Quantifying epigenetic modulation of nucleosome breathing by high-throughput AFM imaging

Abstract

Nucleosomes are the basic units of chromatin and critical for storage and expression of eukaryotic genomes. Chromatin accessibility and gene readout are heavily regulated by epigenetic marks, in which post-translational modifications of histones play a key role. However, the mode of action and the structural implications at the single-molecule level of nucleosomes is still poorly understood. Here we apply a high-throughput atomic force microscopy imaging and analysis pipeline to investigate the conformational landscape of the nucleosome variants three additional methyl groups at lysine 36 of histone H3 (H3K36me3), phosphorylation of H3 histones at serine 10 (H3S10phos), and acetylation of H4 histones at lysines 5, 8, 12, and 16 (H4K5/8/12/16ac). Our data set of more than 25,000 nucleosomes reveals nucleosomal unwrapping steps corresponding to 5-bp DNA. We find that H3K36me3 nucleosomes unwrap significantly more than wild-type nucleosomes and additionally unwrap stochastically from both sides, similar to centromere protein A (CENP-A) nucleosomes and in contrast to the highly anticooperative unwrapping of wild-type nucleosomes. Nucleosomes with H3S10phos or H4K5/8/12/16ac modifications show unwrapping populations similar to wild-type nucleosomes and also retain the same level of anticooperativity. Our findings help to put the mode of action of these modifications into context. Although H3K36me3 likely acts partially by directly affecting nucleosome structure on the single-molecule level, H3S10phos and H4K5/8/12/16ac must predominantly act through higher-order processes. Our analysis pipeline is readily applicable to other nucleosome variants and will facilitate future high-resolution studies of the conformational landscape of nucleoprotein complexes.

Figure 1

DNA and nucleosome structure parameters from automated AFM image analysis. (a) Crystal structure of a canonical nucleosome (PDB: 1KX5). Colored spheres represent the positions of the modified amino acids in the histone tail considered in this work. Among the three histone tail modifications investigated are three additional methyl groups at lysine 36 of histone H3 (H3K36me3, blue spheres), phosphorylation of H3 histones at serine 10 (H3S10phos, red spheres), and acetylation of H4 histones at lysines 5, 8, 12, and 16(H4K5/8/12/16ac, orange spheres). (b) Schematic of the construct used throughout this work. The 486-bp DNA consists of a 147-bp W601 nucleosome positioning sequence that is flanked by a short and a long arm of 106 bp and 233 bp, respectively. Histone octamers contain two copies each of H2A, H2B, H3, and H4. (c) AFM image of bare DNA and nucleosomes with a field of view of 12 × 12 μm at a resolution of 1.46 nm/pixel (8,192² pixels). (d) Traces of 901 bare DNA strands (orange) and 1,624 nucleosomes (yellow), obtained by the automated image analysis pipeline from the image shown in (c). (e) Magnification of a nucleosome image before and after tracing. The magnified area is indicated in (c) and (d). (f) Same nucleosome image as in (e) after Richardson-Lucy deconvolution. The inset displays the estimated shape of the AFM tip deduced from the bare DNA molecules in the same AFM image and used for deconvolution. (g) Opening angle distribution for the same data set, analyzed without and with deconvolution. The deconvolved data show the 20° (5-bp) unwrapping periodicity of nucleosomes (N = 716, only partially unwrapped nucleosomes are shown; see Fig. S3 for the same data set shown with different bin sizes). (h) Bare DNA before and after deconvolution and tracing.

Figure 2

Estimating nucleosome wrapping populations. (a) Wrapped length versus opening angle distribution for canonical nucleosomes. White squares and black circles represent individual nucleosomes (N = 1,035), and the colored contours represent the 2D kernel density estimate (using a Gaussian kernel with bandwidth of 2.5°, 2.5 bp). The inset shows a principal-component analysis—a linear dimensionality reduction to 1D using singular value decomposition of the two parameters wrapped length and volume—that is used to separate the two nucleosome populations (fully versus partially wrapped). (b) 2D Gaussians fit to the density distribution of the partially unwrapped nucleosomes. The Gaussian amplitudes represent the populations of the 5-bp unwrapping substates; the inset shows the residuals of the fit to evaluate the quality of fitting. (c) Representative close-up shots of experimentally measured nucleosomes for the individual unwrapping states. Fully wrapped nucleosomes exceed the expected 147 bp of wrapping because of the overlapping of DNA at the entry/exit site, as described in the text.

Figure 3

DNA wrapping populations of post-translationally modified nucleosomes. (a) Populations of DNA wrapping conformations for unmodified nucleosomes and the three modified nucleosome constructs containing H3K36me3, H3S10phos, or H4K5/8/12/16ac. The populations were determined from high-throughput analysis of AFM images, as shown in Fig. 2; the filled black circles in (a) are from the data set in Fig. 2. For each histone variant, four to five independent measurement repeats were obtained. Circles indicate the populations of the individual data sets and bars and error bars indicate the mean ± SE from the independent repeats. (b) Crystal structure of the canonical nucleosome (PDB: 1KX5). Colored spheres represent the positions of the modified histone tail amino acids. (c) Differences between the wrapping populations of the modified nucleosomes and the unmodified nucleosomes. Significant differences, as determined by two-sample t-tests, are indicated by 1,2 or 3 stars at the p < 0.05, < 0.01, and < 0.001 level, respectively.

Figure 4

Unwrapping pathways of post-translationally modified nucleosomes. (a) 2D kernel density profile (bandwidth, 2.5°; 2.5 bp) of short arm length and opening angle for H3 nucleosomes. A bimodal distribution for opening angles greater than 80° is apparent, consistent with anticooperative unwrapping of the nucleosome core particle (N = 1,035). The distribution of fully wrapped nucleosomes (30.8% of all nucleosomes, indicated by the black ellipse) was omitted from the plot for clarity. (b) 2D kernel density profile (bandwidth, 2.5°; 2.5 bp) of short arm length and opening angle for H3K36me3 nucleosomes (N = 1,155). (c) Quantification of the tendency of the different epigenetically modified nucleosomes to unwrap anticooperatively or not (Fig. S4). Unmodified, H3S10phos, and H4K5/8/12/16ac nucleosomes show similar high levels of anticooperative unwrapping; in contrast, H3K36me3 and CENP-A nucleosomes unwrap less anticooperatively. Differences were tested for significance by two-sample t-tests: n.s. indicates no significant difference at the p = 0.05 level, three stars indicate p < 0.001.