Quantitative Mass Spectrometry of Histones H3.2 and H3.3 in Suz12-deficient Mouse Embryonic Stem Cells Reveals Distinct, Dynamic Post-translational Modifications at Lys-27 and Lys-36
ABSTRACT 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.
- SourceAvailable from: Jamie Denizio
[Show abstract] [Hide abstract]
- "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. "
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]
- "While there is some evidence for an in-vivo interaction between PcG proteins and the NuRD complex in various organisms (Kehle et al, 1998; Unhavaithaya et al, 2002; Morey et al, 2008; Aichinger et al, 2009), the precise nature of this interaction has not been characterised. Nevertheless, the importance of a balance between the acetylation and methylation state of H3K27 has been shown in both mammalian cells and flies (Tie et al, 2009; Jung et al, 2010; Pasini et al, 2010b) and could provide the link between these two complexes in stem cell function. By comparing levels of specific chromatin modifications in ES cells with or without functional NuRD complex, we demonstrate the role played by NuRD in regulating the balance between acetylation and methylation state of H3K27. "
ABSTRACT: Pluripotent cells possess the ability to differentiate into any cell type. Commitment to differentiate into specific lineages requires strict control of gene expression to coordinate the downregulation of lineage inappropriate genes while enabling the expression of lineage-specific genes. The nucleosome remodelling and deacetylation complex (NuRD) is required for lineage commitment of pluripotent cells; however, the mechanism through which it exerts this effect has not been defined. Here, we show that histone deacetylation by NuRD specifies recruitment for Polycomb Repressive Complex 2 (PRC2) in embryonic stem (ES) cells. NuRD-mediated deacetylation of histone H3K27 enables PRC2 recruitment and subsequent H3K27 trimethylation at NuRD target promoters. We propose a gene-specific mechanism for modulating expression of transcriptionally poised genes whereby NuRD controls the balance between acetylation and methylation of histones, thereby precisely directing the expression of genes critical for embryonic development.The EMBO Journal 12/2011; 31(3):593-605. DOI:10.1038/emboj.2011.431 · 10.75 Impact Factor
[Show abstract] [Hide abstract]
- "K27me3/ K36me1, K27me1/K36me3, and K27me2/K36me3); the complete compilation of all these species could not be uniquely characterized in our analysis. However, Jensen and co-workers recently showed that new MS instruments, such as LTQ Orbitrap Velos, allow the acquisition of MS/ MS spectra in high resolution mode, boosting the capability to resolve near-isobaric modifications coexisting on multiple PTM sites within the same peptide (Jung et al. 2010). Although bottom-up approach is sensitive and specific, it falls short in assessing combinations of PTMs, evaluating their relative stoichiometry and measuring the overall abundance of differently modified intact protein species. "
ABSTRACT: In living cells, the N-terminal tails of core histones, the proteinaceous component of nucleosomes, are subjected to a range of covalent post-translational modifications (PTMs), which have specific roles in modulating chromatin structure and function. A growing body of evidence suggests that deregulation of histone modification patterns, upstream or downstream of DNA methylation, is a critical event in cancer initiation and progression. However, a comprehensive description of how histone modifications, singly or in combination, is disrupted in transformed cells is missing; consequently the issue whether and how specific changes in histone PTMs patterns correlate to particular tumor features is still elusive. In the present study, we focused on human breast cancer and comprehensively analyzed PTMs on histone H3 and H4 from four cancer cell lines (MCF7, MDA-MB231, MDA-MB453 and T-47D), in comparison with normal epithelial breast cells. We performed high-resolution mass spectrometry analysis of histones, in combination with stable isotope labeling with amino acids in cell culture (SILAC), to quantitatively track the modification changes in cancer cells, as compared to their normal counterpart. Our investigation focuses on lysine acetylation and methylation on fourteen distinct sites in H3 and H4. We observed significant changes for several modifications in cancer cells: while in a few cases those modifications had been previously described as a hallmark of human tumors, we could identify novel modifications, whose abundance is significantly altered in breast cancer cells. Overall, these modifications may represent part of a "breast cancer-specific epigenetic signature", with implications in the characterization of histone-related biomarkers. This work demonstrates that SILAC-based proteomics is a powerful tool to study qualitatively and quantitatively histone PTMs patterns, contributing significantly to the comprehension of epigenetic phenomena in cancer biology.Amino Acids 07/2011; 41(2):387-99. DOI:10.1007/s00726-010-0668-2 · 3.65 Impact Factor