Fig. 5

CENP-B- and CENP-I-APEX extend further than the CENP-A-bound centromere domain. Representative images of chromatin fibers prepared from cells expressing CENP-I-APEX (a) or APEX-CENP-B (c), stained for CENP-A and biotin. Scale bar: 2.5 μm. Intensity plots for CENP-A and biotin gray values along the length of the fiber (in μm) are shown on the right. For presentation purposes, CENP-A gray values in APEX-CENP-B plot were subjected to a fourfold increase. Distribution of biotin peaks compared to APEX-CENP-A (mean with SEM), for CENP-I (b) and CENP-B (d). Dark gray: percentage of peaks co-localizing with CENP-A, light gray: percentage of peaks not co-localizing with CENP-A inside the CENP-A-bound domain, black: percentage of peaks found outside the CENP-A domain. e Mean percentages of total fibers analyzed showing spreading for CENP-A, CENP-B and CENP-I APEX fusion proteins. FDR adjusted unpaired Student’s t test p values ≤ 0.05 are represented by *. n = four experiments for CENP-A and five experiments for CENP-B and CENP-I APEX fusion proteins. Error bars: SD. f Scatter plot depicting the distance of spreading of biotin peaks from the CENP-A domain. Each fiber is represented by two dots, for the distance of spreading left and right of the centromere domain. Error bars represent mean with SD. g Mean ratio of biotin to CENP-A domain size. Error bars: SD. FDR adjusted Kolmogorov–Smirnov p values are represented as follows: ns for p values > 0.05, * for p values ≤ 0.05 and ** for p values < 0.001. n = 42 fibers for CENP-A (from four experiments), 24 for CENP-B (from five experiments) and 30 for CENP-I (from five experiments). (H) Summary of the three association pattern categories. The first category includes CENP-C, -N and -T, which are confined inside the centromere domain but are differently organized than APEX-CENP-A. The second category includes CENP-P, -M and -K which are organized similarly to APEX-CENP-A. The last category includes CENP-I and CENP-B, which extend further than the centromere domain