Abstract
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.
Highlights
From the ‡Centre for Epigenetics and VILLUM Center for Bioanalytical Sciences, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark; §Biotech Research and Innovation Centre and Centre for Epigenetics, University of Copenhagen, DK-2200, Copenhagen, Denmark; ¶The Danish Stem Cell Centre (Danstem), University of Copenhagen, DK-2200, Copenhagen, Denmark
We set out to detect and reveal novel features of combinatorial histone H3 post-translational modifications (PTMs) regulation and crosstalk by studying four different mouse embryonic stem cells (mESCs) cell lines, three of which were perturbed in the epigenetic machinery
In order to investigate the global changes of the PTM landscape in mESCs, we selected four model systems, i.e. wild type mESC, Suz12Ϫ/Ϫ (PRC2 perturbation), Ring1A/BϪ/Ϫ (PRC1 perturbation), and Dnmt TKO (DNA methylation perturbation)
Summary
Histone PTMs have been extensively studied, and a large number of histone readers/writers/erasers have been characterized [2] It has been known for about 20 years that combinations of histone modifications can affect histoneenzyme interaction, where nearby or distant PTMs interdependently recruit or release a given enzyme. Crosstalk can be used 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. MESCs were knocked out in three fundamental DNA methyltransferases, Dnmt, Dnmt3a, and Dnmt3b (Dnmt TKO) [16] These cells could maintain stem cells properties and proliferation in the complete absence of CpG methylation, including deposition of histone heterochromatic marks such as H3K9me
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