Single-molecule decoding of combinatorially modified nucleosomes

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Science  06 May 2016:
Vol. 352, Issue 6286, pp. 717-721
DOI: 10.1126/science.aad7701
  • Fig. 1 Single-molecule detection of posttranslational modifications on nucleosomes.

    (A) Experimental approach. Step 1: Nucleosomes from cells are prepared by micrococcal nuclease digestion. The gel shows nucleosomal DNA fragments of expected lengths. Step 2: Free DNA ends are ligated to fluorescent biotinylated oligonucleotide adaptors (Bio, biotin). Step 3: Adaptor-ligated mononucleosomes are purified on a glycerol gradient and captured on PEG-streptavidin–coated slides. Step 4: Nucleosome positions on the surface are imaged by TIRF microscopy, and the fluorophore is then cleaved from the adaptor. Step 5: Attached nucleosomes are incubated with fluorescently labeled antibodies to histone modifications. Time-lapse images detect repeated binding and dissociation events and are integrated to score modified nucleosomes. Throughout the diagram, red and green circles represent histone modifications, and the corresponding stars represent fluorophores conjugated to antibodies or oligonucleotide adaptors. In (B) to (D), HEK293 cells were treated with HDAC inhibitor (HDACi) (NT, nontreated; h, hours). (B) Single-molecule detection of labeled nucleosomes (Alexa555, green) bound by labeled H3K9ac antibodies (Alexa647, red). (C) The percentage of nucleosomes marked by H3K9ac under each condition was determined by single-molecule counting (error bars, SD). (D) Western blot confirms increased H3K9ac in treated cells. In (E) and (F), recombinant unmodified nucleosomes and H3K27me3-modified peptide were probed with the indicated antibodies (Ab). (E) The percentage of bound nucleosomes or peptides detected. (F) Single-molecule detection of labeled H3K27me3 peptide [TAMRA (5-carboxytetramethylrhodamine) green] with labeled H3K27me3 antibodies (Alexa647, red) at a single time point. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; G, Gly; K, Lys; L, Leu; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; and V, Val.

  • Fig. 2 Single-molecule imaging of symmetric and asymmetric bivalent nucleosomes.

    (A) We investigated modifications on nucleosomes from pluripotent ESCs, EBs, and lung fibroblasts. On the left, colored bars indicate percentages of nucleosomes with H3K27me3 (red) or H3K4me3 (green) (error bars, SD). On the right, black bars indicate relative over- or underrepresentation of bivalent nucleosomes. Results on the right are presented as the log2 ratio of the observed proportion of bivalent nucleosomes to the proportion of nucleosomes with these two marks that would be expected from random association (the latter is calculated as the fraction with H3K27me3 multiplied by the fraction with H3K4me3; fig. S10). (B) Nucleosomes from a T cell acute lymphoblastic leukemia line, from HEK293 cells, and from glioblastoma stem cells were decoded as in (A). (C) Nucleosomes from an acute leukemia line with an EZH2 LOF mutation, a lymphoma line with an EZH2 GOF mutation, and the lymphoma cells treated with EZH2 inhibitor GSK126. (D) A magnified TIRF image overlay reveals three nucleosomes, one with H3K27me3 (red), one with H3K4me3 (green), and one with concomitant bivalent modifications (arrow). (E) The diagram and image show H3K27me3 and H3K4me3 antibodies binding to individual histones isolated from ESCs. 94% of bivalent nucleosomes are asymmetric, whereas just 6% are modified on the same tail. In (F) and (G), the lymphoma cell line with the EZH2 GOF mutation was treated with GSK126 for 3 days. (F) Nucleosomes were decoded for H3K27me3 and H3K4me3. The image shown is of pretreated samples, with arrows highlighting bivalent nucleosomes. (G) Plot showing the percentages of H3K4me3-negative (H3K27me3 only; left) and H3K4me3-positive (bivalent; right) nucleosomes carrying H3K27me3 (error bars, SD). Bivalent nucleosomes were more likely to lose H3K27me3 after treatment with GSK126.

  • Fig. 3 Higher-order modification states across cellular states and inhibitor treatments.

    (A) Individual nucleosomes from ESCs and lung fibroblasts were decoded for H3K4me3, H3K27me3, H3K27me2, and H3K27ac, as described in Fig. 1. Bars depict the percentage of nucleosomes with the indicated modification (error bars, SD). (B) Bars indicate the relative over- or underrepresentation of each possible modification pair, relative to random expectation, as in Fig. 2A. Opposing modifications were relatively more likely to coexist in ESCs than in lung fibroblasts. (C) ESCs were treated with dimethyl sulfoxide (control), HDAC inhibitor (sodium butyrate), or p300 inhibitor (C646). Nucleosomes were isolated and decoded for H3K27me3, H3K4me3, H3K9ac, and H3K27ac. The plot shows the effects of the inhibitors on each single modification and on the combination of H3K27ac and H3K4me3. ***P < 0.001.

  • Fig. 4 Single-molecule sequencing determines genomic positions of modified nucleosomes.

    (A) Experimental approach. Step 1: Nucleosomes are captured and probed for their modification state, as in Fig. 1A. Histones are evicted by increasing salt concentration. Step 2: The enzyme USER (uracil-specific excision reagent) is applied to excise uracil bases incorporated into the nonbiotinylated adaptor strand and expose a known sequence. Step 3: Complementary primer is hybridized to the adaptor. The image shows single-molecule detection of nucleosomal DNA (Alexa647, red) and primer (Alexa555, green). Step 4: Direct single-molecule DNA sequencing-by-synthesis is performed (22). The images reflect two sequencing cycles, covering the incorporation of thymine, the cleavage of the fluorophore and terminator, and the incorporation of cytosine. Step 5: For each x,y coordinate on the surface, sequence data are analyzed and integrated with the initial images that scored the antibody binding and modification states of the corresponding nucleosomes. (B) Single-molecule reads were aligned to the genome. The plot shows percentages of H3K27me3-modified nucleosome reads (detected) or unmodified nucleosome reads (undetected) that aligned to H3K27me3-enriched regions per conventional ChIP-seq. (C) Percentages of H3K4me3-modified nucleosome reads that aligned to H3K4me3-enriched regions per conventional ChIP-seq. (D) Gene annotations (blue) for the HOXC gene cluster are shown with H3K27me3 and H3K4me3 ChIP-seq tracks. Single-molecule reads that aligned to these regions are indicated, along with the modification status of the corresponding nucleosome. (E) Analogous data for other developmental loci for which bivalent nucleosomes were definitively identified.

Supplementary Materials

  • Single-molecule decoding of combinatorially modified nucleosomes

    Efrat Shema, Daniel Jones, Noam Shoresh, Laura Donohue, Oren Ram, Bradley E. Bernstein

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S14
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