Fig 2: A model for the CENPA/CENPB/CENPC/CENPT chromatin complex. (Left) Hypothesized arrangement of CENPA, B, C, and T along a single 340-bp Cen1-like dimeric unit. (Middle) During interphase, the CENPT complex occupies the CENPB box-containing linker on young 340-bp α-satellite dimers between tandem CENPA nucleosomes. Each CENPA particle binds a CENPC on one side and wraps ∼100-bp DNA with right-handed chirality. (Right) At anaphase, tension causes the positively supercoiled DNA to overwind and tighten up around the complex, favoring stacking of tandem dimers to form a stiff platform that spreads pulling forces.

Fig 3: CENPA, CENPC, and CENPT cross-link in a single chromatin complex. (A) Close correspondence between X-ChIP of CENPA, CENPC, and CENPT data sets. X-ChIP fragments were clustered based on sequence identity, and frequency distributions of fragments within clusters between data sets were compared. Scatter plots show the regression of the number of distinct mapped merged pairs for the CENPC, CENPT, and input X-ChIP data sets on the CENPA X-ChIP data set. (B) Real-time PCR analysis of DNA obtained by sequential ChIP with indicated pairs of antibodies. The S1 primer pair was designed as described in the legend to Figure 2, and the S3 primer pair was designed from the Cen1-like (SF1 family) sequences and is also present on Chromosomes 1, 5, and 19 (Supplemental Table 1). Fold enrichment over input was calculated relative to a control noncentromeric sequence from the 5S rDNA locus.

Fig 4: Sequential ChIP-seq data sets of CCAN components are enriched for centromeric α-satellites and are highly correlated. (A) Sequential ChIP-seq data sets show that abundant centromeric α-satellite arrays (D5Z2 and Cen1-like) are enriched for CENPA, CENPT, CENPC, and CENPB, whereas representative noncentromeric satellites (Xmono and D19Z1) are not. The reads were mapped to previously characterized α-satellite arrays (Hayden et al. 2013; Henikoff et al. 2015), and the number of reads mapped to an array were calculated (using Trim Galore!) as a fraction of the total number of mapped reads processed. (B) Close correspondence between sequential ChIP-seq data sets: CENPA/CENPA, CENPA/CENPC, and CENPA/CENPT. Scatter plots show the regression of the number of mapped reads for the CENPA/CENPA, CENPA/CENPC, CENPA/CENPT, and CENPA/GFP sequential ChIP data sets on the CENPA X-ChIP data set.

Fig 5: CENPT and CENPC bridge the gap between adjacent CENPA nucleosomes over the CENPB box on young α-satellite dimers. (A) Mapping of X-ChIP merged pairs to previously characterized α-satellite arrays (Hayden et al. 2013; Henikoff et al. 2015). The profiles display normalized counts as described in Methods, in which entire fragments are “stacked,” and for each base-pair position the total number of counts is measured on the y-axis. A single α-satellite dimer from each array is shown. The CENPB box region is indicated by the magenta box. Cen1-like and D5Z2 arrays contain CENPB boxes in every α-satellite dimer (dense) (Henikoff et al. 2015). The DXZ1 tandem array, which is a 12-copy HOR, contains CENPB boxes only in a subset of dimeric units of the array (sparse), and a single CENPB box-containing dimer, which is embedded in α-satellite units that lack a CENPB box, is shown. The D5Z1 array does not contain CENPB boxes. The relative scale is the area of the indicated profile divided by the area of the D5Z1 profile, setting the D5Z1 value to 1, in which the numbers reflect the product of the total sequence abundance and enrichment. For example, Cen1-like is 60-fold enriched in the X-ChIP input, reflecting higher sequence abundance, and 235-fold enriched in CENPA ChIP, which implies that ChIP enrichment per copy is 235/60 or approximately fourfold. Biological replicates using different CENPT antibodies gave nearly identical patterns and relative scale values (Supplemental Fig. S2). (B) Fragment ends from native and X-ChIP data sets were mapped to the Cen1-like consensus and DXZ1 sequences. The CENPB box region is indicated by the magenta box. The top panel shows ends from CENPA, CENPC, CENPT X-ChIP, and input. The bottom panel compares ends of CENPA reads in N-ChIP (solid line) and X-ChIP (dotted line).