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Systems Level Analysis of Histone H3 Post-Translational Modifications (Ptms) Reveals Features of PTM Crosstalk in Chromatin Regulation University of Southern Denmark Systems Level Analysis of Histone H3 Post-translational Modifications (PTMs) Reveals Features of PTM Crosstalk in Chromatin Regulation Schwämmle, Veit; Sidoli, Simone; Ruminowicz, Chrystian; Wu, Xudong; Lee, Chung-Fan; Helin, Kristian; Jensen, Ole Nørregaard Published in: Molecular and Cellular Proteomics DOI: 10.1074/mcp.M115.054460 Publication date: 2016 Document version: Final published version Citation for pulished version (APA): Schwämmle, V., Sidoli, S., Ruminowicz, C., Wu, X., Lee, C-F., Helin, K., & Jensen, O. N. (2016). Systems Level Analysis of Histone H3 Post-translational Modifications (PTMs) Reveals Features of PTM Crosstalk in Chromatin Regulation. Molecular and Cellular Proteomics, 15(8), 2715-2729. https://doi.org/10.1074/mcp.M115.054460 Go to publication entry in University of Southern Denmark's Research Portal Terms of use This work is brought to you by the University of Southern Denmark. Unless otherwise specified it has been shared according to the terms for self-archiving. If no other license is stated, these terms apply: • You may download this work for personal use only. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying this open access version If you believe that this document breaches copyright please contact us providing details and we will investigate your claim. Please direct all enquiries to [email protected] Download date: 01. Oct. 2021 crossmark Research © 2016 by The American Society for Biochemistry and Molecular Biology, Inc. This paper is available on line at http://www.mcponline.org Systems Level Analysis of Histone H3 Post- translational Modifications (PTMs) Reveals Features of PTM Crosstalk in Chromatin Regulation*□S Veit Schwa¨mmle‡ʈ**, Simone Sidoli‡§§**, Chrystian Ruminowicz‡, Xudong Wu§‡‡, Chung-Fan Lee§, Kristian Helin§¶, and Ole N. Jensen‡ Histones are abundant chromatin constituents carrying vealed correlations between PTM and enzyme levels. We numerous post-translational modifications (PTMs). Such conclude that middle-down proteomics is a powerful tool PTMs mediate a variety of biological functions, including to determine conserved or dynamic interdependencies recruitment of enzymatic readers, writers and erasers between histone marks, which paves the way for detailed that modulate DNA replication, transcription and repair. investigations of the histone code. Histone H3 PTM data Individual histone molecules contain multiple coexisting is publicly available in the CrossTalkDB repository at PTMs, some of which exhibit crosstalk, i.e. coordinated or http://crosstalkdb.bmb.sdu.dk. Molecular & Cellular mutually exclusive activities. Here, we present an inte- Proteomics 15: 10.1074/mcp.M115.054460, 2715–2729, grated experimental and computational systems level 2016. molecular characterization of histone PTMs and PTM crosstalk. Using wild type and engineered mouse embry- onic stem cells (mESCs) knocked out in components of the Polycomb Repressive Complex 2 (PRC2, Suz12؊/؊), Chromatin is a dynamic fiber mainly composed of DNA and -PRC1 (Ring1A/B؊/؊) and (Dnmt1/3a/3b؊/؊) we performed histone proteins. Although DNA stores the genomic informa comprehensive PTM analysis of histone H3 tails (50 aa) by tion of the organism, histones and their post-translational utilizing quantitative middle-down proteome analysis by modifications (PTMs) are responsible for maintaining the tandem mass spectrometry. We characterized combina- chromatin structure. Histone PTMs play a fundamental role in torial PTM features across the four mESC lines and then transcriptional regulation and epigenetics, as they contribute applied statistical data analysis to predict crosstalk be- in recruiting enzymes involved in chromatin remodeling and tween histone H3 PTMs. We detected an overrepresenta- gene activity, and can be inherited through the cell cycle tion of positive crosstalk (codependent marks) between thereby maintaining a memory of gene expression profiles (1). adjacent mono-methylated and acetylated marks, and Histone PTMs have been extensively studied, and a large negative crosstalk (mutually exclusive marks) among most of the seven characterized di- and tri-methylated number of histone readers/writers/erasers have been charac- lysine residues in the H3 tails. We report novel features of terized (2). It has been known for about 20 years now that PTM interplay involving hitherto poorly characterized ar- combinations of histone modifications can affect histone- ginine methylation and lysine methylation sites, including enzyme interaction, where nearby or distant PTMs interde- H3R2me, H3R8me and H3K37me. Integration of the H3 pendently recruit or release a given enzyme. This phenome- data with RNAseq data by coabundance clustering anal- non is described as PTM crosstalk (3, 4). Originally, crosstalk ysis of histone PTMs and histone modifying enzymes re- is the disturbance caused by the electric or magnetic fields of one telecommunication signal affecting a signal in an adjacent From the ‡Centre for Epigenetics and VILLUM Center for Bioana- circuit. Thus, it defines something that generates interference lytical Sciences, Department of Biochemistry and Molecular Biology, to a functional mechanism. In chromatin biology, crosstalk University of Southern Denmark, Campusvej 55, DK-5230 Odense M, can be used also in positive terms, as one single PTM might Denmark; §Biotech Research and Innovation Centre and Centre for Epigenetics, University of Copenhagen, DK-2200, Copenhagen, Den- require the presence of a second distinct PTM within the mark; ¶The Danish Stem Cell Centre (Danstem), University of Copen- same protein in order to mediate a biological function. This hagen, DK-2200, Copenhagen, Denmark phenomenon is typically described as positive crosstalk (5). Received August 7, 2015, and in revised form, June 13, 2016 For instance, the combinatorial histone mark H3S10phK14ac Published, MCP Papers in Press, June 14, 2016, DOI within the p21 (CDKN1A) gene activates transcription, which 10.1074/mcp.M115.054460 Author contributions: V.S., S.S., K.H., and O.N.J. designed re- does not occur when only one of the two PTMs are present search; V.S., S.S., X.W., and C.L. performed research; V.S., S.S., and (6), implying that the two modifications need to be deposited C.R. analyzed data; V.S., S.S., and O.N.J. wrote the paper. in a coordinated manner. Also, the histone lysine demethylase Molecular & Cellular Proteomics 15.8 2715 Conserved Crosstalk Patterns of Post-translational Histone Modifications PHF8 has its highest binding efficiency to the nucleosome perturbed states where global changes of the PTM landscape when the three marks H3K4me3K9acK14ac are present (7). are induced. Such a study has not been carried out to date. On the other hand, negative crosstalk involves two PTMs that Mass spectrometry (MS) is extensively used to study his- are mutually exclusive, either in the same amino acid residue tone PTMs (17), as it is currently the most suitable technology or on two different residues. For instance, the protein HP1 to characterize proteins containing unknown modifications recognizes H3K9me2/me3 and it spreads this mark along (18). We recently optimized a middle-down proteomics work- chromatin to compact it, but it releases the binding in case flow to quantify coexisting histone H3 marks from mouse H3S10 is phosphorylated (8). Also, acetylation of H3K4 was stem cells (19) using high mass resolution MS equipped with found to inhibit the binding of the protein spChp1 to electron transfer dissociation (ETD) MS/MS capability. ETD H3K9me2/me3 in S. pombe (9). provides efficient fragmentation of histone N-terminal tails The Polycomb Repressive Complex (PRC) group proteins (20), which leads to precise mapping of PTMs within the are involved in histone modifications and PRC2, involved in protein sequence. We also developed bioinformatics tools to histone H3K27 methylation, was recently found to crosstalk process MS/MS data from hypermodified peptides. We sub- with the other major histone H3 methyltransferases G9a/GLP, sequently proposed a simple quantifier approach to estimate responsible for H3K9 methylation (10). Moreover, PRC1 rec- the crosstalk between two histone PTMs; by comparing the ognizes H3K27me3 with its subunit CBX and catalyze ubiq- observed and theoretical frequency of two PTMs to coexist on uitination of H2AK119 (11). The epigenetic machinery is also the same histone tail the tool can identify conserved interplays characterized by interconnection between numerous compo- between binary marks in different cell cultures (21). The inter- nents; for instance, the silencing of the inactive X chromo- play was calculated by predicting the frequency of two PTMs some in female organisms is mediated by interacting DNA given their individual relative abundance as calculated from methylation, noncoding RNA and histone PTMs (12). Similarly, peptide ion intensities. Using our in-house developed data DNA methylation is required in embryonic stem cells (ESCs) repository, CrossTalkDB (21), we can now analyze large data for the deposition of H3K9me3 and it antagonizes deposition sets of extensively modified proteins and reveal PTM interde- of H3K27me3 (13). Because of this, a number of engineered pendencies and crosstalk. cell lines were developed with mESC genomes knocked out Here, we report the application of an improved analytical for crucial components of these complexes in order to inves-
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