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

Centre for Epigenetics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
Molecular &amp Cellular Proteomics (Impact Factor: 7.25). 05/2010; 9(5):838-50. DOI: 10.1074/mcp.M900489-MCP200
Source: PubMed

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.

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    • "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. "
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    • "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. "
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    • "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. "
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