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. 2012 Sep 28;151(1):181-93.
doi: 10.1016/j.cell.2012.09.002.

Asymmetrically modified nucleosomes

Philipp Voigt  1 , Gary LeRoy, William J Drury 3rd, Barry M Zee, Jinsook Son, David B Beck, Nicolas L Young, Benjamin A Garcia, Danny Reinberg
Affiliations

Asymmetrically modified nucleosomes

Philipp Voigt (VSports手机版) et al. Cell. .

Abstract

Mononucleosomes, the basic building blocks of chromatin, contain two copies of each core histone VSports手机版. The associated posttranslational modifications regulate essential chromatin-dependent processes, yet whether each histone copy is identically modified in vivo is unclear. We demonstrate that nucleosomes in embryonic stem cells, fibroblasts, and cancer cells exist in both symmetrically and asymmetrically modified populations for histone H3 lysine 27 di/trimethylation (H3K27me2/3) and H4K20me1. Further, we obtained direct physical evidence for bivalent nucleosomes carrying H3K4me3 or H3K36me3 along with H3K27me3, albeit on opposite H3 tails. Bivalency at target genes was resolved upon differentiation of ES cells. Polycomb repressive complex 2-mediated methylation of H3K27 was inhibited when nucleosomes contain symmetrically, but not asymmetrically, placed H3K4me3 or H3K36me3. These findings uncover a potential mechanism for the incorporation of bivalent features into nucleosomes and demonstrate how asymmetry might set the stage to diversify functional nucleosome states. .

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Figures

Figure 1
Figure 1. Analysis of Histone Modification Symmetry by Immunoaffinity Purification and Mass Spectrometry
A, Outline of the experimental approach. See main text for details. B, For a hypothetical binary (unmodified/modified) mark, asymmetric nucleosomes give rise to equal amounts of modified and unmodified peptide, whereas the symmetric case yields only the modified peptide. C, If a mixed population is present, the abundance of unmodified peptide decreases with increasing proportions of symmetric nucleosomes. The peptide quantification yields the relative amount of symmetric and asymmetric populations through interpolation between the limiting cases. D, Modifications covered in MS analysis. Tryptic peptides generated from propionylated histones H3 and H4 are shown along with detected acetylation and methylation sites. See also Figure S1.
Figure 2
Figure 2. Nucleosomes are modified both symmetrically and asymmetrically with H3K27me2/3 and H4K20me1
A, Example of a mononucleosome affinity purification and specificity controls for an H3K27me2/3-specific antibody. B, C, Determination of symmetric and asymmetric populations for H3K27me2/3 (B) and H4K20me1 (C) based on quantitative LC-MS/MS data. Left panels show data for E14 ES cells, tables summarize data for the indicated cell types. Results represent average and SEM of at least two independent experiments based on two different nucleosome preparations per cell type. See also Figures S1, S2, and S3 and Table S2.
Figure 3
Figure 3. Co-occurrence of histone marks with H3K27me2/3 and H3K4me3 in ES, MEF, and HeLa cells
A, B, Modification profile of mononucleosomes prepared from E14 ES cells by H3K27m2/3 (A) or H3K4me3 (B) antibody-based affinity purification. Given are fold changes over input for the indicated sites and modification states on H3 and H4. For H4 acetylation, abundance of unacetylated – tetraacetylated species (H4ac0–4) as well as site-specific acetylation status (H4Kac5/8/12/16) is shown. Positive values denote co-enrichment, while negative values indicate an inverse correlation with the targeted site. Results represent average and SEM of at least two independent experiments each. C, D, Modification state of H3K27me2/3 and H3K4me3 mononucleosomes from ES cells, MEFs, and HeLa cells. Abundances of methylation and acetylation states are shown as stacked columns normalized to 100% for each site. Shown are averages of at least two independent experiments each. See also Figure S4 and Table S2.
Figure 4
Figure 4. Sequential ChIP analysis of bivalent promoters in ES cells and modification state of H3K27me2/3 nucleosomes in differentiated ES cells
A, B, ChIP analysis of the indicated promoters with H3K4me3 and H3K27me3 antibodies. Right panel shows sequential ChIP with the H3K27me3 followed by H3K4me3 antibody. Assays were performed on untreated E14 cells (A) and E14 ES cells differentiated with retinoic acid (RA) for 6 days (B). The re-ChIP data is given as fold over IgG control in the second IP. Shown are means and SEM from two independent experiments. C, Mononucleosomes were affinity-purified from retinoic acid-treated cells with H3K27me2/3 antibody to assess their symmetry state as described in Figure 2. D, Modification profile of H3K27me2/3 nucleosomes immunopurified from retinoic acid (RA)-differentiated cells. For comparison, the modification state of E14 ES cells is given. Results represent mean and SEM of three independent experiments. See also Figure S2H, I.
Figure 5
Figure 5. PRC2 is inhibited by symmetric, but not asymmetric, presence of H3K4me3 or H3K36me3
A, Histone methyltransferase assays on oligonucleosome substrates containing H3 with trimethyl-lysine analogues at the indicated positions either on both (s) or only one tail (as) per nucleosome. The asymmetric cases were generated using differentially tagged modified and unmodified histone H3 and double affinity purification. B, Titration of symmetrically and asymmetrically modified substrates. Panels shown are representative of three independent assays. See also Figure S5.
Figure 6
Figure 6. Bivalent Nucleosomes Contain H3K27me3 and H3K4me3/H3K36me3 on Opposing Tails In Vivo
A, Illustration of the experimental design. H3K27me2/3 nucleosomes are immunoaffinity-purified and subjected to Bottom Up LC-MS/MS analysis. H3K27 and H3K36 remain on the same peptide, allowing assessment of their interplay on single tails versus nucleosomes. B, The relative abundance of peptides detected is grouped according to modification status at H3K27 (open bars, unmodified or me1; red bars, me2, me3). Note the difference in y axis scale between the panels. Shown are means and SEM of at least three experiments each. C, Alternative representation of the data shown in B. To assess the relative abundance of H3K36 states in the presence or absence of H3K27me2/3, the percentage of H3K36 states was normalized to the total abundance of peptides with or without H3K27me2/3. D, Illustration of the Middle Down MS approach. Acid-extracted nuclear histones were subjected to Glu-C digest and the resulting H3.1(1–50) peptide was analyzed. E, Abundance of peptides containing H3K4me3 and H3K4me2 plotted as a function of H3K27 modification state on the same peptides. Peptides were assigned with custom-made software and manually validated in some cases. Shown are means and SEM of three experiments each. The error reflects both technical error of measurement and mis-assignment of peptide species by the software. F, Alternative representation of the data in E with peptide abundances normalized to the sum of all H3K4me3/H3K4me2 peptides.
Figure 7
Figure 7. Model for the generation of symmetric and asymmetric nucleosomes by PRC2
Schematic model showing modulation of PRC2 activity by active marks present on one or both copies of H3 per nucleosome as well as their possible fates in the transition from ES cells (ESC) to more differentiated lineages (such as MEFs). Scale of nucleosome symbols in the ESC and MEF panels reflects their relative abundance.
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Comment in

  • Chromatin: Histone sibling rivalry.
    Schuldt A. Schuldt A. Nat Rev Mol Cell Biol. 2012 Nov;13(11):683. doi: 10.1038/nrm3462. Epub 2012 Oct 17. Nat Rev Mol Cell Biol. 2012. PMID: 23072886 No abstract available.

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