High-throughput AFM analysis reveals unwrapping pathways of H3 and CENP-A nucleosomes

Nucleosomes, the fundamental units of chromatin, regulate readout and expression of eukaryotic genomes. Single-molecule experiments have revealed force-induced transient nucleosome accessibility, but a high-resolution unwrapping landscape in the absence of external forces is currently lacking. Here, we introduce a high-throughput pipeline for the analysis of nucleosome conformations based on atomic force microscopy and automated, multi-parameter image analysis. Our data set of ~10,000 nucleosomes reveals multiple unwrapping states corresponding to steps of 5 bp DNA. For canonical H3 nucleosomes, we observe that dissociation from one side impedes unwrapping from the other side, but unlike to force-induced unwrapping, we find only a weak sequence-dependent asymmetry. Centromeric CENP-A nucleosomes do not unwrap anti-cooperatively, in stark contrast to H3 nucleosomes, likely due to their shorter N-terminal α-helix. Finally, our results reconcile previously conflicting findings about the differences in height between H3 and CENP-A nucleosomes. We expect our approach to enable critical insights into epigenetic regulation of nucleosome structure and stability.

Identifying wrapping intermediates by multi-parameter analysis and image simulations. To quantify nucleosome wrapping, we first evaluated the average contour length of bare DNA molecules and found lc = 152.9 ± 6.3 nm (mean ± std from 5651 molecules, Supplementary Fig. 1) corresponding to a length per bp of 0.314 ± 0.013 nm, in excellent agreement with previous measurements by AFM 34,35 and solution X-ray scattering 36 . Similarly, we analyzed the DNA arm lengths of nucleosomes. By subtracting the combined nucleosome arm lengths from the contour length of bare DNA molecules, we obtain the distribution of wrapped lengths, i.e. the length of DNA confined in the nucleosome core particle. For a representative data set of H3 nucleosomes in buffer with 200 mM NaCl, we obtain a wrapped length of 135 ± 27 bp (mean ± std from 1011 molecules), in good agreement with previously reported values 33 . However, in contrast to previous reports 33,37,38 we observed a bimodal distribution rather than a single peak for the wrapping of H3 nucleosomes (Fig. 2a). Fitting the wrapped length distribution to the sum of two Gaussians yields populations centered at 120 ± 14 bp and at 168 ± 12 bp. The distributions of opening angles ( Fig. 2b) and of nucleosome core particle volumes (Fig. 2c) similarly suggest at least two different populations. However, the opening angle distribution is relatively flat, indicating that a large range of opening angles is sampled. To obtain a full quantitative view of nucleosome conformations, we exploit the fact that our analysis pipeline measures multiple parameters for each nucleosome particle to go beyond 1D distributions. Because of the solenoidal winding of nucleosomal DNA, we expect wrapped length and opening angle to be correlated and we indeed find that nucleosomes at wrapped lengths below 150 bp show a negative correlation between opening angle and wrapped length (Fig. 2d), suggesting that these nucleosomes populate states of partial unwrapping. The remaining nucleosomes at wrapped lengths between 160 bp and 190 bp (Fig. 2d) exceed the expected wrapping of the canonical nucleosome by ̴ 20 bp.   Fig. 3c). Our simulations rationalize why the apparent wrapped lengths for fully wrapped nucleosomes exceed the 147 bp expected from the crystal structure: the DNA arms that leave the nucleosome entry/exit site overlap close to the nucleosome core particle due to their initial directionality and the bending stiffness of DNA (Fig.   3a). AFM tip convolution obscures the crossing DNA strands and the software routine interprets the DNA crossover as being part of the nucleosome core, which in turn results in the apparent wrapped length values >160 bp. The simulated images of fully wrapped nucleosomes also reproduce the positive correlation between opening angle and wrapped length (Fig. 3b, state 1). Taken together, the results from simulated nucleosome structures strongly suggest that the population at ̴ 165 bp wrapped length corresponds to fully wrapped nucleosomes (Fig. 3c, top left population), with the DNA arms crossing close to the nucleosome core particle.  Histone H3 nucleosomes unwrap anti-cooperatively. Building on the ability to precisely quantify nucleosome wrapping from high-throughput analysis of large AFM data sets, we next investigated the cooperativity in unwrapping behavior of H3 nucleosomes. The two sides of DNA exiting the nucleosome are distinguishable in our assay since the W601 sequence is placed asymmetrically, giving rise to a longer and a shorter DNA arm (Fig. 1a). The 2D distribution of short nucleosome arm length versus opening angle reveals a population at opening angles <80° and a bimodal distribution of short arm lengths for opening angles >80° (Fig. 5a). The population at opening angles To quantify the propensity to unwrap via the distinct pathways, we simulated AFM images of nucleosomes featuring different levels of anti-cooperative unwrapping (Fig. 5b). Again, we fitted a linear combination of 2D Gaussians to the experimental density distribution of the partially unwrapped H3 nucleosomes using the expected positions based on the simulations to obtain population sizes along the different unwrapping pathways ( Fig. 5c and Supplementary Fig. 3). We observed a small but significant (two-sample t-test p = 0.015) preference for long arm opening over short arm opening with probabilities of (53.7 ± 1.6) % and (46.3 ± 1.6) % respectively (mean ± SEM from four biological repeats). This preference for long arm opening reflects the non-palindromic nature of the W601 nucleosome positioning sequence: The DNA flanking the nucleosome dyad is less flexible on the long arm side compared to the short arm side (Supplementary Fig. 4) leading to the energetically more favorable unwrapping from the stiffer side, in line with previous forceinduced experimental 17 and computational 48 unwrapping studies. However, in these studies nucleosomes almost exclusively unwrapped from the stiffer side, in contrast to the 54% to 46% partitioning that we observe. The difference might be caused by the experimental configurations: Force-induced nucleosome unwrapping with constrained DNA arms requires a nucleosome flip of 180° during unspooling and thus induces strong deformations in the DNA 48 . In contrast, thermal fluctuations and electrostatic interactions of the free DNA arms that drive nucleosome unwrapping in our study might be less susceptible to DNA flexibility. Quantifying the 5 bp unwrapping substates, we observe small, but significant differences in the unwrapping profiles for the two sides: unwrapping from the short arm side tends to occur by 20-25 bp, while the long arm side favors unwrapping by 30-35 bp (Fig 5c). These differences are in line with the free energy profile of W601 nucleosome unwrapping from a previous force-induced unzipping study 47,49 .   Fig. 6b,c). Second, CENP-A nucleosomes exhibit a shift of the partially unwrapped population towards more unwrapped states compared to H3 nucleosomes. Surprisingly, the clear negative correlation of wrapped length with opening angle for H3 nucleosomes is not apparent for CENP-A nucleosomes (Fig. 6a). We note that in our analysis, opening angles >180° are "folded back" and appear at smaller values (Fig. 6a, inset). To resolve higher unwrapping states, we examined again the first component of a PCA of volume and wrapped length and observed three populations (instead of two for H3 nucleosomes) separated by local minima (Fig. 6b, inset). One population corresponds to the fully wrapped state, as for H3. We assign the remaining two populations to partially unwrapped CENP-A nucleosomes with opening angles Fitting the populations of unwrapping states by 5 bp steps in the CENP-A nucleosome data (Fig. 6c), we find that the most frequented states of CENP-A nucleosomes lie between 25 bp and 35 bp unwrapping, similar to H3 nucleosomes under the same conditions (Fig. 4c). However, in contrast to H3 nucleosomes, CENP-A nucleosomes also significantly populate unwrapping states between 40 bp to 60 bp (35.4 ± 4.6% of the total population).
Third, in addition to the overall shift to less wrapped states, our CENP-A nucleosome data reveal a striking difference to H3 nucleosomes in wrapping pathways: While the H3 nucleosome data feature a clearly bimodal distribution of short arm length vs. opening angle for angles >80° (Fig. 5a), indicative of anti-cooperative unwrapping, the CENP-A data exhibit no such branching and a broad distribution of short arm lengths instead (Fig. 6d), suggesting that unwrapping of the two arms is not anti-cooperative for CENP-A nucleosomes.
change of the adjacent H3 N, which in turn leads to rearrangement of H3 N on the opposite side, resulting in stabilization of the DNA contact on the wrapped side. Together, these allosteric changes constitute a latch mechanism that likely contributes to the asymmetric unwrapping of H3 nucleosomes. In CENP-A nucleosomes the N helix is shortened 20 compared to H3. The lack of anticooperative unwrapping revealed by our data suggest that the reduced N helix in CENP-A nucleosomes is insufficient for the latch mechanism, leading to stochastic unwrapping of DNA from CENP-A nucleosomes from both sides, in line with a recent molecular dynamics study 52 .
Interestingly, we observe that the most unwrapped configurations (Fig. 6d, opening 53 . However, this finding was subsequently challenged and follow up studies found no height difference for CENP-A and H3 nucleosomes [54][55][56] . In our measurements we find that overall H3 nucleosomes are significantly higher than CENP-A nucleosomes (two-sample t-test p = 1.610 -60 ; Fig. 6e), with mean heights of (2.04 ± 0.01) nm for H3 (mean ± SEM from N = 1011 molecules) and (1.83 ± 0.01) nm for CENP-A nucleosomes (N = 1645 molecules). Our results are in agreement both regarding the significant height difference between H3 and CENP-A nucleosomes and in terms of the absolute height values with the first study that reported the height differences 53 . However, we observe a significant correlation between wrapped length and measured height ( Fig. 6f; Pearson's R = 0.45 and p = 7.1  10 -52 for H3 nucleosomes and R = 0.40 and p = 2.4  10 -65 for CENP-A nucleosomes). Since CENP-A nucleosomes are on average less wrapped (Fig. 6c), the correlation between wrapping and height implies that differences in wrapping might account for the reduced height of CENP-A nucleosomes. Comparing only fully wrapped H3 and CENP-A nucleosomes (classified based on the PCA results, see Fig. 4a and Fig. 6b), we indeed find mean heights of (2.28 ± 0.01) nm (N = 315 molecules) and (2.24 ± 0.02) nm (N = 205 molecules), respectively, corresponding to no significant difference in the mean heights (two-sample t-test p = 0.11).
In summary, we find that if we average over all wrapping states, CENP-A nucleosomes are lower than H3 nucleosomes, in agreement with an initial report of nucleosome height difference in AFM 53 . However, if we only consider fully wrapped nucleosomes, we do not detect a significant difference, similar to follow up work motivated by the initial study 54,55 . The apparently conflicting findings might be explained by differences in imaging conditions. In particular, the three studies used different salt conditions (10 mM NaCl and 1 mM EDTA vs. 150 mM NaCl + 2 mM MgCl2 vs. 1 mM EDTA only) [53][54][55] , which can significantly alter unwrapping (Fig. 4). In addition, they used different underlying DNA constructs (nucleosome arrays on linear DNA vs. nucleosome arrays on supercoiled plasmids vs. mononucleosomes on short DNA), which also might affect wrapping. We conclude that in addition to precisely controlling imaging parameters, it is important to compare AFM measured heights for nucleosomes occupying the same state of wrapping, highlighting the power of our multi-parametric analysis approach.

Discussion
Quantitative assessment of nucleosome conformations is a key to understanding regulation of DNA accessibility and the binding of transcription factors 8  In contrast to their canonical H3 counterparts, we find centromeric CENP-A nucleosomes to be substantially less wrapped than H3 nucleosomes, with 2-fold fewer nucleosomes being fully wrapped. This result is in agreement with previous high-resolution structural studies by X-ray crystallography 22 and cryo-EM 20 that assigned the shortened N-terminal -helix of histone H3 to weakened interaction with DNA at the exit points of the nucleosome. More than 30% of the population of CENP-A nucleosomes unwrapped >35 bp. Unwrapping >40 bp requires partial opening of both arms, which is suppressed in H3 nucleosomes. Our data are in line with results from force-induced unzipping of nucleosomal DNA: peak-forces associated with the strong barrier between superhelical locations 3.5 and 6.5, i.e. up to 40 bp unwrapping, were significantly reduced in yeast centromeric versus H3 nucleosomes 39 . However, DNA-histone interactions closer to the dyad were equally strong for yeast centromeric and H3 nucleosomes preventing unwrapping of more than 40 bp from one side.
Our data show no anti-cooperativity in the unwrapping of CENP-A nucleosomes, in contrast to H3 nucleosomes. We propose that the stabilizing latch mechanism that contributes to anticooperativity 18 in H3 nucleosome unwrapping is missing in CENP-A nucleosomes due to the shortened N helix. Since CENP-A is a key epigenetic mark to maintain structural integrity of the centromer, we speculate that both stochastic unwrapping and overall decreased DNA wrapping of CENP-A nucleosomes might facilitate binding of proteins to specific DNA sequences in the centromer. For example, the centromer-DNA binding protein complex CBF3 is essential for chromosome segregation and binds selectively to the highly conserved CDEIII DNA sequence found in centromers 61 .
In summary, we have developed a high-throughput automated analysis platform and used it to uncover thermally activated pathways of H3 and CENP-A nucleosome wrapping in unprecedented detail. Our methodology will facilitate future (high-speed) AFM studies that involve structure and interactions of nucleoprotein complexes by either using fast imaging of large molecular ensembles or by time-lapsed imaging of the molecular dynamics at the single molecule level 27 .
(0.01% w/v) coated muscovite mica for 30 seconds and subsequently rinsed with 20 mL of milliQ water before drying with a gentle stream of filtered N2 gas.
We used two different commercial AFM instruments for imaging. All AFM images were acquired in tapping mode at room temperature. One set of images was acquired on a Multimode VIII AFM (Bruker) using silicon tips (AC160TS, drive frequency of 300-350 kHz, Olympus). Images were scanned over a field of view of 3 m x 3 m at 2048 x 2048 pixels with a scanning speed of 1 Hz.
Independent measurement repeats were performed on a Nanowizard Ultraspeed 2 (JPK) with reflex gold coated tips (USC-F5-k30, drive frequency 5000 kHz, Nanoworld). Here, images were scanned over a field of view of 6 m x 6 m at 4096 x 4096 pixels with a scanning speed of 3 Hz or over a field of view of 12 m x 12 m at 8192 x 8192 pixels at 3 Hz (Fig. 1b). For H3 nucleosomes, four data sets were acquired at 200 mM NaCl and 10 mM Tris over three separate nucleosome reconstitutions. For CENP-A nucleosomes, three data sets were acquired at 200 mM NaCl and 10 mM Tris over two separate nucleosome reconstitutions.

AFM image analysis.
We developed an automated image analysis pipeline to analyze the flattened AFM images. For the AFM data analyzed in this work, a background height threshold of 0.16 nm was applied. The bare DNA strands were traced with 5 nm segments 32 from both sides separately and the mean value was used as contour length. Over 95% of the viable molecules in the images were detected automatically. Here, viable molecules are defined as molecules that do not have overlaps with other molecules and can be analyzed by manual tracing. To achieve an even higher detection rate, manual input allowed the separation of unclassified objects (for example for two DNA arms that slightly overlap and thus prevent automated detection). This way, 98% of all viable nucleosomes of the example image were detected ( Supplementary Fig. 1 Fig. 2d, Supplementary   Fig. 2). Consecutively, the DNA was dilated to its expected width of 2 nm and random noise in 22 combination with a Gaussian filter ( = 2 nm) was applied to mimic the effect of tip convolution.
Partially unwrapped nucleosomes were simulated by adding base pairs to one end of the simulated chains in 5 bp steps, increasing the opening angle by 4.45° per base pair of unwrapping (based on 147 bp wrapped over a total of 654° in the crystal structure) and adjusting the direction of the protruding DNA arms. Similarly, synthetic images of bare DNA were simulated with a 2D worm-like chain of 486 bp and the same steps of dilation and tip convolution. The synthetic bare DNA images were analyzed for their average DNA contour length that is needed to calculate the wrapped length in nucleosomes in our automated readout pipeline. The simulated images were analyzed with the same automated readout software as the experimental images.

Data availability
Data supporting the findings of this manuscript are available from the corresponding authors upon reasonable request.

Code availability
Code is available on https://github.com/SKonrad-Science/AFM_nucleoprotein_readout including a detailed installation guide and an example image.