SUZ12 is a core component of the polycomb repressive complex 2 (PRC2) and is required for the differentiation of mouse embryonic stem cells (ESCs). PRC2 is associated with transcriptional repression via methylation of H3 Lys-27. We applied quantitative mass spectrometry to investigate the effects of Suz12 deficiency on H3.2 and H3.3 from mouse ESCs. Using high mass accuracy MS combined with CID or electron transfer dissociation (ETD) tandem mass spectrometry, we identified a total of 81 unique modified peptides from H3.2 and H3.3 and assigned 46 modifications at 22 different positions, including distinct coexisting modifications. In certain cases, high mass accuracy LTQ-Orbitrap MS/MS allowed precise localization of near isobaric coexisting PTMs such as trimethylation and acetylation within individual peptides. ETD MS/MS facilitated sequencing and annotation of phosphorylated histone peptides. The combined use of ETD and CID MS/MS increased the total number of identified modified peptides. Comparative quantitative analysis of histones from wild type and Suz12-deficient ESCs using stable isotope labeling with amino acids in cell culture and LC-MS/MS revealed a dramatic reduction of H3K27me2 and H3K27me3 and an increase of H3K27ac, thereby uncovering an antagonistic methyl/acetyl switch at H3K27. The reduction in H3K27 methylation and increase in H3K27 acetylation was accompanied by H3K36 acetylation and methylation. Estimation of the global isoform percentage of unmodified and modified histone peptides (amino acids 27-40) showed the relative distribution of distinct coexisting histone marks. Our study revealed limitations of antibody-based Western blotting methods for detection of coexisting protein modifications and demonstrated the utility of quantitative tandem mass spectrometry for detailed analysis of the dynamics of coexisting post-translational modifications in proteins.
"The discovery of variants of histone H3 that are enriched in PTMs associated with either active or repressed chromatin has led to the notion that histone variant exchange facilitates plasticity at the nucleosome level (4). In mammals, histone H3 non-centromeric variants, H3.1, H3.2 and H3.3, differ in their chromatin deposition patterns and PTMs despite having a high degree of sequence similarity (4,5). H3.1 and H3.2, which are enriched in repressive chromatin marks, are predominantly expressed during S-Phase and deposited in DNA synthesis-coupled fashion during DNA replication and repair (replication-dependent, RD). "
[Show abstract][Hide abstract] ABSTRACT: The epigenome is defined as a type of information that can be transmitted independently of the DNA sequence, at the chromatin level, through post-translational modifications present on histone tails. Recent advances in the identification of histone 3 variants suggest a new model of information transmission through deposition of specific histone variants. To date, several non-centromeric histone 3 variants have been identified in mammals. Despite protein sequence similarity, specific deposition complexes have been characterized for both histone 3.1 (H3.1) and histone 3.3 (H3.3), whereas no deposition complex for histone 3.2 (H3.2) has been identified to date. Here, we identified human H3.2 partners by immunopurification of nuclear H3.2 complexes followed by mass spectrometry analysis. Further biochemical analyses highlighted two major complexes associated with H3.2, one containing chromatin associated factor-1 subunits and the other consisting of a subcomplex of mini chromosome maintenance helicases, together with Asf1. The purified complexes could associate with a DNA template in vitro.
Nucleic Acids Research 01/2014; 42(6). DOI:10.1093/nar/gkt1355 · 9.11 Impact Factor
"Results indicated that Suz12- deficient cells experience a dramatic reduction of H3K27me2 and H3K27me3 and an increase in H3K27ac, highlighting this residue as an acetyl/methyl switch. H3K27ac was accompanied by a corresponding increase in H3K36ac, a combination that had not been previously described in mammalian embryonic stem cells (Jung et al., 2010). Top-down MS analysis has been used to thoroughly characterize all canonical histones and a vast majority of their known variants. "
[Show abstract][Hide abstract] ABSTRACT: Histone proteins are dynamically modified to mediate a variety of cellular processes including gene transcription, DNA damage repair, and apoptosis. Regulation of these processes occurs through the recruitment of non-histone proteins to chromatin by specific combinations of histone post-translational modifications (PTMs). Mass spectrometry has emerged as an essential tool to discover and quantify histone PTMs both within and between samples in an unbiased manner. Developments in mass spectrometry that allow for characterization of large histone peptides or intact protein has made it possible to determine which modifications occur simultaneously on a single histone polypeptide. A variety of techniques from biochemistry, biophysics, and chemical biology have been employed to determine the biological relevance of discovered combinatorial codes. This review first describes advancements in the field of mass spectrometry that have facilitated histone PTM analysis and then covers notable approaches to probe the biological relevance of these modifications in their nucleosomal context.
Frontiers in Genetics 12/2013; 4:264. DOI:10.3389/fgene.2013.00264
[Show abstract][Hide abstract] ABSTRACT: H3K27me3 is deposited at promoters by the preferential association of Polycomb repressive complex 2 (PRC2) with CpG-rich DNA elements regulating development by repressing gene transcription. H3K27 is also present in mono- and dimethylated states; however, the functional roles of H3K27me1 and H3K27me2 deposition remain poorly characterized. Here, we show that PRC2 activity is not only associated with H3K27me3 but also regulates all forms of H3K27 methylation in a spatially defined manner, contributing to different genomic functions in mouse embryonic stem cells. H3K27me1 accumulates within transcribed genes, promotes transcription, and is regulated by Setd2-dependent H3K36me3 deposition. Contrarily, H3K27me2 is present on approximately 70% of total histone H3 and is distributed in large chromatin domains, exerting protective functions by preventing firing of non-cell-type-specific enhancers. Considering that only 5%-10% of deregulated genes in PRC2-deficient cells are direct H3K27me3 targets, our data support an active role for all H3K27 methylated forms in regulating transcription and determining cell identity.
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