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DNA accessibility of chromatosomes quantified by automated image analysis of AFM data.
Würtz M
,
Aumiller D
,
Gundelwein L
,
Jung P
,
Schütz C
,
Lehmann K
,
Tóth K
,
Rohr K
.
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DNA compaction and accessibility in eukaryotes are governed by nucleosomes and orchestrated through interactions between DNA and DNA-binding proteins. Using QuantAFM, a method for automated image analysis of atomic force microscopy (AFM) data, we performed a detailed statistical analysis of structural properties of mono-nucleosomes. QuantAFM allows fast analysis of AFM images, including image preprocessing, object segmentation, and quantification of different structural parameters to assess DNA accessibility of nucleosomes. A comparison of nucleosomes reconstituted with and without linker histone H1 quantified H1's already described ability of compacting the nucleosome. We further employed nucleosomes bearing two charge-modifying mutations at position R81 and R88 in histone H2A (H2A R81E/R88E) to characterize DNA accessibility under destabilizing conditions. Upon H2A mutation, even in presence of H1, the DNA opening angle at the entry/exit site was increased and the DNA wrapping length around the histone core was reduced. Interestingly, a distinct opening of the less bendable DNA side was observed upon H2A mutation, indicating an enhancement of the intrinsic asymmetry of the Widom-601 nucleosomes. This study validates AFM as a technique to investigate structural parameters of nucleosomes and highlights how the DNA sequence, together with nucleosome modifications, can influence the DNA accessibility.
Figure 1. Schematic overview of the automated image analysis method and extracted parameters. (a) Example results illustrating the algorithmic steps of the image analysis method QuantAFM: (i) Original input image, (ii) denoising step with Non-Local Means filtering resulting in less variation of background noise53, (iii) additional mean and low-pass filtering for further removing the noise levels of the AFM tip, (iv) initial binarization yielding a rough segmentation result, (v) refined segmentation result after background equalization and removal of small as well as large objects, (vi) extraction of nucleosome candidates with the Hough Transform57 indicated by red circles, and (vii) simultaneous thinning step. (viii) Results for the thinned fragments and nucleosomes are merged based on physical proximity. Subsequently, length and angles are determined for valid fragments. (b) Enlarged sample nucleosome object. Determined parameters include nucleosome radius r (black), opening angle θ (yellow), and long (blue) and short (magenta) filament protruding DNA arm lengths. If no nucleosome is present, the total contour length is determined instead.
Figure 2. AFM imaging of chromatosomes. Nucleosome complexes deposited on PL coated surface. (a and b) Example images of wt nucleosomes reconstituted on 464 bp DNA, without and with histone H1, respectively. The images show that nucleosomes are clearly distinguishable from background and free DNA (black circle). Example images for mutated nucleosomes are shown in Supplementary Fig. S8. (c) Enlarged sections for each nucleosome class, selected from different images, with representative DNA wrapping length (lw) and trend of the opening angle θ. All selected complexes show a central positioning on the DNA. Mutated nucleosomes show a more open conformation. For mut +H1 a highly wrapped and less wrapped object is shown.
Figure 3. DNA opening angles are affected by mutation and histone H1 interaction. Normalized probabilities of the opening angles θ were plotted in 15° binned sectors for the different cases. The small example images in (a) represent nucleosome objects with the corresponding DNA opening angle. For each case, (a) wt, (b) wt +H1, (c) mut and (d) mut +H1 all opening angles are plotted (nwt = 444, nwt +H1 = 445, nmut = 444, nmut +H1 = 448). Diagrams for samples with histone H1 are shown in red. Linker histone H1 induces a distinct population between 60° and 90° in the distribution of the opening angle for wt. Compared to wt, mutated nucleosomes show higher opening angles.
Figure 4. Linker histone affects both DNA wrapping length and opening angle. Top: Scatter plots of the opening angle θ and protruding DNA arm contour length of wt nucleosomes (a) without (nwtâ=â444, black) and (b) with histone H1 (nwt +H1â=â445, red); centroids are highlighted in blue. Scatter plots for mutated nucleosomes are shown in Supplementary Fig. S8. The histograms show the normalized probabilities for the opening angle (30° bin width) and the protruding DNA arm contour length (bin width 10ânm). Histone H1 induced a decreased protruding DNA length together with a preference of a distinct opening angle. Bottom: The protruding DNA arm contour length was converted into the wrapping length for (c) wt nucleosome objects and (d) mutated nucleosome objects without and with histone H1 (nwtâ=â444, nwt +H1â=â445, nmutâ=â444, nmut +H1â=â448). Normalized histograms of wrapping length are plotted from 60âbpâ225âbp (bin width 15âbp). Bars of samples with linker histone H1 are indicated by red edges. DNA wrapping is lower for mutated nucleosomes than wt, but increased in the presence of histone H1 in both cases.
Figure 5. Nucleosome positioning. The positioning of the nucleosome objects on the DNA fragment is dominated by the Widom-601 positioning sequence. (a) The theoretical length of the DNA arms protruding from the octamer, consisting of a (H3–H4)2 tetramer (turquoise) and two H2A-H2B dimers (red and yellow) when positioned on the 147 bp long Widom- 601 sequence. The α-side corresponds for 464 bp nucleosomes to the longer and for 599 bp nucleosomes to the shorter DNA arm. This illustration displays the approximate length ratios between nucleosome core and protruding DNA arms. As nucleosome cores appear on the AFM images roughly twice the theoretical size (Table 2), ratios between the DNA arms were corrected (see Methods AFM data evaluation). (b) Cumulative frequency plot of the sa-ratioc of all nucleosome samples reconstituted on 464 bp and 599 bp DNA. The cumulative frequency values of sa-ratioc, were normalized and binned (bin wide 0.01). (c) shows the median values with 95% confidence interval (CI) of the sa- ratioc for the samples in (b). The theoretical values for positioning on the Widom-601 sequences are indicated by black arrows. For (b and c) only objects with a sa-ratioc of at least 0.3 were considered: nwt/464 = 443, nwt +H1/464 = 443, nmut/464 = 439, nmut +H1/464 = 439, nwt/599 = 174, nwt +H1/599 = 185, nmut/599 = 183, nmut +H1/599 = 162. The shift between wt and mut for both 464 bp, and 599 bp DNA fragments indicate an asymmetric unwrapping of the α-side for the mutants.
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