmed in postmortem human brain. We demonstrate that the bromodomain and extraterminal (BET) protein, BRD4, is a bona fide ‘reader’ of H2A.Z acetylation, and further provide evidence that BET family protein inhibition ameliorates transcriptional abnormalities in patient-derived neurons. Thus, treatments aimed at alleviating BET protein interactions with hyperacetylated histones may aid in the prevention or treatment of SZ.

Characterizing cellular heterogeneity in chromatin state with scCUT&Tag-pro.

Technologies that profile chromatin modifications at single-cell resolution offer enormous promise for functional genomic characterization, but the sparsity of the measurements and integrating multiple binding maps represent substantial challenges. Here we introduce scCUT&Tag-pro, a multimodal assay for profiling protein-DNA interactions coupled with the abundance of surface proteins in single cells. In addition, we introduce scChromHMM, which integrates data from multiple experiments to infer and annotate chromatin states based on combinatorial histone modification patterns. We apply these tools to perform an integrated analysis across nine different molecular modalities in circulating human immune cells. We demonstrate how these two approaches can characterize dynamic changes in the function of individual genomic elements across both discrete cell states and continuous developmental trajectories, nominate associated motifs and regulators that establish chromatin states, and identify extensive and cell type-specific regulatory priming. Finally, we demonstrate how our integrated reference can serve as a scaffold to map and improve the interpretation of additional scCUT&Tag datasets.

An explainable artificial intelligence approach for decoding the enhancer histone modifications code and identification of novel enhancers in Drosophila

BackgroundEnhancers are non-coding regions of the genome that control the activity of target genes. Recent efforts to identify active enhancers experimentally and in silico have proven effective. While these tools can predict the locations of enhancers with a high degree of accuracy, the mechanisms underpinning the activity of enhancers are often unclear.ResultsUsing machine learning (ML) and a rule-based explainable artificial intelligence (XAI) model, we demonstrate that we can predict the location of known enhancers in Drosophila with a high degree of accuracy. Most importantly, we use the rules of the XAI model to provide insight into the underlying combinatorial histone modifications code of enhancers. In addition, we identified a large set of putative enhancers that display the same epigenetic signature as enhancers identified experimentally. These putative enhancers are enriched in nascent transcription, divergent transcription and have 3D contacts with promoters of transcribed genes. However, they display only intermediary enrichment of mediator and cohesin complexes compared to previously characterised active enhancers. We also found that 10–15% of the predicted enhancers display similar characteristics to super enhancers observed in other species.ConclusionsHere, we applied an explainable AI model to predict enhancers with high accuracy. Most importantly, we identified that different combinations of epigenetic marks characterise different groups of enhancers. Finally, we discovered a large set of putative enhancers which display similar characteristics with previously characterised active enhancers.

Chemical biology approaches to study histone interactors.

Protein-protein interactions (PPIs) in the nucleus play key roles in transcriptional regulation and ensure genomic stability. Critical to this are histone-mediated PPI networks, which are further fine-tuned through dynamic post-translational modification. Perturbation to these networks leads to genomic instability and disease, presenting epigenetic proteins as key therapeutic targets. This mini-review will describe progress in mapping the combinatorial histone PTM landscape, and recent chemical biology approaches to map histone interactors. Recent advances in mapping direct interactors of histone PTMs as well as local chromatin interactomes will be highlighted, with a focus on mass-spectrometry based workflows that continue to illuminate histone-mediated PPIs in unprecedented detail.

NucHMM: a method for quantitative modeling of nucleosome organization identifying functional nucleosome states distinctly associated with splicing potentiality

We develop a novel computational method, NucHMM, to identify functional nucleosome states associated with cell type-specific combinatorial histone marks and nucleosome organization features such as phasing, spacing and positioning. We test it on publicly available MNase-seq and ChIP-seq data in MCF7, H1, and IMR90 cells and identify 11 distinct functional nucleosome states. We demonstrate these nucleosome states are distinctly associated with the splicing potentiality of skipping exons. This advances our understanding of the chromatin function at the nucleosome level and offers insights into the interplay between nucleosome organization and splicing processes.

Combinatorial Histone H3 Modifications Are Dynamically Altered in Distinct Cell Cycle Phases.

The cell cycle is a highly regulated and evolutionary conserved process that results in the duplication of cell content and the equal distribution of the duplicated chromosomes into a pair of daughter cells. Histones are fundamental structural components of chromatin in eukaryotic cells, and their post-translational modifications (PTMs) benchmark DNA readout and chromosome condensation. Aberrant regulation of the cell cycle associated with dysregulation of histone PTMs is the cause of critical diseases such as cancer. Monitoring changes of histone PTMs could pave the way to understanding the molecular mechanisms associated with epigenetic regulation of cell proliferation. Previously, our lab established a novel middle-down workflow using porous graphitic carbon (PGC) as a stationary phase to analyze histone PTMs, which utilizes the same reversed-phase chromatography for gradient separation as canonical proteomics coupled with online mass spectrometry (MS). Here, we applied this novel workflow for high-throughput analysis of histone modifications of H3.1 and H3.2 during the cell cycle. Collectively, we identified 1133 uniquely modified canonical histone H3 N-terminal tails. Consistent with previous findings, histone H3 phosphorylation increased significantly during the mitosis (M) phase. Histone H3 variant-specific and cell-cycle-dependent expressions of PTMs were observed, underlining the need to not combine H3.1 and H3.2 together as H3. We confirmed previously known H3 PTM crosstalk (e.g., K9me-S10ph) and revealed new information in this area as well. These findings imply that the combinatorial PTMs play a role in cell cycle control, and they may serve as markers for proliferation.

Expeditious Extraction of Histones from Limited Cells or Tissue Samples and Quantitative Top-Down Proteomic Analysis.

Histones are the primary protein component of chromatin and are involved in virtually all DNA-templated processes. Histones are abundantly post-translationally modified by a variety of chromatin-modifying machinery. These post-translational modifications (PTMs) are recognized by a range of "reader" proteins, which recruit additional proteins to specific locations on chromatin and impart precise and powerful effects on gene regulation. Each PTM typically exerts a positive or negative effect on transcription, and recent studies have shown that histone PTMs function in a combinatorial histone code: that is, histone PTMs function in combination to exert precise DNA-templated regulation. Thus, there is a need to identify and understand proteoforms, or unambiguously defined single protein molecules with all combinations of modifications. Top-down proteomics is currently the only viable approach for identifying and quantitating histone proteoforms, and mass spectrometry instruments have become sufficiently powerful to perform these quantitative analyses in a robust and high-throughput fashion. These recent innovations have enabled new experimental directions in chromatin research but have also introduced temporal and other constraints. This has led us to develop the protocols described here, which increase throughput, reduce sample requirements, and maintain robust quantitation. Although originally designed for high-throughput quantitative top-down proteomics, the protocols described here are useful for a wide range of chromatin biology applications. Starting with small amounts of cells or tissue, we describe two basic protocols for exceptionally rapid and efficient nuclei isolation, acid extraction of histones, and high-performance liquid chromatography fractionation of histones into histone families. We additionally describe the quantitative top-down proteomic analysis of histone H4 proteoforms. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Nuclei isolation and acid extraction of histones from mammalian cells in culture/tissues Basic Protocol 2: HPLC fractionation of histones and histone H4 HPLC-MS/MS Support Protocol: Preparation of intact H3 histone tails by Glu-C digestion.

The Use of Mononucleosome Immunoprecipitation for Analysis of Combinatorial Histone Post-translational Modifications and Purification of Nucleosome-Interacting Proteins.

The nucleosome is the principal structural unit of chromatin. Although many studies focus on individual histone post-translational modifications (PTMs) in isolation, it is important to recognize that multiple histone PTMs can function together or cross-regulate one another within the nucleosome context. In addition, different modifications or histone-binding surfaces can synergize to stabilize the binding of nuclear factors to nucleosomes. To facilitate these types of studies, we present here a step-by-step protocol for isolating high yields of mononucleosomes for biochemical analyses. Furthermore, we discuss differences and variations of the basic protocol used in different publications and characterize the relative abundance of selected histone PTMs and chromatin-binding proteins in the different chromatin fractions obtained by this method.

Bullet points to evaluate the performance of the middle-down proteomics workflow for histone modification analysis.

Middle-down proteomics has emerged as the method of choice to study combinatorial histone post translational modifications (PTMs). In the common bottom-up workflow, histones are digested into relatively short peptides (4-20 aa), separated using reversed-phase chromatography and analyzed using typical proteomics methods in mass spectrometry. In middle-down, histones are cleaved into longer polypeptides (50-60 aa) mostly corresponding to their N-terminal tails, resolved using weak cation exchange-hydrophilic interaction liquid chromatography (WCX-HILIC) and analyzed with less conventional mass spectrometry, i.e. using Electron Transfer Dissociation (ETD) for analyte fragmentation. Middle-down is not nearly as utilized as bottom-up for PTM analysis, partially due to its limited reproducibility and robustness. This has also limited the establishment of rigorous benchmarks to discriminate good vs poor quality experiments. Here, we describe critical aspects of the middle-down workflow to assist the user in evaluating the presence of biased and misleading results. Specifically, we tested the use of porous graphitic carbon (PGC) during the desalting step, demonstrating that desalting using only C18 material leads to sample loss. We also tested different salts in the WCX-HILIC buffers for their effect on retention, selectivity, and reproducibility of analysis of variants of histone tail fragments, in particular replacing ammonium ion with ethylenediammonium ion in buffer A. These substitutions had marked effects on selectivity and retention. Our results provide a streamlined way to evaluate middle-down performance to identify and quantify combinatorial histone PTMs.

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

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.

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.

Epiproteomic Landscape and Histone Code of Cutaneous T-Cell Lymphoma/Sézary Syndrome

Background: The genetic basis and the genome-wide abnormalities underlying most forms of cancer are being comprehensively annotated in many human malignancies. However, the contribution of epigenetic aberrations, particularly the post-translational modifications (PTM) of histone tails has not been investigated. This is largely due to lack of approaches to comprehensively interrogate the status of the numerous PTMs that define histone marks which control gene expression and complex biologic processes such as cancer. Sézary Syndrome (SS) is an aggressive form of cutaneous T-cell lymphoma (CTCL) characterized by poor outcomes and complex genetic alterations frequently targeting epigenetic regulators and chromatin remodelers. CTCLs represent the first FDA-approved disease for treatment using histone deacetylase inhibitors (HDACi) such as romidepsin, but the direct consequences of this treatment on histone PTMs remain unknown. Here we define the histone PTM signatures of CTCL/SS by using a novel unbiased strategy leveraging tandem mass spectrometry (MS/MS)-based quantitative proteomics to study the comprehensive combinatorial histone PTM code in a cohort of primary SS samples, and 4 CTCL/SS cell lines in comparison with normal reactive CD4+ T-lymphocytes from healthy individuals.Methods: CD4+ T-cells were isolated using CD4 immunomagnetic beads from PBMC's of healthy volunteers (n=8) or patients with diagnosed SS (n=20). CD4+ cells were frozen and subsequently analyzed in batches. The CTCL/SS-derived cell lines (HH and MJ, HuT78 and H9) were also analyzed each with three biological replicates. Histones were acid extracted from isolated nuclei. Total bulk histones were propionylated and trypsinized prior to MS analysis. Nano-scale liquid chromatography followed by -tandem mass spectrometry (nano LC-MS/MS) data acquisition was performed in an Orbitrap FusionTM TribridTM MS in technical triplicates for each sample with a data-independent acquisition (DIA)-method using a 50 m/z quadrupole-isolation windows that steps across the 200-1500 m/z ranges. Data analysis was performed using EpiProfile for single and combinatorial histone PTM analysis and quantification. Orthogonal validation for selected modifications was performed using western blotting and single-cell mass cytometry by time of flight (CyTOF) analysis.Results: Our quantitative epiproteomic strategy interrogated the relative ratios of a total of 228 histone PTM combinations, and 20 histone variants in a well-characterized cohort of SS patient samples, four CTCL cell lines and a cohort of normal primary CD4+ T-lymphocytes isolated from healthy individuals. With this approach, we were able to identify and quantify 23 unique histone peptides on histone H4, 105 on histone H3, 80 on histone H2A, 12 on histone H2B and 28 on histone H1 across every sample. Pearson correlation and principal component analysis of overall histone PTMs profiles across all samples provided accurate discrimination. We found a distinct pattern of histone PTMs in both SS patient samples and CTCL cell lines that were strikingly different from CD4+ T-cells obtained from healthy individuals. Notably, differences between cell lines and primary patient samples are more marked than those within individual SS primary patient samples, implying that cell lines may poorly recapitulate some aspects of in vivo biology. Unsupervised hierarchical clustering of the primary patient samples, cell lines and the normal CD4+ T-cells performed based on abundances of the identified histone PTMs revealed disease-specific ubiquitous marks as well as disease-specific unique marks for SS. Among the analyzed histone PTMs, H3K27ac, H3.3K27ac, H4K8ac, H4K20ac, H3K4me3, H3K18me1, H3K79me3 and H4K20me3 were distinct between healthy and CTCL CD4+ T-cells. Selected differential marks such as H4K20me3 were orthogonally corroborated by Western blotting and CyTOF mass cytometry analysis.Conclusions: For the first time, we have defined the histone code of CTCL/SS using global mass spectrometry based quantitative proteomics with high specificity and sensitivity. The results of our MS-based epiproteomic profiling revealed disease-specific histone PTM signatures and opportunities to exploit tractable changes induced by HDACi treatment as clinically actionable molecular biomarkers and therapeutic targets. DisclosuresNo relevant conflicts of interest to declare.

Understanding the Histone DNA Repair Code: H4K20me2 Makes Its Mark.

Chromatin is a highly compact structure that must be rapidly rearranged in order for DNA repair proteins to access sites of damage and facilitate timely and efficient repair. Chromatin plasticity is achieved through multiple processes, including the posttranslational modification of histone tails. In recent years, the impact of histone posttranslational modification on the DNA damage response has become increasingly well recognized, and chromatin plasticity has been firmly linked to efficient DNA repair. One particularly important histone posttranslational modification process is methylation. Here, we focus on the regulation and function of H4K20 methylation (H4K20me) in the DNA damage response and describe the writers, erasers, and readers of this important chromatin mark as well as the combinatorial histone posttranslational modifications that modulate H4K20me recognition. Finally, we discuss the central role of H4K20me in determining if DNA double-strand breaks (DSB) are repaired by the error-prone, nonhomologous DNA end joining pathway or the error-free, homologous recombination pathway. This review article discusses the regulation and function of H4K20me2 in DNA DSB repair and outlines the components and modifications that modulate this important chromatin mark and its fundamental impact on DSB repair pathway choice. Mol Cancer Res; 16(9); 1335-45. ©2018 AACR.