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Centromeres are defined by the presence of CENP-A nucleosomes in chromatin and are essential for accurate chromosome segregation. Centromeric chromatin epigenetically seeds new CENP-A nucleosome formation, thereby maintaining functional centromeres as cells divide. The features within centromeric chromatin that direct new CENP-A assembly remain unclear. Here, we developed a cell-free CENP-A assembly system that enabled the study of chromatin-bound CENP-A and soluble CENP-A separately. We show that two distinct domains of CENP-A within existing CENP-A nucleosomes are required for new CENP-A assembly and that CENP-A nucleosomes recruit the CENP-A assembly factors CENP-C and M18BP1 independently. Furthermore, we demonstrate that the mechanism of CENP-C recruitment to centromeres is dependent on the density of underlying CENP-A nucleosomes.
Figure 1. Reconstitution of CENP-A assembly in vitro. (a) Schematic of in vitro CENP-A assembly assay. (b) In vitro FLAGâxCENP-A assembly requires HJURP and mitotic exit. Extracts were supplemented with calcium, xHJURP RNA, or both. The top graph shows the means ± SEM; n = 5. The bottom images show FLAGâxCENP-A staining (FLAG) and bead autofluorescence (Beads). Bar, 5 µm. (c) Characterization of FLAGâxCENP-A assembly. (left) The levels of CENP-A (hCENP-A), histone H3, and histone H4 on the beads before extract addition. (right) The levels of FLAGâxCENP-A, CENP-C, and histone H4 in the extract before chromatin bead addition and after bead recovery (Extract Samples), and the chromatin beadâbound proteins after their recovery from the extract (Chromatin Samples). (d) Summary of quantitative Western blots (see Fig. S1 c) estimating the number of input nucleosomes (Myc-H4 signal) and newly assembled FLAGâxCENP-A nucleosomes per chromatin array; bars show the means ± SEM; n = 5.
Figure 2. CENP-C depletion prevents in vitro CENP-A assembly but not M18BP1 recruitment. (a) Levels of CENP-C, hCENP-A, and M18BP1 on chromatin beads after bead incubation in mock-depleted (â) or CENP-Câdepleted (Î) extracts. All bar graphs represent means ± SEM; n = 4. (b) The levels of CENP-C protein in egg extracts before (Input extracts) or after (Postchromatin extracts) incubation of chromatin beads in the extract and CENP-A assembly. The extracts were mock depleted (â), CENP-C depleted (Î), or complemented with CENP-C (+). Tubulin is shown as a loading control. (c) CENP-C signal on chromatin was assessed after the experiment described in b; n = 3. (d) Representative images showing FLAGâxCENP-A assembly on chromatin beads as described in b. The FLAGâxCENP-A signal (top row) and the bead autofluorescence (bottom row) are shown. Bar, 5 µm. (e) Quantification of FLAGâxCENP-A assembly assays described in d; n = 3.
Figure 3. CENP-A assembly on chromatin arrays requires M18BP1 isoform 2. (a, top) M18BP1 levels in egg extract after mock depletion (â), M18BP1 depletion (Î), or complementation with M18BP1 isoform 1 (bottom band of doublet), isoform 2 (top band of doublet) or both (1, 2, and 1/2, respectively). (bottom) A nonspecific band shown as a loading control. (b) M18BP1 (left) and FLAGâxCENP-A (right) levels on chromatin beads after M18BP1 depletion from egg extracts as described in a. All graphs show the means ± SEM, normalized to the mock-depleted CENP-A signal, n = 4. (c) CENP-C levels on chromatin beads after M18BP1 depletion as described in a.
Figure 4. CAC-mediated CENP-C recruitment is dependent on high nucleosome density. (a) Schematic of the histone chimeras used and the amino acid residues of human CENP-A (blue) and histone H3 (gray), respectively. (b) CENP-C (top) and M18BP1 (bottom) levels on low saturation chimeric nucleosome arrays in interphase. Signals are compared with the amount of recruitment to wild-type CENP-A beads and internally normalized in each sample to the levels of Myc-H4 on the beads. Bars in all panels represent the means ± SEM; n = 3. (c) Fluorescent images of CENP-C recruitment to low-saturation CENP-A, H3(CAC), and H3(CATD+CAC) chromatin arrays. The Myc-H4, CENP-C, and bead autofluorescence signals are shown. (d) CENP-C levels on CENP-A/H3 chimeric nucleosome arrays reconstituted at 0.5 µM nucleosome concentration in CSF-arrested extract. Signals are normalized as in b. (e) CENP-C (top) and M18BP1 (bottom) levels on high-saturation chimeric nucleosome arrays. Signals are normalized as in b. The signal on low-saturation CENP-A arrays (CENP-A, 0.5 µM) are shown for comparison. (f) Fluorescence images of CENP-C levels on high-saturation CENP-A, H3(CAC), and H3(CATD+CAC) chromatin arrays. Images labeled as in c. Bars, 5 µm.
Figure 5. FLAGâxCENP-A assembly requires the CATD and CAC. (a) Fluorescence images of FLAGâxCENP-A assembly on low saturation chimeric chromatin. Myc-H4, FLAGâxCENP-A, and bead autofluorescence are shown. (b) Quantification of FLAGâxCENP-A assembly as shown in a; all bars represent means ± SEM normalized to the signal on CENP-A arrays; n = 4. (c) FLAGâxCENP-A assembly on high saturation CENP-A/H3 chimeric chromatin. Normalized as in b. n = 4. (d) Fluorescence images of FLAGâxCENP-A assembly on high saturation CENP-A/H3 chimeric chromatin. Labeled as in a. (e) Protein levels of CENP-C, FLAGâxCENP-A, Myc-H3, hCENP-A, and H3 with son saturated chromatin arrays containing 80% hCENP-A nucleosomes and 20% Myc-H3 nucleosomes after incubation in extract supplemented with buffer (â) or with FLAG_xCENP-A and xHJURP RNA (+). Bars, 5 µm.
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