Combinatorial Post-translational Modifications Research Articles

BRAF/MAPK and GSK3 signaling converges to control MITF nuclear export

The close integration of the MAPK, PI3K, and WNT signaling pathways underpins much of development and is deregulated in cancer. In principle, combinatorial posttranslational modification of key lineage-specific transcription factors would be an effective means to integrate critical signaling events. Understanding how this might be achieved is central to deciphering the impact of microenvironmental cues in development and disease. The microphthalmia-associated transcription factor MITF plays a crucial role in the development of melanocytes, the retinal pigment epithelium, osteoclasts, and mast cells and acts as a lineage survival oncogene in melanoma. MITF coordinates survival, differentiation, cell-cycle progression, cell migration, metabolism, and lysosome biogenesis. However, how the activity of this key transcription factor is controlled remains poorly understood. Here, we show that GSK3, downstream from both the PI3K and Wnt pathways, and BRAF/MAPK signaling converges to control MITF nuclear export. Phosphorylation of the melanocyte MITF-M isoform in response to BRAF/MAPK signaling primes for phosphorylation by GSK3, a kinase inhibited by both PI3K and Wnt signaling. Dual phosphorylation, but not monophosphorylation, then promotes MITF nuclear export by activating a previously unrecognized hydrophobic export signal. Nonmelanocyte MITF isoforms exhibit poor regulation by MAPK signaling, but instead their export is controlled by mTOR. We uncover here an unanticipated mode of MITF regulation that integrates the output of key developmental and cancer-associated signaling pathways to gate MITF flux through the import-export cycle. The results have significant implications for our understanding of melanoma progression and stem cell renewal.

Dissecting Structure-Encoded Determinants of Allosteric Cross-Talk between Post-Translational Modification Sites in the Hsp90 Chaperones

Post-translational modifications (PTMs) represent an important regulatory instrument that modulates structure, dynamics and function of proteins. The large number of PTM sites in the Hsp90 proteins that are scattered throughout different domains indicated that synchronization of multiple PTMs through a combinatorial code can be invoked as an important mechanism to orchestrate diverse chaperone functions and recognize multiple client proteins. In this study, we have combined structural and coevolutionary analysis with molecular simulations and perturbation response scanning analysis of the Hsp90 structures to characterize functional role of PTM sites in allosteric regulation. The results reveal a small group of conserved PTMs that act as global mediators of collective dynamics and allosteric communications in the Hsp90 structures, while the majority of flexible PTM sites serve as sensors and carriers of the allosteric structural changes. This study provides a comprehensive structural, dynamic and network analysis of PTM sites across Hsp90 proteins, identifying specific role of regulatory PTM hotspots in the allosteric mechanism of the Hsp90 cycle. We argue that plasticity of a combinatorial PTM code in the Hsp90 may be enacted through allosteric coupling between effector and sensor PTM residues, which would allow for timely response to structural requirements of multiple modified enzymes.

Direct Profiling the Post-Translational Modification Codes of a Single Protein Immobilized on a Surface Using Cu-free Click Chemistry.

Combinatorial post-translational modifications (PTMs), which can serve as dynamic “molecular barcodes”, have been proposed to regulate distinct protein functions. However, studies of combinatorial PTMs on single protein molecules have been hindered by a lack of suitable analytical methods. Here, we describe erasable single-molecule blotting (eSiMBlot) for combinatorial PTM profiling. This assay is performed in a highly multiplexed manner and leverages the benefits of covalent protein immobilization, cyclic probing with different antibodies, and single molecule fluorescence imaging. Especially, facile and efficient covalent immobilization on a surface using Cu-free click chemistry permits multiple rounds (>10) of antibody erasing/reprobing without loss of antigenicity. Moreover, cumulative detection of coregistered multiple data sets for immobilized single-epitope molecules, such as HA peptide, can be used to increase the antibody detection rate. Finally, eSiMBlot enables direct visualization and quantitative profiling of combinatorial PTM codes at the single-molecule level, as we demonstrate by revealing the novel phospho-codes of ligand-induced epidermal growth factor receptor. Thus, eSiMBlot provides an unprecedentedly simple, rapid, and versatile platform for analyzing the vast number of combinatorial PTMs in biological pathways.

Abstract A10: Integrated proteome and epigenetic analysis reveals crosstalk between chromatin and cancer pathways in malignant peripheral nerve sheath tumors

Abstract Malignant peripheral nerve sheath tumors (MPNST) are rare, aggressive sarcomas. Recent work has identified loss-of-function mutations in PRC2 components in a subset of these tumors. PRC2 mutation leads to a loss of trimethylation at lysine 27 of histone H3 (H3K27me3), and is linked to transcriptional upregulation of the Ras pathway in cell culture models of MPNST. We hypothesized that PRC2 loss would result in global alterations in histone post-translational modifications (PTMs), and such changes would be associated with altered protein expression in oncogenic pathways. We thus implemented protein and histone extraction methods directly on formalin-fixed, paraffin-embedded human tumor samples and performed parallel histone PTM and proteome analysis. Data were merged using our in-house developed chromatin and proteomics bioinformatics tools. Methods: Human tumors were obtained from pathology archives. Total protein was isolated from 3-mm tissue cores, following deparaffinization, antigen retrieval, lysis, and homogenization. Histone bands were excised following SDS-PAGE of total lysate. For cell lines, total protein was obtained following lysis in urea and histones were obtained via nuclear isolation and acid precipitation. Cell proteome and histone PTMs were analyzed using nano-liquid chromatography and tandem mass spectrometry. The proteome was characterized using MaxQuant. Histone PTMs were identified and quantified using lab-developed software. Statistical analysis was performed using Student's t-test, Gene Ontology enrichment was assessed using hypergeometric distribution annotations, and GSEA analysis was used to identify signatures of pathway activation. Results: PRC2 loss was associated with decreased H3K27me3 and increased H3K27 acetylation (p=0.02 and p=0.01). This was accompanied by increased hyperacetylation of H4, a marker of open chromatin. We also observed an increase in overall H3K36me2 (p=0.0001). By quantifying combinatorial PTMs, we observed that tandem H3K27me2K36me2 and H3K27me3K36me2 marks were markedly decreased in MPNST with PRC2 loss (p=0.04 and p=0.004). These alterations result in chromatin in which the active H3K36me2 mark is not flanked by repressive PTMs. At the level of the proteome, PRC2 loss was associated with increased expression of chromatin remodelers, particularly members of the SWI/SNF complex. We confirmed increased Ras pathway expression and noted increases in other tumor pathways, and decreases in proteins associated with nerve sheath differentiation and immune surveillance. To assess whether PRC2 function was directly linked to the proteome changes seen in the human tumors, we restored PRC2 function in PRC2-mutant MPNST cell lines and measured the proteome response. PRC2 reconstitution downregulated pathways that were upregulated with PRC2 loss in human tumors and restored expression of markers of nerve sheath differentiation and immune surveillance. The same effects were observed in PRC2 mutant cell lines upon shRNA silencing of NSD2, the methyltransferase responsible for H3K36me2, suggesting that, for a subset of genes, H3K27me3 and H3K36me2 function in tandem to control expression. These results link PRC2 loss to increased active chromatin signatures that promote pathways associated with aggressive tumor behavior. Significantly, the effects of PRC2 loss were ameliorated by restoration of PRC2 function, or inhibition of NSD2 function, revealing crosstalk between H3K27me and H3K36me marks, and suggesting that multiple agents targeting components of the active chromatin state may be beneficial in this rare and deadly disease. Citation Format: John B. Wojcik, Simone Sidoli, Kumarasen Cooper, Benjamin A. Garcia. Integrated proteome and epigenetic analysis reveals crosstalk between chromatin and cancer pathways in malignant peripheral nerve sheath tumors [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A10.

Metabolic labeling in middle-down proteomics allows for investigation of the dynamics of the histone code

BackgroundMiddle-down mass spectrometry (MS), i.e., analysis of long (~50–60 aa) polypeptides, has become the method with the highest throughput and accuracy for the characterization of combinatorial histone posttranslational modifications (PTMs). The discovery of histone readers with multiple domains, and overall the cross talk of PTMs that decorate histone proteins, has revealed that histone marks have synergistic roles in modulating enzyme recruitment and subsequent chromatin activities. Here, we demonstrate that the middle-down MS strategy can be combined with metabolic labeling for enhanced quantification of histone proteins and their combinatorial PTMs in a dynamic manner.MethodsWe used a nanoHPLC-MS/MS system consisting of hybrid weak cation exchange–hydrophilic interaction chromatography combined with high resolution MS and MS/MS with ETD fragmentation. After spectra identification, we filtered confident hits and quantified polypeptides using our in-house software isoScale.ResultsWe first verified that middle-down MS can discriminate and differentially quantify unlabeled from heavy labeled histone N-terminal tails (heavy lysine and arginine residues). Results revealed no bias toward identifying and quantifying unlabeled versus heavy labeled tails, even if the heavy labeled peptides presented the typical skewed isotopic pattern typical of long protein sequences that hardly get 100% labeling. Next, we plated epithelial cells into a media with heavy methionine-(methyl-13CD3), the precursor of the methyl donor S-adenosylmethionine and stimulated epithelial to mesenchymal transition (EMT). We assessed that results were reproducible across biological replicates and with data obtained using the more widely adopted bottom-up MS strategy, i.e., analysis of short tryptic peptides. We found remarkable differences in the incorporation rate of methylations in non-confluent cells versus confluent cells. Moreover, we showed that H3K27me3 was a critical player during the EMT process, as a consistent portion of histones modified as H3K27me2K36me2 in epithelial cells were converted into H3K27me3K36me2 in mesenchymal cells.ConclusionsWe demonstrate that middle-down MS, despite being a more scarcely exploited MS technique than bottom-up, is a robust quantitative method for histone PTM characterization. In particular, middle-down MS combined with metabolic labeling is currently the only methodology available for investigating turnover of combinatorial histone PTMs in dynamic systems.

Systems Level Analysis of Histone H3 Post-translational Modifications (PTMs) Reveals Features of PTM Crosstalk in Chromatin Regulation

Histones are abundant chromatin constituents carrying numerous post-translational modifications (PTMs). Such PTMs mediate a variety of biological functions, including recruitment of enzymatic readers, writers and erasers that modulate DNA replication, transcription and repair. Individual histone molecules contain multiple coexisting PTMs, some of which exhibit crosstalk, i.e. coordinated or mutually exclusive activities. Here, we present an integrated experimental and computational systems level molecular characterization of histone PTMs and PTM crosstalk. Using wild type and engineered mouse embryonic stem cells (mESCs) knocked out in components of the Polycomb Repressive Complex 2 (PRC2, Suz12(-/-)), PRC1 (Ring1A/B(-/-)) and (Dnmt1/3a/3b(-/-)) we performed comprehensive PTM analysis of histone H3 tails (50 aa) by utilizing quantitative middle-down proteome analysis by tandem mass spectrometry. We characterized combinatorial PTM features across the four mESC lines and then applied statistical data analysis to predict crosstalk between histone H3 PTMs. We detected an overrepresentation of positive crosstalk (codependent marks) between adjacent mono-methylated and acetylated marks, and negative crosstalk (mutually exclusive marks) among most of the seven characterized di- and tri-methylated lysine residues in the H3 tails. We report novel features of PTM interplay involving hitherto poorly characterized arginine methylation and lysine methylation sites, including H3R2me, H3R8me and H3K37me. Integration of the H3 data with RNAseq data by coabundance clustering analysis of histone PTMs and histone modifying enzymes revealed correlations between PTM and enzyme levels. We conclude that middle-down proteomics is a powerful tool to determine conserved or dynamic interdependencies between histone marks, which paves the way for detailed investigations of the histone code. Histone H3 PTM data is publicly available in the CrossTalkDB repository at http://crosstalkdb.bmb.sdu.dk.

Analyses of Histone Proteoforms Using Front-end Electron Transfer Dissociation-enabled Orbitrap Instruments

Histones represent a class of proteins ideally suited to analyses by top-down mass spectrometry due to their relatively small size, the high electron transfer dissociation-compatible charge states they exhibit, and the potential to gain valuable information concerning combinatorial post-translational modifications and variants. We recently described new methods in mass spectrometry for the acquisition of high-quality MS/MS spectra of intact proteins (Anderson, L. C., English, A. M., Wang, W., Bai, D. L., Shabanowitz, J., and Hunt, D. F. (2015) Int. J. Mass Spectrom. 377, 617-624). Here, we report an extension of these techniques. Sequential ion/ion reactions carried out in a modified Orbitrap Velos Pro/Elite(TM) capable of multiple fragment ion fills of the C-trap, in combination with data-dependent and targeted HPLC-MS experiments, were used to obtain high resolution MS/MS spectra of histones from butyrate-treated HeLa cells. These spectra were used to identify several unique intact histone proteoforms with up to 81% sequence coverage. We also demonstrate that parallel ion parking during ion/ion proton transfer reactions can be used to separate species of overlapping m/z that are not separated chromatographically, revealing previously indiscernible signals. Finally, we characterized several truncated forms of H2A and H2B found within the histone fractions analyzed, achieving up to 93% sequence coverage by electron transfer dissociation MS/MS. Results of follow-up in vitro experiments suggest that some of the truncated histone H2A proteoforms we observed can be generated by cathepsin L, an enzyme known to also catalyze clipping of histone H3.

Quantitative Mass Spectrometry Reveals that Intact Histone H1 Phosphorylations are Variant Specific and Exhibit Single Molecule Hierarchical Dependence

Breast cancer was the second leading cause of cancer related mortality for females in 2014. Recent studies suggest histone H1 phosphorylation may be useful as a clinical biomarker of breast and other cancers because of its ability to recognize proliferative cell populations. Although monitoring a single phosphorylated H1 residue is adequate to stratify high-grade breast tumors, expanding our knowledge of how H1 is phosphorylated through the cell cycle is paramount to understanding its role in carcinogenesis. H1 analysis by bottom-up MS is challenging because of the presence of highly homologous sequence variants expressed by most cells. These highly basic proteins are difficult to analyze by LC-MS/MS because of the small, hydrophilic nature of peptides produced by tryptic digestion. Although bottom-up methods permit identification of several H1 phosphorylation events, these peptides are not useful for observing the combinatorial post-translational modification (PTM) patterns on the protein of interest. To complement the information provided by bottom-up MS, we utilized a top-down MS/MS workflow to permit identification and quantitation of H1 proteoforms related to the progression of breast cells through the cell cycle. Histones H1.2 and H1.4 were observed in MDA-MB-231 metastatic breast cells, whereas an additional histone variant, histone H1.3, was identified only in nonneoplastic MCF-10A cells. Progressive phosphorylation of histone H1.4 was identified in both cell lines at mitosis (M phase). Phosphorylation occurred first at S172 followed successively by S187, T18, T146, and T154. Notably, phosphorylation at S173 of histone H1.2 and S172, S187, T18, T146, and T154 of H1.4 significantly increases during M phase relative to S phase, suggesting that these events are cell cycle-dependent and may serve as markers for proliferation. Finally, we report the observation of the H1.2 SNP variant A18V in MCF-10A cells.

Top-down characterization of histone H4 proteoforms with ProteinGoggle 2.0

Top-down characterization of combinatorial and dense post-translational modifications (PTMs) on core histones has long been a big analytical challenge because of enormous putative proteoforms for identification and simultaneously enormous putative sites of each individual PTM for localization. ProteinGoggle 2.0, as implemented with the isotopic mass-to-charge ratio and envelope fingerprinting algorithm, has multiple unique strengths for top-down characterization of histone PTMs together with high-resolution tandem mass spectrometry. Here we report our database search and proteoform identification of HeLa core histone H4 using ProteinGoggle 2.0. The Theoretical database containing all putative proteoforms was created with shotgun annotation from the human H4 flat text downloaded from UniProt; information including the amino acid sequence, putative PTMs (methylation, di-methylation, tri-methylation, acetylation and phosphorylation) and amino acid variation (A77 to P) was adopted from the flat text file. A total of 426 proteoforms were confidently identified with a spectrum level false discovery rate of less than 1%, which represents the most comprehensive H4 proteoforms reported so far. Side-by-side comparison of these proteoforms with those identified by ProSightPC 2.0 was also made. By and large, ProteinGoggle 2.0 can be adopted for database search and proteoform identification of proteins with multiple combinatorial PTMs as well as amino acid variation.

Combinatorial Histone Acetylation Patterns Are Generated by Motif-Specific Reactions

Post-translational modifications (PTMs) are pivotal to cellular information processing, but how combinatorial PTM patterns ("motifs") are set remains elusive. We develop a computational framework, which we provide as open source code, to investigate the design principles generating the combinatorial acetylation patterns on histone H4 in Drosophila melanogaster. We find that models assuming purely unspecific or lysine site-specific acetylation rates were insufficient to explain the experimentally determined motif abundances. Rather, these abundances were best described by an ensemble of models with acetylation rates that were specific to motifs. The model ensemble converged upon four acetylation pathways; we validated three of these using independent data from a systematic enzyme depletion study. Our findings suggest that histone acetylation patterns originate through specific pathways involving motif-specific acetylation activity.

Reading the Combinatorial Histone Language.

Histones are subject to frequent combinatorial post-translational modifications (PTMs), forming a complex chemical "language" that is interpreted by PTM-specific histone-interacting protein modules (reader domains). These specific interactions are thought to instruct gene expression and downstream biological functions. While the majority of studies have focused on individual modifications, our current understanding of the combinatorial PTM patterns on histones is starting to emerge, benefiting from the convergence of multiple technologies. Here, we review the key technical advances and progress on discovery and characterization of combinatorial histone PTM patterns. We focus on the interactions between reader domains and combinatorial PTMs, which is essential for understanding the mechanism and biological meaning of establishing and interpreting information embedded in histone PTM patterns.

Writing and Reading the CTD Code: In Vitro Reconstruction of Post‐Translational Modifications on RNA Polymerase II

Information flow within the cell is filtered through a vast array of codes. Vital biological data is compiled at the DNA, RNA, and protein levels with additional information relayed through an ever‐expanding repertoire of post‐transcriptional and post‐translational modifications (PTM). The PTM state of proteins can impact their recognition, structure, and function as well as relay important signals to influence cellular events, as found in histone code. A newly characterized coding system in eukaryotic transcription is “CTD code.” The C‐terminal Domain of RNA Polymerase II (CTD) is a unique structure composed of repeats of the consensus heptad YSPTSPS. These residues are subject to a variety of PTMs and specific factors are recruited depending on the PTM state of CTD. Particular PTM marks are linked to transcriptional events including initiation, elongation, and termination. The PTM state of CTD is generated and altered through the action of PTM enzymes such as kinases and phosphatases. Current technologies allow the determination of isolated and simplistic PTM marks along the CTD but lack the potential for the investigation of a combinatorial code or the identification of novel PTM marks. To overcome this we propose a bottom up approach to in vitro reconstruct the CTD code. We treat CTD substrate with PTM enzymes and characterize resultant modifications using a combination of molecular biology and mass spectrometry. Our novel mass spectrometry methodologies allow for heptad level resolution and characterization of individual and combinatorial PTM marks along CTD. Using this interdisciplinary cipher we can determine the lexicon for CTD code and better understand the impact of PTM on cellular communication.

MARCC (Matrix‐Assisted Reader Chromatin Capture): ChIP‐less Analysis Of Chromatin States

Histone post-translational modifications (PTMs) are key epigenetic regulators in chromatin-based processes. The combinations of histone PTMs rather than individual PTMs are thought to define functional chromatin states. Here we demonstrate MARCC, an antibody-free method for interrogating epigenetic states. At the heart of the workflow are recombinant chromatin reader domains, which target distinct chromatin states with combinatorial PTM patterns. Utilizing combinatorial histone peptide microarray, we showed three reader domains displayed greater specificity towards combinatorial PTM patterns than corresponding commercial antibodies. Such specific recognitions were employed to develop MARCC. We successfully applied MARCC to capture unique chromatin states and quantitatively profiled inter-connections between nucleosomal histone PTMs. A newly identified signature that harbored H3K4me0, H3K9me2/3, H3K79me0 and H4K20me2/3 within the same nucleosome was enriched in heterochromatin, as revealed by the associated DNA. Our results suggest the broad utility of recombinant reader domains as an enrichment tool specific to combinatorial PTM patterns. The reader affinity platform is compatible with downstream analyses to investigate the physical co-existence of nucleosomal PTM states associated with specific genomic loci. Collectively, the reader-based workflow will greatly facilitate our understanding of how distinct chromatin states and reader domains function in gene regulatory mechanisms. This work was supported by National Institutes of Health (Grant #GM059785-15/P250VA).

Bottom-up and middle-down proteomics have comparable accuracies in defining histone post-translational modification relative abundance and stoichiometry.

Histone proteins are key components of chromatin. Their N-terminal tails are enriched in combinatorial post-translational modifications (PTMs), which influence gene regulation, DNA repair, and chromosome condensation. Mass spectrometry (MS)-based middle-down proteomics has emerged as a technique to analyze co-occurring PTMs, as it allows for the characterization of intact histone tails (>50 aa) rather than short (<20 aa) peptides analyzed by bottom-up. However, a demonstration of its reliability is still lacking. We compared results obtained with the middle-down and the bottom-up strategy in calculating PTM relative abundance and stoichiometry. Since bottom-up was proven to have biases in peptide signal detection such as uneven ionization efficiency, we performed an external correction using a synthetic peptide library with known peptide relative abundance. Corrected bottom-up data were used as reference. Calculated abundances of single PTMs showed similar deviations from the reference when comparing middle-down and uncorrected bottom-up results. Moreover, we show that the two strategies provided similar performance in defining accurate PTM stoichiometry. Collectively, we evidenced that the middle-down strategy is at least equally reliable to bottom-up in quantifying histone PTMs.

Intrinsically disordered proteins in cellular signalling and regulation.

Intrinsically disordered proteins (IDPs) are important components of the cellular signalling machinery, allowing the same polypeptide to undertake different interactions with different consequences. IDPs are subject to combinatorial post-translational modifications and alternative splicing, adding complexity to regulatory networks and providing a mechanism for tissue-specific signalling. These proteins participate in the assembly of signalling complexes and in the dynamic self-assembly of membrane-less nuclear and cytoplasmic organelles. Experimental, computational and bioinformatic analyses combine to identify and characterize disordered regions of proteins, leading to a greater appreciation of their widespread roles in biological processes.

In-line separation by capillary electrophoresis prior to analysis by top-down mass spectrometry enables sensitive characterization of protein complexes.

Intact protein analysis via top-down mass spectrometry (MS) provides a bird’s eye view over the protein complexes and complex protein mixtures with the unique capability of characterizing protein variants, splice isoforms, and combinatorial post-translational modifications (PTMs). Here we applied capillary electrophoresis (CE) through a sheathless CE–electrospray ionization interface coupled to an LTQ Velos Orbitrap Elite mass spectrometer to analyze the Dam1 complex from Saccharomyces cerevisiae. We achieved a 100-fold increase in sensitivity compared to a reversed-phase liquid chromatography coupled MS analysis of recombinant Dam1 complex with a total loading of 2.5 ng (12 amol). N-terminal processing forms of individual subunits of the Dam1 complex were observed as well as their phosphorylation stoichiometry upon Mps1p kinase treatment.

Sheathless capillary electrophoresis-tandem mass spectrometry for top-down characterization of Pyrococcus furiosus proteins on a proteome scale.

Intact protein analysis via top-down mass spectrometry (MS) provides the unique capability of fully characterizing protein isoforms and combinatorial post-translational modifications (PTMs) compared to the bottom-up MS approach. Front-end protein separation poses a challenge for analyzing complex mixtures of intact proteins on a proteomic scale. Here we applied capillary electrophoresis (CE) through a sheathless capillary electrophoresis-electrospray ionization (CESI) interface coupled to an Orbitrap Elite mass spectrometer to profile the proteome from Pyrococcus furiosus. CESI-top-down MS analysis of Pyrococcus furiosus cell lysate identified 134 proteins and 291 proteoforms with a total sample consumption of 270 ng in 120 min of total analysis time. Truncations and various PTMs were detected, including acetylation, disulfide bonds, oxidation, glycosylation, and hypusine. This is the largest scale analysis of intact proteins by CE-top-down MS to date.

ChIP-less analysis of chromatin states.

BackgroundHistone post-translational modifications (PTMs) are key epigenetic regulators in chromatin-based processes. Increasing evidence suggests that vast combinations of PTMs exist within chromatin histones. These complex patterns, rather than individual PTMs, are thought to define functional chromatin states. However, the ability to interrogate combinatorial histone PTM patterns at the nucleosome level has been limited by the lack of direct molecular tools.ResultsHere we demonstrate an efficient, quantitative, antibody-free, chromatin immunoprecipitation-less (ChIP-less) method for interrogating diverse epigenetic states. At the heart of the workflow are recombinant chromatin reader domains, which target distinct chromatin states with combinatorial PTM patterns. Utilizing a newly designed combinatorial histone peptide microarray, we showed that three reader domains (ATRX-ADD, ING2-PHD and AIRE-PHD) displayed greater specificity towards combinatorial PTM patterns than corresponding commercial histone antibodies. Such specific recognitions were employed to develop a chromatin reader-based affinity enrichment platform (matrix-assisted reader chromatin capture, or MARCC). We successfully applied the reader-based platform to capture unique chromatin states, which were quantitatively profiled by mass spectrometry to reveal interconnections between nucleosomal histone PTMs. Specifically, a highly enriched signature that harbored H3K4me0, H3K9me2/3, H3K79me0 and H4K20me2/3 within the same nucleosome was identified from chromatin enriched by ATRX-ADD. This newly reported PTM combination was enriched in heterochromatin, as revealed by the associated DNA.ConclusionsOur results suggest the broad utility of recombinant reader domains as an enrichment tool specific to combinatorial PTM patterns, which are difficult to probe directly by antibody-based approaches. The reader affinity platform is compatible with several downstream analyses to investigate the physical coexistence of nucleosomal PTM states associated with specific genomic loci. Collectively, the reader-based workflow will greatly facilitate our understanding of how distinct chromatin states and reader domains function in gene regulatory mechanisms.

A quantitative atlas of histone modification signatures from human cancer cells

BackgroundAn integral component of cancer biology is the understanding of molecular properties uniquely distinguishing one cancer type from another. One class of such properties is histone post-translational modifications (PTMs). Many histone PTMs are linked to the same diverse nuclear functions implicated in cancer development, including transcriptional activation and epigenetic regulation, which are often indirectly assayed with standard genomic technologies. Thus, there is a need for a comprehensive and quantitative profiling of cancer lines focused on their chromatin modification states.ResultsTo complement genomic expression profiles of cancer lines, we report the proteomic classification of 24 different lines, the majority of which are cancer cells, by quantifying the abundances of a large panel of single and combinatorial histone H3 and H4 PTMs, and histone variants. Concurrent to the proteomic analysis, we performed transcriptomic analysis on histone modifying enzyme abundances as a proxy for quantifying their activity levels. While the transcriptomic and proteomic results were generally consistent in terms of predicting histone PTM abundance from enzyme abundances, several PTMs were regulated independently of the modifying enzyme expression. In addition, combinatorial PTMs containing H3K27 methylation were especially enriched in breast cell lines. Knockdown of the predominant H3K27 methyltransferase, enhancer of zeste 2 (EZH2), in a mouse mammary xenograft model significantly reduced tumor burden in these animals and demonstrated the predictive utility of proteomic techniques.ConclusionsOur proteomic and genomic characterizations of the histone modification states provide a resource for future investigations of the epigenetic and non-epigenetic determinants for classifying and analyzing cancer cells.

Understanding the combinatorial post‐translational modifications associated with histone H3 methylation in yeast

Histone post‐translational modifications (PTMs) recruit effector molecules to chromatin, which in‐turn regulate DNA‐templated processes including replication, gene expression, and DNA repair. Many recent reports have highlighted the importance of combinatorial PTMs, on one or more histone tail, for coordinating the binding of protein factors. However, the patterns of PTMs that might exist within a nucleosome are still not well understood. Lysine methylation on histone H3 is known to recruit both lysine acetyltransferases and deacetylases to chromatin. In this study, we examined what overlap exists between lysine methylation and acetylation on all four histones within a single nucleosome in budding yeast. Variant combinations of modifications found suggest recruitment of effector molecules is dictated by interaction with more than one histone within a nucleosome.