PDGF-BB overexpression in p53 null oligodendrocyte progenitors increases H3K27me3 and induces transcriptional changes which favor proliferation.

Proneural gliomas are brain tumors characterized by enrichment of oligodendrocyte progenitor cell (OPC) transcripts and genetic alterations. In this study we sought to identify transcriptional and epigenetic differences between OPCs with Trp53 deletion and PDGF-BB overexpression (BB-p53n), which form tumors when transplanted in mouse brains, and those carrying only p53 deletion (p53n), which do not. We used unbiased histone proteomics and RNA-seq analysis on these two genetically modified OPC populations and detected higher levels of H3K27me3 in BB-p53n compared to p53n OPCs. The BB-p53n OPC were characterized by higher levels of transcripts related to proliferation and lower levels of those related to differentiation. Pharmacological inhibition of histone H3K27 trimethylation in BB-p53n OPC reduced cell cycle transcripts and increased the expression of differentiation markers. These data suggest that PDGF-BB overexpression in p53 null OPC results in histone post-translational modifications and consequent transcriptional changes favoring proliferation while halting differentiation, thereby promoting the early stages of transformation.

Comparative Analysis of Drug-like EP300/CREBBP Acetyltransferase Inhibitors.

The human acetyltransferase paralogues EP300 and CREBBP are master regulators of lysine acetylation whose activity has been implicated in various cancers. In the half-decade since the first drug-like inhibitors of these proteins were reported, three unique molecular scaffolds have taken precedent: an indane spiro-oxazolidinedione (A-485), a spiro-hydantoin (iP300w), and an aminopyridine (CPI-1612). Despite increasing use of these molecules to study lysine acetylation, the dearth of data regarding their relative biochemical and biological potencies makes their application as chemical probes a challenge. To address this gap, here we present a comparative study of drug-like EP300/CREBBP acetyltransferase inhibitors. First, we determine the biochemical and biological potencies of A-485, iP300w, and CPI-1612, highlighting the increased potencies of the latter two compounds at physiological acetyl-CoA concentrations. Cellular evaluation shows that inhibition of histone acetylation and cell growth closely aligns with the biochemical potencies of these molecules, consistent with an on-target mechanism. Finally, we demonstrate the utility of comparative pharmacology by using it to investigate the hypothesis that increased CoA synthesis caused by knockout of PANK4 can competitively antagonize the binding of EP300/CREBBP inhibitors and demonstrate proof-of-concept photorelease of a potent inhibitor molecule. Overall, our study demonstrates how knowledge of the relative inhibitor potency can guide the study of EP300/CREBBP-dependent mechanisms and suggests new approaches to target delivery, thus broadening the therapeutic window of these preclinical epigenetic drug candidates.

Acetyl-methyllysine marks chromatin at active transcription start sites.

Lysine residues in histones and other proteins can be modified by post-translational modifications that encode regulatory information1. Lysine acetylation and methylation are especially important for regulating chromatin and gene expression2-4. Pathways involving these post-translational modifications are targets for clinically approved therapeutics to treat human diseases. Lysine methylation and acetylation are generally assumed to be mutually exclusive at the same residue. Here we report cellular lysine residues that are both methylated and acetylated on the same side chain to form Nε-acetyl-Nε-methyllysine (Kacme). We show that Kacme is found on histone H4 (H4Kacme) across a range of species and across mammalian tissues. Kacme is associated with marks of active chromatin, increased transcriptional initiation and is regulated in response to biological signals. H4Kacme can be installed by enzymatic acetylation of monomethyllysine peptides and is resistant to deacetylation by some HDACs in vitro. Kacme can be bound by chromatin proteins that recognize modified lysine residues, as we demonstrate with the crystal structure of acetyllysine-binding protein BRD2 bound to a histone H4Kacme peptide. These results establish Kacme as a cellular post-translational modification with the potential to encode information distinct from methylation and acetylation alone and demonstrate that Kacme has all the hallmarks of a post-translational modification with fundamental importance to chromatin biology.

Comparative analysis of drug-like EP300/CREBBP acetyltransferase inhibitors.

The human acetyltransferase paralogs EP300 and CREBBP are master regulators of lysine acetylation whose activity has been implicated in various cancers. In the half-decade since the first drug-like inhibitors of these proteins were reported, three unique molecular scaffolds have taken precedent: an indane spiro-oxazolidinedione (A-485), a spiro-hydantoin (iP300w), and an aminopyridine (CPI-1612). Despite increasing use of these molecules to study lysine acetylation, the dearth of data regarding their relative biochemical and biological potencies makes their application as chemical probes a challenge. To address this gap, here we present a comparative study of drug-like EP300/CREBBP acetyltransferase inhibitors. First, we determine the biochemical and biological potencies of A-485, iP300w, and CPI-1612, highlighting the increased potency of the latter two compounds at physiological acetyl-CoA concentrations. Cellular evaluation shows that inhibition of histone acetylation and cell growth closely aligns with the biochemical potencies of these molecules, consistent with an on-target mechanism. Finally, we demonstrate the utility of comparative pharmacology by using it to investigate the hypothesis that increased CoA synthesis caused by knockout of PANK4 can competitively antagonize binding of EP300/CREBBP inhibitors and demonstrate proof-of-concept photorelease of a potent inhibitor molecule. Overall, our study demonstrates how knowledge of relative inhibitor potency can guide the study of EP300/CREBBP-dependent mechanisms and suggests new approaches to target delivery, thus broadening the therapeutic window of these preclinical epigenetic drug candidates.

NSD1 mediates antagonism between SWI/SNF and polycomb complexes and is required for transcriptional activation upon EZH2 inhibition.

Disruption of antagonism between SWI/SNF chromatin remodelers and polycomb repressor complexes drives the formation of numerous cancer types. Recently, an inhibitor of the polycomb protein EZH2 was approved for the treatment of a sarcoma mutant in the SWI/SNF subunit SMARCB1, but resistance occurs. Here, we performed CRISPR screens in SMARCB1-mutant rhabdoid tumor cells to identify genetic contributors to SWI/SNF-polycomb antagonism and potential resistance mechanisms. We found that loss of the H3K36 methyltransferase NSD1 caused resistance to EZH2 inhibition. We show that NSD1 antagonizes polycomb via cooperation with SWI/SNF and identify co-occurrence of NSD1 inactivation in SWI/SNF-defective cancers, indicating in vivo relevance. We demonstrate that H3K36me2 itself has an essential role in the activation of polycomb target genes as inhibition of the H3K36me2 demethylase KDM2A restores the efficacy of EZH2 inhibition in SWI/SNF-deficient cells lacking NSD1. Together our data expand the mechanistic understanding of SWI/SNF and polycomb interplay and identify NSD1 as the key for coordinating this transcriptional control.

Provision of a local anaesthetic minor procedures service by surgical advanced clinical practitioners: 5-year study.

BACKGROUND: Surgical advanced clinical practitioners (SACPs) form part of the extended surgical workforce drawn from a variety of allied healthcare backgrounds. The primary aim of this study was to determine whether there was a financial benefit in having minor surgical procedures undertaken by dedicated SACPs compared with operating lists assigned to consultant surgeons. METHODS: This was a retrospective cohort study including all patients who had minor 'lumps and bumps' procedures undertaken between April 2014 and August 2019 at East Lancashire Hospitals NHS Trust under local anaesthetic by the general surgery team. Clinical patient information, including lesion type, was collected along with operating room staffing levels and duration of operation. The cost of the procedure was calculated as operating time multiplied by cost of staff per minute according to local banding. RESULTS: A total of 1399 patients had a lesion excised; 907 procedures were carried out by a doctor, and the rest independently by a SACP. The majority of lesions excised were lipomas and cysts. There was no difference in the median surgical time taken between SACPs and doctors (20 (i.q.r. 14-28) min). Minor procedures carried out on consultant surgeon lists cost 62.3 per cent (€25.33) more on average than those on SACP lists (median €65.96 versus 40.63 respectively; P < 0.001). CONCLUSION: A dedicated and independent SACP 'lumps and bumps' list was financially beneficial. Operating times were similar to those of doctors. These lists safely free trainee and consultant surgeons to undertake more complex work.

Epigenetic and Transcriptional Control of the Epidermal Growth Factor Receptor Regulates the Tumor Immune Microenvironment in Pancreatic Cancer.

Although immunotherapy has revolutionized cancer care, patients with pancreatic ductal adenocarcinoma (PDA) rarely respond to these treatments, a failure that is attributed to poor infiltration and activation of T cells in the tumor microenvironment (TME). We performed an in vivo CRISPR screen and identified lysine demethylase 3A (KDM3A) as a potent epigenetic regulator of immunotherapy response in PDA. Mechanistically, KDM3A acts through Krueppel-like factor 5 (KLF5) and SMAD family member 4 (SMAD4) to regulate the expression of the epidermal growth factor receptor (EGFR). Ablation of KDM3A, KLF5, SMAD4, or EGFR in tumor cells altered the immune TME and sensitized tumors to combination immunotherapy, whereas treatment of established tumors with an EGFR inhibitor, erlotinib, prompted a dose-dependent increase in intratumoral T cells. This study defines an epigenetic-transcriptional mechanism by which tumor cells modulate their immune microenvironment and highlights the potential of EGFR inhibitors as immunotherapy sensitizers in PDA. SIGNIFICANCE: PDA remains refractory to immunotherapies. Here, we performed an in vivo CRISPR screen and identified an epigenetic-transcriptional network that regulates antitumor immunity by converging on EGFR. Pharmacologic inhibition of EGFR is sufficient to rewire the immune microenvironment. These results offer a readily accessible immunotherapy-sensitizing strategy for PDA.This article is highlighted in the In This Issue feature, p. 521.

Global Regulation of the Histone Mark H3K36me2 Underlies Epithelial Plasticity and Metastatic Progression.

Epithelial plasticity, reversible modulation of a cell's epithelial and mesenchymal features, is associated with tumor metastasis and chemoresistance, leading causes of cancer mortality. Although different master transcription factors and epigenetic modifiers have been implicated in this process in various contexts, the extent to which a unifying, generalized mechanism of transcriptional regulation underlies epithelial plasticity remains largely unknown. Here, through targeted CRISPR/Cas9 screening, we discovered two histone-modifying enzymes involved in the writing and erasing of H3K36me2 that act reciprocally to regulate epithelial-to-mesenchymal identity, tumor differentiation, and metastasis. Using a lysine-to-methionine histone mutant to directly inhibit H3K36me2, we found that global modulation of the mark is a conserved mechanism underlying the mesenchymal state in various contexts. Mechanistically, regulation of H3K36me2 reprograms enhancers associated with master regulators of epithelial-to-mesenchymal state. Our results thus outline a unifying epigenome-scale mechanism by which a specific histone modification regulates cellular plasticity and metastasis in cancer. SIGNIFICANCE: Although epithelial plasticity contributes to cancer metastasis and chemoresistance, no strategies exist for pharmacologically inhibiting the process. Here, we show that global regulation of a specific histone mark, H3K36me2, is a universal epigenome-wide mechanism that underlies epithelial-to-mesenchymal transition and mesenchymal-to-epithelial transition in carcinoma cells. These results offer a new strategy for targeting epithelial plasticity in cancer.This article is highlighted in the In This Issue feature, p. 747.

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 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.

PHF19 promotes multiple myeloma tumorigenicity through PRC2 activation and broad H3K27me3 domain formation.

Dysregulation of polycomb repressive complex 2 (PRC2) promotes oncogenesis partly through its enzymatic function for inducing trimethylation of histone H3 lysine 27 (H3K27me3). However, it remains to be determined how PRC2 activity is regulated in normal and diseased settings. We here report a PRC2-associated cofactor, PHD finger protein 19 (PHF19; also known as polycomb-like 3), as a crucial mediator of tumorigenicity in multiple myeloma (MM). Overexpression and/or genomic amplification of PHF19 is found associated with malignant progression of MM and plasma cell leukemia, correlating to worse treatment outcomes. Using various MM models, we demonstrated a critical requirement of PHF19 for tumor growth in vitro and in vivo. Mechanistically, PHF19-mediated oncogenic effect relies on its PRC2-interacting and chromatin-binding functions. Chromatin immunoprecipitation followed by sequencing profiling showed a critical role for PHF19 in maintaining the H3K27me3 landscape. PHF19 depletion led to loss of broad H3K27me3 domains, possibly due to impaired H3K27me3 spreading from cytosine guanine dinucleotide islands, which is reminiscent to the reported effect of an "onco"-histone mutation, H3K27 to methionine (H3K27M). RNA-sequencing-based transcriptome profiling in MM lines also demonstrated a requirement of PHF19 for optimal silencing of PRC2 targets, which include cell cycle inhibitors and interferon-JAK-STAT signaling genes critically involved in tumor suppression. Correlation studies using patient sample data sets further support a clinical relevance of the PHF19-regulated pathways. Lastly, we show that MM cells are generally sensitive to PRC2 inhibitors. Collectively, this study demonstrates that PHF19 promotes MM tumorigenesis through enhancing H3K27me3 deposition and PRC2's gene-regulatory functions, lending support for PRC2 blockade as a means for MM therapeutics.

Phosphorylation of TIP60 Suppresses 53BP1 Localization at DNA Damage Sites.

A proper balance between the repair of DNA double-strand breaks (DSBs) by homologous recombination and nonhomologous end joining is critical for maintaining genome integrity and preventing tumorigenesis. This balance is regulated and fine-tuned by a variety of factors, including cell cycle and the chromatin environment. The histone acetyltransferase TIP60 was previously shown to suppress pathological end joining and promote homologous recombination. However, it is unknown how regulatory posttranslational modifications impact TIP60 acetyltransferase activity to influence the outcome of DSB responses. In this study, we report that phosphorylation of TIP60 on serines 90 and 86 is important for limiting the accumulation of the pro-end joining factor 53BP1 at DSBs in S and G2 cell cycle phases. Mutation of these sites disrupts histone acetylation changes in response to DNA damage, BRCA1 localization to DSBs, and poly(ADP-ribose) polymerase (PARP) inhibitor resistance. These findings reveal that phosphorylation directs TIP60-dependent acetylation to promote homologous recombination and maintain genome stability.

Targeting CDK9 Reactivates Epigenetically Silenced Genes in Cancer.

Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50 = 5 nM) that has broad anti-cancer activity in vitro and is effective in in vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.

Association and expression of the antigen-processing gene PSMB8, coding for low-molecular-mass protease 7, with vitiligo in North India: case-control study.

BACKGROUND: Vitiligo is a multifactorial, autoimmune, depigmenting disorder of the skin where aberrant presentation of autoantigens may have a role. OBJECTIVES: To study the association of two antigen-processing genes, PSMB8 and PSMB9, with vitiligo. METHODS: In total 1320 cases of vitiligo (1050 generalized and 270 localized) and 752 healthy controls were studied for the PSMB9 exon 3 G/A single-nucleotide polymorphism (SNP), PSMB8 exon 2 C/A SNP and PSMB8 intron 6 G/T SNP at site 37 360 using polymerase chain reaction (PCR)-restriction fragment length polymorphism. Real-time PCR was used for transcriptional expression of PSMB8 and cytokines. Expression of ubiquitinated proteins and phosphorylated-p38 (P-p38) was studied by Western blotting. RESULTS: Significant increases in PSMB8 exon 2 allele A (P < 2.07 × 10-6 , odds ratio 1·93) and genotypes AA (P < 1.03 × 10-6 , odds ratio 2·51) and AC (P < 1.29 × 10-6 , odds ratio 1·63) were observed in patients with vitiligo. Interferon-γ stimulation induced lower expression of PSMB8 in peripheral blood mononuclear cells of cases compared with controls, suggesting impaired antigen processing, which was confirmed by accumulation of ubiquitinated proteins in both lesional and nonlesional skin of patients with vitiligo. Expression of proinflammatory cytokines - interleukin (IL)-6, IL-1ß and IL-8 - was higher in the lesional skin. P-p38 expression was variable but correlated with the amount of ubiquitinated proteins in the lesional and nonlesional skin, suggesting that the inflammatory cytokine responses in lesional skin could be a result of both P-p38-dependent and -independent pathways. CONCLUSIONS: The PSMB8 exon 2 SNP is significantly associated with vitiligo. Accumulation of ubiquitinated proteins in skin of cases of vitiligo suggests their aberrant processing, which may promote the development of the disease.

Naked Mole Rat Cells Have a Stable Epigenome that Resists iPSC Reprogramming.

Naked mole rat (NMR) is a valuable model for aging and cancer research due to its exceptional longevity and cancer resistance. We observed that the reprogramming efficiency of NMR fibroblasts in response to OSKM was drastically lower than that of mouse fibroblasts. Expression of SV40 LargeT antigen (LT) dramatically improved reprogramming of NMR fibroblasts. Inactivation of Rb alone, but not p53, was sufficient to improve reprogramming efficiency, suggesting that NMR chromatin may be refractory to reprogramming. Analysis of the global histone landscape revealed that NMR had higher levels of repressive H3K27 methylation marks and lower levels of activating H3K27 acetylation marks than mouse. ATAC-seq revealed that in NMR, promoters of reprogramming genes were more closed than mouse promoters, while expression of LT led to massive opening of the NMR promoters. These results suggest that NMR displays a more stable epigenome that resists de-differentiation, contributing to the cancer resistance and longevity of this species.

Taxane-Platin-Resistant Lung Cancers Co-develop Hypersensitivity to JumonjiC Demethylase Inhibitors.

Although non-small cell lung cancer (NSCLC) patients benefit from standard taxane-platin chemotherapy, many relapse, developing drug resistance. We established preclinical taxane-platin-chemoresistance models and identified a 35-gene resistance signature, which was associated with poor recurrence-free survival in neoadjuvant-treated NSCLC patients and included upregulation of the JumonjiC lysine demethylase KDM3B. In fact, multi-drug-resistant cells progressively increased the expression of many JumonjiC demethylases, had altered histone methylation, and, importantly, showed hypersensitivity to JumonjiC inhibitors in vitro and in vivo. Increasing taxane-platin resistance in progressive cell line series was accompanied by progressive sensitization to JIB-04 and GSK-J4. These JumonjiC inhibitors partly reversed deregulated transcriptional programs, prevented the emergence of drug-tolerant colonies from chemo-naive cells, and synergized with standard chemotherapy in vitro and in vivo. Our findings reveal JumonjiC inhibitors as promising therapies for targeting taxane-platin-chemoresistant NSCLCs.

Melt-compounded composites of ethylene vinyl acetate with magnesium sulfate as flexible EPR dosimeters: Mechanical properties, manufacturing process feasibility and dosimetric characteristics.

Novel polymeric composites for radiation dosimetry were developed. The composites were prepared by solvent-free melt compounding of ethylene vinyl acetate (EVA) (40% vinyl) and magnesium sulfate (MgSO4). Mechanical properties, melt flow characteristics and dosimetric properties were investigated. The composites with up to 50% (wt) of MgSO4 were flexible and capable of flow. The dose response of the EPR signal of the composites was studied in the dose range 3Gy-4kGy and found to be linear between 18Gy and 4kGy. The reproducibility of dose measurements was good. The signal fading rate and the energy dependence of the dose response were found to be acceptable.

Multi-faceted quantitative proteomics analysis of histone H2B isoforms and their modifications.

BACKGROUND: Histone isoforms and their post-translational modifications (PTMs) play an important role in the control of many chromatin-related processes including transcription and DNA damage. Variants of histones H2A and H3 have been studied in depth and have been found to have distinct functions. Although 13 somatic histone H2B isoforms have been identified by various biochemical and mass spectrometric (MS) approaches, the distinct roles of these isoforms within human cells are as yet unknown. Here, we have developed quantitative MS techniques to characterize isoform-specific H2B expression across the cell cycle, in differentiated myogenic cells, and in different cancer cell lines to illuminate potential functional roles. RESULTS: Using the MS strategies that we developed, we identified differences in H2B isoform levels between different cancer cell types, suggesting cancer or tissue-specific H2B isoform regulation. In particular, we found large variations in the levels of isoforms H2B1B and H2B1M across the panel of cell lines. We also found that, while individual H2B isoforms do not differ in their acetylation levels, trends in the acetylation on all H2B isoforms correlated with acetylation on other histone family members in the cancer cell line panel. We also used the MS strategies to study H2B protein expression across the cell cycle and determined that H2B isoforms that are alternatively spliced to carry a polyadenylation signal rather than the standard histone downstream element are expressed independently of the cell cycle. However, the level of protein produced from the polyadenylated transcripts does not contribute significantly to the total pool of H2B isoforms translated across the cell cycle or in non-cycling myogenic cells. CONCLUSIONS: Our results show that H2B isoforms are expressed at varying levels in different cells, suggesting isoform-specific, and possibly cell-type-specific, H2B gene regulation. The bottom-up mass spectrometry technique we developed for H2B quantification is compatible with the current standard histone H3 and H4 bottom-up 'one-pot' analysis platform so that H2B isoforms and their modifications can be studied in future experiments at the same time as histone H3 and H4 modifications. Therefore, we have expanded the histone landscape that can be interrogated in future experiments.

EpiProfile Quantifies Histone Peptides With Modifications by Extracting Retention Time and Intensity in High-resolution Mass Spectra.

Histone post-translational modifications contribute to chromatin function through their chemical properties which influence chromatin structure and their ability to recruit chromatin interacting proteins. Nanoflow liquid chromatography coupled with high resolution tandem mass spectrometry (nanoLC-MS/MS) has emerged as the most suitable technology for global histone modification analysis because of the high sensitivity and the high mass accuracy of this approach that provides confident identification. However, analysis of histones with this method is even more challenging because of the large number and variety of isobaric histone peptides and the high dynamic range of histone peptide abundances. Here, we introduce EpiProfile, a software tool that discriminates isobaric histone peptides using the distinguishing fragment ions in their tandem mass spectra and extracts the chromatographic area under the curve using previous knowledge about peptide retention time. The accuracy of EpiProfile was evaluated by analysis of mixtures containing different ratios of synthetic histone peptides. In addition to label-free quantification of histone peptides, EpiProfile is flexible and can quantify different types of isotopically labeled histone peptides. EpiProfile is unique in generating layouts (i.e. relative retention time) of histone peptides when compared with manual quantification of the data and other programs (such as Skyline), filling the need of an automatic and freely available tool to quantify labeled and non-labeled modified histone peptides. In summary, EpiProfile is a valuable nanoflow liquid chromatography coupled with high resolution tandem mass spectrometry-based quantification tool for histone peptides, which can also be adapted to analyze nonhistone protein samples.

Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH) Analysis for Characterization and Quantification of Histone Post-translational Modifications.

Histone post-translational modifications (PTMs) have a fundamental function in chromatin biology, as they model chromatin structure and recruit enzymes involved in gene regulation, DNA repair, and chromosome condensation. High throughput characterization of histone PTMs is mostly performed by using nano-liquid chromatography coupled to mass spectrometry. However, limitations in speed and stochastic sampling of data dependent acquisition methods in MS lead to incomplete discrimination of isobaric peptides and loss of low abundant species. In this work, we analyzed histone PTMs with a data-independent acquisition method, namely SWATH™ analysis. This approach allows for MS/MS-based quantification of all analytes without upfront assay development and no issues of biased and incomplete sampling. We purified histone proteins from human embryonic stem cells and mouse trophoblast stem cells before and after differentiation, and prepared them for MS analysis using the propionic anhydride protocol. Results on histone H3 peptides verified that sequential window acquisition of all theoretical mass spectra could accurately quantify peptides (<9% average coefficient of variation, CV) over four orders of magnitude, and we could discriminate isobaric and co-eluting peptides (e.g. H3K18ac and H3K23ac) using MS/MS-based quantification. This method provided high sensitivity and precision, supported by the fact that we could find significant differences for remarkably low abundance PTMs such as H3K9me2S10ph (relative abundance <0.02%). We performed relative quantification for few sample peptides using different fragment ions and observed high consistency (CV <15%) between the fragments. This indicated that different fragment ions can be used independently to achieve the same peptide relative quantification. Taken together, sequential window acquisition of all theoretical mass spectra proved to be an easy-to-use MS acquisition method to perform high quality MS/MS-based quantification of histone-modified peptides.