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. 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. Chromatin is a dynamic fiber mainly composed of DNA and histone proteins. Although DNA stores the genomic information of the organism, histones and their post-translational modifications (PTMs) are responsible for maintaining the chromatin structure. Histone PTMs play a fundamental role in transcriptional regulation and epigenetics, as they contribute in recruiting enzymes involved in chromatin remodeling and gene activity, and can be inherited through the cell cycle thereby maintaining a memory of gene expression profiles (1.Kouzarides T. Chromatin modifications and their function.Cell. 2007; 128: 693-705Abstract Full Text Full Text PDF PubMed Scopus (8067) Google Scholar). Histone PTMs have been extensively studied, and a large number of histone readers/writers/erasers have been characterized (2.Bannister A.J. Kouzarides T. Regulation of chromatin by histone modifications.Cell Res. 2011; 21: 381-395Crossref PubMed Scopus (3458) Google Scholar). It has been known for about 20 years now that combinations of histone modifications can affect histone-enzyme interaction, where nearby or distant PTMs interdependently recruit or release a given enzyme. This phenomenon is described as PTM crosstalk (3.Fischle W. Wang Y. Allis C.D. Histone and chromatin cross-talk.Current Opinion Cell Biol. 2003; 15: 172-183Crossref PubMed Scopus (985) Google Scholar, 4.Lee J.S. Smith E. Shilatifard A. The language of histone crosstalk.Cell. 2010; 142: 682-685Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). Originally, crosstalk is the disturbance caused by the electric or magnetic fields of one telecommunication signal affecting a signal in an adjacent circuit. Thus, it defines something that generates interference to a functional mechanism. In chromatin biology, crosstalk can be used also in positive terms, as one single PTM might require the presence of a second distinct PTM within the same protein in order to mediate a biological function. This phenomenon is typically described as positive crosstalk (5.Hunter T. The age of crosstalk: phosphorylation, ubiquitination, and beyond.Mol. Cell. 2007; 28: 730-738Abstract Full Text Full Text PDF PubMed Scopus (682) Google Scholar). For instance, the combinatorial histone mark H3S10phK14ac within the p21 (CDKN1A) gene activates transcription, which does not occur when only one of the two PTMs are present (6.Simboeck E. Sawicka A. Zupkovitz G. Senese S. Winter S. Dequiedt F. Ogris E. Di Croce L. Chiocca S. Seiser C. A phosphorylation switch regulates the transcriptional activation of cell cycle regulator p21 by histone deacetylase inhibitors.J. Biol. Chem. 2010; 285: 41062-41073Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), implying that the two modifications need to be deposited in a coordinated manner. Also, the histone lysine demethylase PHF8 has its highest binding efficiency to the nucleosome when the three marks H3K4me3K9acK14ac are present (7.Vermeulen M. Eberl H.C. Matarese F. Marks H. Denissov S. Butter F. Lee K.K. Olsen J.V. Hyman A.A. Stunnenberg H.G. Mann M. Quantitative interaction proteomics and genome-wide profiling of epigenetic histone marks and their readers.Cell. 2010; 142: 967-980Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar). On the other hand, negative crosstalk involves two PTMs that are mutually exclusive, either in the same amino acid residue or on two different residues. For instance, the protein HP1 recognizes H3K9me2/me3 and it spreads this mark along chromatin to compact it, but it releases the binding in case H3S10 is phosphorylated (8.Hirota T. Lipp J.J. Toh B.H. Peters J.M. Histone H3 serine 10 phosphorylation by Aurora B causes HP1 dissociation from heterochromatin.Nature. 2005; 438: 1176-1180Crossref PubMed Scopus (519) Google Scholar). Also, acetylation of H3K4 was found to inhibit the binding of the protein spChp1 to H3K9me2/me3 in S. pombe (9.Xhemalce B. Kouzarides T. A chromodomain switch mediated by histone H3 Lys 4 acetylation regulates heterochromatin assembly.Genes Development. 2010; 24: 647-652Crossref PubMed Scopus (74) Google Scholar). The Polycomb Repressive Complex (PRC) group proteins are involved in histone modifications and PRC2, involved in histone H3K27 methylation, was recently found to crosstalk with the other major histone H3 methyltransferases G9a/GLP, responsible for H3K9 methylation (10.Mozzetta C. Pontis J. Fritsch L. Robin P. Portoso M. Proux C. Margueron R. Ait-Si-Ali S. The histone H3 lysine 9 methyltransferases G9a and GLP regulate polycomb repressive complex 2-mediated gene silencing.Mol. Cell. 2014; 53: 277-289Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Moreover, PRC1 recognizes H3K27me3 with its subunit CBX and catalyze ubiquitination of H2AK119 (11.Margueron R. Reinberg D. The Polycomb complex PRC2 and its mark in life.Nature. 2011; 469: 343-349Crossref PubMed Scopus (2244) Google Scholar). The epigenetic machinery is also characterized by interconnection between numerous components; for instance, the silencing of the inactive X chromosome in female organisms is mediated by interacting DNA methylation, noncoding RNA and histone PTMs (12.Heard E. Delving into the diversity of facultative heterochromatin: the epigenetics of the inactive X chromosome.Current Opinion Genetics Development. 2005; 15: 482-489Crossref PubMed Scopus (164) Google Scholar). Similarly, DNA methylation is required in embryonic stem cells (ESCs) for the deposition of H3K9me3 and it antagonizes deposition of H3K27me3 (13.Murphy P.J. Cipriany B.R. Wallin C.B. Ju C.Y. Szeto K. Hagarman J.A. Benitez J.J. Craighead H.G. Soloway P.D. Single-molecule analysis of combinatorial epigenomic states in normal and tumor cells.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 7772-7777Crossref PubMed Scopus (68) Google Scholar). Because of this, a number of engineered cell lines were developed with mESC genomes knocked out for crucial components of these complexes in order to investigate their role in embryonic cell development. Specifically, mESCs were knocked out in Suppressor of Zeste 12 (Suz12−/−) to unravel its role in PCR2, revealing that this protein is essential for the complex activity and affects regulation of proliferation and embryogenesis (14.Pasini D. Bracken A.P. Jensen M.R. Lazzerini Denchi E. Helin K. Suz12 is essential for mouse development and for EZH2 histone methyltransferase activity.EMBO J. 2004; 23: 4061-4071Crossref PubMed Scopus (672) Google Scholar). PRC1 was deprived of the protein Ring1A and its homolog Ring1B (Ring1A/B−/−), showing that these proteins play a central role in genome-wide ubiquitylation of histone H2A, most likely functioning as H2A-specific E3 ligases (15.de Napoles M. Mermoud J.E. Wakao R. Tang Y.A. Endoh M. Appanah R. Nesterova T.B. Silva J. Otte A.P. Vidal M. Koseki H. Brockdorff N. Polycomb group proteins Ring1A/B link ubiquitylation of histone H2A to heritable gene silencing and X inactivation.Developmental Cell. 2004; 7: 663-676Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar). In particular, this study focuses in proving that the double knockout cells cannot maintain ubiquitination on histone H2A on the inactive X chromosome. Finally, mESCs were also knocked out in three fundamental DNA methyltransferases, Dnmt1, Dnmt3a, and Dnmt3b (Dnmt TKO) (16.Tsumura A. Hayakawa T. Kumaki Y. Takebayashi S. Sakaue M. Matsuoka C. Shimotohno K. Ishikawa F. Li E. Ueda H.R. Nakayama J. Okano M. Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b.Genes Cells. 2006; 11: 805-814Crossref PubMed Scopus (410) Google Scholar). These cells could maintain stem cells properties and proliferation in the complete absence of CpG methylation, including deposition of histone heterochromatic marks such as H3K9me3. Quantitative proteomics analysis by mass spectrometry is a powerful tool to determine the existence and function of crosstalk patterns in proteins, as it can be used to determine the abundance of specific PTM combinations and thus potentially reveal the underlying rules for the deposition of these coexisting marks. Hence, deciphering crosstalk between histone PTMs might reveal the mechanisms underlying the biological processes responsible for the formation and maintenance of chromatin states. Accurate characterization of these mechanisms is achievable by combining large-scale studies of the wild-type cell system and a sufficiently large number of perturbed states where global changes of the PTM landscape are induced. Such a study has not been carried out to date. Mass spectrometry (MS) is extensively used to study histone PTMs (17.Sidoli S. Cheng L. Jensen O.N. Proteomics in chromatin biology and epigenetics: Elucidation of post-translational modifications of histone proteins by mass spectrometry.J. Proteomics. 2012; 75: 3419-3433Crossref PubMed Scopus (109) Google Scholar), as it is currently the most suitable technology to characterize proteins containing unknown modifications (18.Walther T.C. Mann M. Mass spectrometry-based proteomics in cell biology.J. Cell Biol. 2010; 190: 491-500Crossref PubMed Scopus (307) Google Scholar). We recently optimized a middle-down proteomics workflow to quantify coexisting histone H3 marks from mouse stem cells (19.Jung H.R. Sidoli S. Haldbo S. Sprenger R.R. Schwammle V. Pasini D. Helin K. Jensen O.N. Precision mapping of coexisting modifications in histone H3 tails from embryonic stem cells by ETD-MS/MS.Anal. Chem. 2013; 85: 8232-8239Crossref PubMed Scopus (63) Google Scholar) using high mass resolution MS equipped with electron transfer dissociation (ETD) MS/MS capability. ETD provides efficient fragmentation of histone N-terminal tails (20.Mikesh L.M. Ueberheide B. Chi A. Coon J.J. Syka J.E. Shabanowitz J. Hunt D.F. The utility of ETD mass spectrometry in proteomic analysis.Biochim. Biophys. Acta. 2006; 1764: 1811-1822Crossref PubMed Scopus (464) Google Scholar), which leads to precise mapping of PTMs within the protein sequence. We also developed bioinformatics tools to process MS/MS data from hypermodified peptides. We subsequently proposed a simple quantifier approach to estimate the crosstalk between two histone PTMs; by comparing the observed and theoretical frequency of two PTMs to coexist on the same histone tail the tool can identify conserved interplays between binary marks in different cell cultures (21.Schwammle V. Aspalter C.M. Sidoli S. Jensen O.N. Large scale analysis of co-existing post-translational modifications in histone tails reveals global fine structure of cross-talk.Mol. Cell. Proteomics. 2014; 13: 1855-1865Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The interplay was calculated by predicting the frequency of two PTMs given their individual relative abundance as calculated from peptide ion intensities. Using our in-house developed data repository, CrossTalkDB (21.Schwammle V. Aspalter C.M. Sidoli S. Jensen O.N. Large scale analysis of co-existing post-translational modifications in histone tails reveals global fine structure of cross-talk.Mol. Cell. Proteomics. 2014; 13: 1855-1865Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), we can now analyze large data sets of extensively modified proteins and reveal PTM interdependencies and crosstalk. Here, we report the application of an improved analytical and computational workflow for consistent systems level analysis of histone H3 PTMs for determination of the impact of epigenetic perturbations on coexisting PTMs and their interplay and crosstalk in histone H3 tails. We analyzed H3 tails isolated from mouse embryonic stem cells (mESCs, strain E14) and mESCs that were perturbed in three different components of the epigenetic reprogramming machinery, namely Suz12, Ring1A and Ring1B, and the three DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. We demonstrate that our middle-down proteomics platform combined with advanced computational and statistical data analysis reveal histone PTM crosstalk at the systems level, including rarely studied marks such as arginine methylation. In addition, we performed a systems biology analysis by integrating RNAseq data with our histone-derived proteomics data to study whether observed histone PTM abundance correlated with the cognate enzyme abundances. Cells were grown and prepared as previously described for mouse ESCs wild type, Suz12−/− (14.Pasini D. Bracken A.P. Jensen M.R. Lazzerini Denchi E. Helin K. Suz12 is essential for mouse development and for EZH2 histone methyltransferase activity.EMBO J. 2004; 23: 4061-4071Crossref PubMed Scopus (672) Google Scholar), Ring1A/B−/− (15.de Napoles M. Mermoud J.E. Wakao R. Tang Y.A. Endoh M. Appanah R. Nesterova T.B. Silva J. Otte A.P. Vidal M. Koseki H. Brockdorff N. Polycomb group proteins Ring1A/B link ubiquitylation of histone H2A to heritable gene silencing and X inactivation.Developmental Cell. 2004; 7: 663-676Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar), and Dnmt triple knockout (16.Tsumura A. Hayakawa T. Kumaki Y. Takebayashi S. Sakaue M. Matsuoka C. Shimotohno K. Ishikawa F. Li E. Ueda H.R. Nakayama J. Okano M. Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b.Genes Cells. 2006; 11: 805-814Crossref PubMed Scopus (410) Google Scholar) (strain E14). Wild type and Suz12−/− morphology was extensively described in (22.Pasini D. Bracken A.P. Hansen J.B. Capillo M. Helin K. The polycomb group protein Suz12 is required for embryonic stem cell differentiation.Mol. Cell. Biol. 2007; 27: 3769-3779Crossref PubMed Scopus (543) Google Scholar). Ring1A/1B−/− were donated by Vidal and Koseki and thoroughly described in (23.Endoh M. Endo T.A. Endoh T. Fujimura Y. Ohara O. Toyoda T. Otte A.P. Okano M. Brockdorff N. Vidal M. Koseki H. Polycomb group proteins Ring1A/B are functionally linked to the core transcriptional regulatory circuitry to maintain ES cell identity.Development. 2008; 135: 1513-1524Crossref PubMed Scopus (234) Google Scholar). Dnmt TKO cells were taken from ref. (24.Sakaue M. Ohta H. Kumaki Y. Oda M. Sakaide Y. Matsuoka C. Yamagiwa A. Niwa H. Wakayama T. Okano M. DNA methylation is dispensable for the growth and survival of the extraembryonic lineages.Curr. Biol. 2010; 20: 1452-1457Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Histones were purified using a common protocol with minor modifications (25.Vaidyanathan G. Zalutsky M.R. Synthesis of N-succinimidyl 4-guanidinomethyl-3-[*I]iodobenzoate: a radio-iodination agent for labeling internalizing proteins and peptides.Nat. Protoc. 2007; 2: 282-286Crossref PubMed Scopus (34) Google Scholar). Briefly, cell membrane was disrupted by 30 min incubation with hypotonic solution (10 mm TrisHCl pH 8.0, 1 mm KCl, 1.5 mm MgCl2, and 1 mm DTT) and nuclei were precipitated by centrifugation at (10,000 × g for 10 min). Pellet was resuspended in 0.2 m H2SO4 and incubated for 30 mins at room temperature. After centrifugation (10 min at 16,000 × g) supernatant was recovered and trichloroacetic acid (TCA) was added to the solution to a final concentration of 33%. After 30 min mix histones were spun down by centrifugation (16,000 × g for 10 min at 4 degrees). Pellet was washed twice with cold acetone. Samples were evaporated in speedvac, resuspended in 40 μl of NH4HCO3 100 mm and digested overnight with GluC with a ratio of 1:50 enzyme/sample. Digestion was interrupted by adding 1 μl of 100% trifluoroacetic acid (TFA). Digested histones were analyzed as previously described (19.Jung H.R. Sidoli S. Haldbo S. Sprenger R.R. Schwammle V. Pasini D. Helin K. Jensen O.N. Precision mapping of coexisting modifications in histone H3 tails from embryonic stem cells by ETD-MS/MS.Anal. Chem. 2013; 85: 8232-8239Crossref PubMed Scopus (63) Google Scholar) with few modifications. Briefly, histones were loaded and separated in a Dionex nanoLC Ultimate 3000 (Thermo Scientific). The two column system consisted on a 5 cm pre-column (100 μm ID) packed with C18 bulk material (ReproSil, Pur C18AQ 5 μm; Dr. Maisch) and a 22 cm analytical column with pulled needle (75 μm ID) packed with Polycat A resin (PolyLC, Columbia, MD, 3 μm particles, 1500 Å). Loading buffer was 0.1% formic acid. Buffer A was 75% acetonitrile, 20 mm propionic acid (Fluka), adjusted to pH 6.0 using ammonium hydroxide (Sigma-Aldrich), and solvent B was 25% acetonitrile adjusted to pH 2.5 with formic acid. Histones were run at least in four replicates at a flowrate of 250 nL/min, with a gradient of 5 min 100% solvent A, followed by 55 to 85% solvent B in 150 min and 85–100% in 10 min for column washing. For detection an LTQ-Orbitrap Velos with ETD source (Thermo Scientific) was coupled online with the nanoLC. Nanoelectrospray (Proxeon) was used with a spray voltage of 2.2 kV. No sheath, sweep, and auxiliary gases were used, and capillary temperature was set to 270 °C. Acquisition method was set for not using dynamic exclusion. Acquisition was performed in the Orbitrap for both precursors and products, with a resolution of 60,000 (full-width at half-height) for MS and 30,000 for MS/MS. Precursor charge state 1+, 2+ and 3+ were excluded. Isolation width was set at 2 m/z. The six most intense ions were isolated for fragmentation using ETD with an activation Q value of 0.25, activation time of 90 ms, and supplementary activation. m/z window was set at 450–750 to include only charge states 8–10. Spectra were deconvoluted with Xtract using the following parameters: S/N threshold 0; resolution at 400 m/z 30,000 and monoisotopic mass only true. Database search was performed with Proteome Discoverer 1.4.0.288 (Thermo Scientific). Mascot (v2.5, Matrix Science) was chosen as search engine, with the following search parameters: MS mass tolerance 2.2 Da; MS/MS tolerance 0.01 Da; enzyme GluC with 0 missed cleavages allowed, database was manually curated by filtering for histone proteins from mouse in Uniprot (downloaded October 2012, 43 entries). Variable modifications were: mono- and dimethylation (KR), trimethylation (K) and acetylation (K). Raw files and annotated spectra are available at the ProteomeXchange database (Accession: PXD002560). CSV result files from Mascot were imported and processed in 'isoScale slim′ by using a tolerance of 30 ppm. This tool includes filtering of ambiguously assigned PTMs (only PTMs with at least one ion before and after the assigned modification site were accepted) and quantification (principle of quantification described in Sidoli et al., (26.Sidoli S. Schwammle V. Ruminowicz C. Hansen T.A. Wu X. Helin K. Jensen O.N. Middle-down hybrid chromatography/tandem mass spectrometry workflow for characterization of combinatorial post-translational modifications in histones.Proteomics. 2014; 14: 2200-2211Crossref PubMed Scopus (67) Google Scholar)). isoScale slim is available at http://middle-down.github.io/Software. isoScale slim files can be directly uploaded to the new version of CrossTalkDB. The server summarizes peptides with identical sequences and PTM codes. Abundances are calculated from the summed intensities by normalizing them to the total of a protein family within the data set. The overall aim of this work is to reveal conserved cross-talk patterns of histone PTMs independently of PTM abundance. Therefore, we performed intact histone tails analysis across differently mutated mESCs, which were previously characterized as with altered epigenome and phenotype. As control, we used the mESCs wild type in proliferation. Peptides with similar sequences (Levenshtein distance smaller than 3) are considered members of the same protein family. This normalization allows quantification of the total of e.g. histone H3 variants while preserving their relative abundance and revealing the statistical properties of individual histone variants. Cofrequencies of binary marks are calculated by summing the abundances of peptides containing the respective marks. Interplay values result from cofrequencies according to I=log(f12f1f2) where f12,f1,f2 denote the relative abundance of the binary mark, and both single marks 1 and 2, respectively. This formula yields positive/negative values when the double mark is over-/under-represented in the data set (21.Schwammle V. Aspalter C.M. Sidoli S. Jensen O.N. Large scale analysis of co-existing post-translational modifications in histone tails reveals global fine structure of cross-talk.Mol. Cell. Proteomics. 2014; 13: 1855-1865Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Conservation of interplay values was assessed by a t test. p values were corrected for multiple testing according to Benjamini-Hochberg. Comparison of the results to randomized data sets provides information about conservation of the detected patterns. The Ring1A/B−/− data set was taken and two stages of randomization were introduced. ESRand set: Shuffling of intensity values. ESRandPTM set: Shuffling of PTM codes where only lysine modifications were taken into account. The distribution for the number of PTMs per peptide was maintained. Unrealistic peptides with more than one PTM per residue were removed. Both artificial data sets are available at CrossTalkDB. Transcriptomics data sets were downloaded from (27.Riising E.M. Comet I. Leblanc B. Wu X. Johansen J.V. Helin K. Gene silencing triggers polycomb repressive complex 2 recruitment to CpG islands genome wide.Mol. Cell. 2014; 55: 347-360Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar) (wild type, Suz12−/− and Ring1A/B−/−), and GEO data set GSE61457, which is described in (28.Li Z. Dai H. Martos S.N. Xu B. Gao Y. Li T. Zhu G. Schones D.E. Wang Z. Distinct roles of DNMT1-dependent and DNMT1-independent methylation patterns in the genome of mouse embryonic stem cells.Genome Biol. 2015; 16: 115Crossref PubMed Scopus (57) Google Scholar). Expression profiles over the all four cell lines were merged with abundances of single and binary histone marks and clustered by the fuzzy c-means algorithm (29.Bezdek J.C. Pattern recognition with fuzzy objective function algorithms. Plenum Press, New York1981Crossref Google Scholar). Both parameters of the clustering methods were determined according to (30.Schwammle V. Jensen O.N. A simple and fast method to determine the parameters for fuzzy c-means cluster analysis.Bioinformatics. 2010; 26: 2841-2848Crossref PubMed Scopus (135) Google Scholar). Results were filtered for a minimum membership value of 0.5 to avoid poor assignments. For each of the four clusters, known enzymes from Histome (31.Khare S.P. Habib F. Sharma R. Gadewal N. Gupta S. Galande S. HIstome–a relational knowledgebase of human histone proteins and histone modifying enzymes.Nucleic Acids Res. 2012; 40: D337-D342Crossref PubMed Scopus (102) Google Scholar) and ref (32.Zhao Y. Garcia B.A. Comprehensive Catalog of Currently Documented Histone Modifications.Cold Spring Harb. Perspect. Biol. 2015; 7: a025064Crossref PubMed Scopus (232) Google Scholar). were selected. Moreover, gene names of each cluster were submitted to DAVID (33.Huang da W. Sherman B.T. Lempicki R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.Nat. Protoc. 2009; 4: 44-57Crossref PubMed Scopus (25496) Google Scholar) using the R library clusterProfiler (34.Yu G. Wang L.G. Han Y. He Q.Y. clusterProfiler: an R package for comparing biological themes among gene clusters.OMICS. 2012; 16: 284-287Crossref PubMed Scopus (12094) Google Scholar) for standard analysis (p value cutoff 0.05). GO terms were simplified using the simplify function of clusterProfiler. We set out to detect and reveal novel features of combinatorial histone H3 PTM regulation and crosstalk by studying four different mESC cell lines, three of which were perturbed in the epigenetic machinery. Perturbations of chromatin-modifying enzymes often lead to large-scale changes of chromatin features and markers, such as distinct histone PTMs and DNA methylation. We focused our study on histone H3 which carry many distinct PTMs, including coexisting and combinatorial functional PTMs. Thus, histone H3 is an excellent model for systematic investigations of PTM crosstalk and interplay in proteins. In order to investigate the global changes of the PTM landscape in mESCs, we selected four model systems, i.e. wild type mESC, Suz12−/− (PRC2 perturbation), Ring1A/B−/− (PRC1 perturbation), and Dnmt TKO (DNA methylation perturbation). Specifically, PRC2 and PRC1 are involved in gene repression by directly catalyzing H3K27me2/me3 and H2AK119ub deposition (29.Bezdek J.C. Pattern recognition with fuzzy objective function algorithms. Plenum Press, New York1981Crossref Google Scholar), whereas DNA methylation is also involved in gene repression and for the deposition of H3K9me3 (13.Murphy P.J. Cipriany B.R. Wallin C.B. Ju C.Y. Szeto K. Hagarman J.A. Benitez J.J. Craighead H.G. Soloway P.D. Single-molecule analysis of combinatorial epigenomic states in normal and tumor cells.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 7772-7777Crossref PubMed Scopus (68) Google Scholar). As previously reported, Suz12−/− mESCs survive only up to 10.5 days post coitus, although they are much smaller than wild type ESCs (14.Pasini D. Bracken A.P. Jensen M.R. Lazzerini Denchi E. Helin K. Suz12 is essential for mouse development and for EZH2 histone methyltransferase activity.EMBO J. 2004; 23: 4061-4071Crossref PubMed Scopus (672) Google Scholar); Ring1A/B−/− has defects in the maintenance of ESCs identity, as the silencing of genes that govern differentiation of ESCs is hampered (35.Lapthanasupkul P. Feng J. Mantesso A. Takada-Horisawa Y. Vidal M. Koseki H. Wang L. An Z. Miletich I. Sharpe P.T. Ring1a/b polycomb proteins regulate the mesenchymal stem cell niche in continu
A fair scheduling mechanism called distributed elastic round robin (DERR) is proposed in this letter for IEEE 802.11 wireless LANs operated in a distributed manner. To quantify the fairness, we not only derive its fairness bound, but also observe the fairness through ratios of throughput and weight using a simulation approach. By numerical comparisons among DERR, distributed deficit round robin (DDRR), and IEEE 802.11e, we demonstrate that DERR outperforms the other two mechanisms in performance and fairness.
A fair scheduling mechanism called distributed elastic round robin (DERR) is proposed in this letter for IEEE 802.11 wireless LANs operated in a distributed manner. To quantify the fairness, we not only derive its fairness bound, but also observe the fairness through ratios of throughput and weight using a simulation approach. By numerical comparisons among DERR, distributed deficit round robin (DDRR), and IEEE 802.11e, we demonstrate that DERR outperforms the other two mechanisms in performance and fairness.
In this paper, a packet scheduling algorithm called distributed elastic round robin (DERR) suitable for operation in a distributed environment is proposed to provide fair scheduling for the IEEE 802.11 wireless LAN. By simulations, we show that DERR possesses slightly improved performance in throughput and delay and exhibits better fairness than distributed deficit round robin (DDRR), which is a previously proposed fair scheduling algorithm in the literature.
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