Figure 5. CenH3 nucleosomes protect small DNA fragments.

(A, B, C) Normalized fragment counts in input (A), cenH3 ChIP (B) and insoluble chromatin (C) samples at centromeric sites. MNase fragments were divided into nucleosomal (141–500 bp) and small (21–140 bp) size classes. Average signals within a 1-kb window around all 707 cenH3 sites are plotted. Dashed lines mark the centers of the flanking nucleosomes in (A) (black lines) or the centromeric nucleosome in (B) (blue line) and in (C) (magenta line). (D) Cartoon illustrating how MNase fragment size distributions shown in (E and F) were determined. Fragments that cross the center of the cenH3 nucleosome or of the flanking nucleosomes were counted. (E and F) MNase fragment size distribution after 2 min (E) and 10 min (F) of MNase digestion. Input fragments at flanking nucleosomes (black) and cenH3 ChIP fragments (blue) and insoluble chromatin fragments (magenta) at centromeric nucleosomes are shown. Cartoons of the protected particles are shown below each panel. (G and H) Comparison of worm holocentromere and budding yeast point centromere. (G) C. elegans holocentromere. Centromere model and cenH3 ChIP over input ratio (all size classes; left y-axis) and nucleosomal signal from input (141–500 bp; right y-axis). Average signals within a 1-kb window around all 707 cenH3 sites are shown. (H) Budding yeast point centromere. Centromere model and data from Krassovsky et al. (Krassovsky et al., 2012), cenH3 ChIP over input ratio (left y-axis) and input signal (right y-axis) from all 16 centromeres.

DOI: http://dx.doi.org/10.7554/eLife.02025.016

Figure 5—figure supplement 1. Comparison of input signal at centromeric sites in native and formaldehyde-crosslinked samples.

Nucleosome-size fragments at centromeric sites from native and formaldehyde-crosslinked input samples (top, MNase-seq, this study), and mononucleosome-size fragments from native and formaldehyde-crosslinked samples from the modENCODE database (http://intermine.modencode.org/), derived from microarray data (bottom).

DOI: http://dx.doi.org/10.7554/eLife.02025.017

Figure 5—figure supplement 2. Fragment size distribution for different degrees of MNase digestions.

(A) MNase fragment size distribution of fragments crossing the dyad of the nucleosomes flanking the cenH3 peaks (top) or of all fragments genome wide (bottom). Fragment size distributions for the four indicated MNase digestion time points are shown. (B) MNase fragment size distribution of fragments within cenH3 domains (top) and regions between domains (bottom) for input and cenH3 ChIP after 2 min of MNase digestion. Domains were defined in Gassmann et al. (Gassmann et al., 2012). The MNase fragment size distribution of fragments crossing the center of the cenH3 nucleosome at cenH3 sites is shown in light blue for comparison, where the gray area marks the peak size interval.

DOI: http://dx.doi.org/10.7554/eLife.02025.018

Figure 5—figure supplement 3. Particle size estimation from overdigested insoluble chromatin.

Half-height width of the average signal of insoluble chromatin at cenH3 peaks after 10 min MNase. Average signal within a 1-kb window around all 707 cenH3 sites is shown.

DOI: http://dx.doi.org/10.7554/eLife.02025.019