Bulk chromatin and centromeric chromatin were solubilized by MNase digestion of HeLa nuclei in 0.3 M NaCl. (A) Centromeric proteins CENP-A, -B, and -C were solubilized by MNase digestion. Isolated HeLa nuclei (2 × 10⁸) were suspended with 1 ml of WB containing 0.3 M NaCl (sample a in lane 1 and sample c in lanes 4 to 6) or 0.6 M NaCl (sample b in lanes 2 and 3). Sample c was digested with 60 U of MNase per ml for 10 min at 37°C. Soluble and insoluble materials from each sample were separated by centrifugation. ACA beads were added to the supernatant of sample c and incubated overnight at 4°C. Pellets were resuspended in 1 ml of SDS buffer by extensive sonication and 5 μl of each sample was separated by SDS-7.5% (for CENP-B and CENP-C) or 12.5% (for CENP-A) PAGE, and centromeric proteins were detected by immunostaining with ACA serum. Lane 1, supernatant fraction of a; lane 2, supernatant fraction of b; lane 3, pellet fraction of b; lane 4, supernatant fraction of c before addition of ACA beads; lane 5, supernatant fraction of c after addition of ACA beads; lane 6, pellet fraction of c. Lane M, marker centromeric proteins, CENP-A, CENP-B, and CENP-C. (B) Size distribution of DNA fragments from bulk chromatin after MNase digestion. HeLa nuclei were digested with MNase to various extents. The fragmented DNA in the soluble fractions was extracted with phenol and electrophoresed through 1% agarose gel. DNA was detected with ethidium bromide staining. Lane 1, 20 U/ml for 2 min (40 U/ml × min, sample 1); lane 2, 20 U/ml for 4 min (80 U/ml × min, sample 2); lane 3, 40 U/ml for 5 min (200 U/ml × min, sample 3); lane 4, 80 U/ml for 45 min (3,600 U/ml × min, sample 4). Positions of the DNA size markers are indicated at the left. (C) Detection of core histones and CENP-A in each fraction. Soluble (sup.) and insoluble (pellet) fractions were subjected to SDS-12.5% PAGE, and the separated core histones were stained with Coomassie brilliant blue (upper panel). The proteins were transferred to a membrane and immunolabeled with ACA serum (AK) (lower panel). Lane M in the lower panel is a recombinant CENP-A marker protein. Lanes 1 to 4 correspond to samples 1 to 4 of the soluble (sup.) fractions, and lanes 5 to 8 to samples 1 to 4 of the pellet fractions.

CENP-A, CENP-B, and CENP-C were coprecipitated by CHIP using anti-CENP-A and/or anti-CENP-C antibodies. (A) Indirect immunofluorescence microscopy using newly prepared anti-CENP-A (top right) and anti-CENP-C (top left) antibodies. Mitotic chromosomes from HeLa cells were stained with anti-CENP-A or anti-CENP-C antibodies. Second antibodies were anti-mouse IgG-fluorescein isothiocyanate (FITC) conjugate for CENP-A, and anti-guinea pig IgG-rhodamine isothiocyanate (RITC) conjugate for CENP-C. Chromosomes were stained with DAPI (4′,6′-diamidino-3-phenylindole, bottom left). The three panels were merged (bottom right). CENP-A, green; CENP-C, red; DNA, white. (B) CHIP of the solubilized chromatin. Isolated HeLa nuclei were digested with 40 U of MNase per ml for 5 min (lanes 1 and 3) or 80 U/ml for 45 min (lanes 2 and 4), and the soluble fractions were subjected to CHIP using anti-CENP-A (lanes 1 and 2) or anti-CENP-C (lanes 3 and 4) antibodies. The precipitated proteins were separated by SDS-7.5% (for CENP-B and -C) or 12.5% (for CENP-A) PAGE, and the centromere proteins were detected by Western blotting using ACA serum (AK). Lane M shows the positions of CENP-A, CENP-B, and CENP-C. Positions of molecular size markers are indicated at the right.

α-Satellite DNA fragments with CENP-B boxes were concentrated by CHIP using anti-CENP-A, anti-CENP-B, or anti-CENP-C antibodies. Before IP, clones prepared from the solubilized bulk chromatin before immunoprecipitation. IP, DNA fragments immunoprecipitated by anti-CENP-A (αCA), anti-CENP-B (αCB), or anti-CENP-C (αCC) antibodies were cloned into T vector and sequenced. More than 80% of the cloned fragments were longer than 500 bp. The black bars show the number of clones with α-satellite DNA that contained the CENP-B box. The open bars show the number of clones that were unique and contained no other repetitive sequence, not even α-satellite DNA without the CENP-B box.

Mapping of hypersensitive sites for MNase digestion on αI-type arrays. (A) Strategy for in vivo mapping of cleavage sites by MNase digestion using CENP-B box primers or mid-primers. CENP-B boxes were located mainly in α-satellite dimer subfamilies. Dimer repeats are marked with thick vertical bars. CENP-B boxes and mid sequences are shown with solid and open boxes, respectively. The distances from the CENP-B box or the mid sequence to the hypersensitive sites for MNase digestion were measured by primer extension analyses using CENP-B boxes or mid sequences as primers. The primer for each sequence is indicated by a thick bar, and the direction of DNA synthesis is shown with arrows. Owing to the occasional deletion of 2 to 4 bases to the right of the CENP-B box (indicated by x in the figure), direct sequencing reactions were sometimes unreadable because of overlapping of the sequence ladder. (B) Agarose gel electrophoresis of HeLa DNA after MNase digestion. Purified DNA (20 μg, 0.1 ml; lanes 2 to 4) or nuclei (5 × 10⁶, 0.1 ml; lanes 1 and 5 to 8) extracted from HeLa cells were digested with MNase (0.05 U for purified DNA and 15 U for nuclei) for 0 min (lane 1), 1 min (lane 2), 5 min (lane 3), 15 min (lane 4), 1 min (lane 5), 2 min (lane 6), 5 min (lane 7), and 20 min (lane 8). DNA from each sample was extracted with phenol and electrophoresed through a 1.7% agarose gel. Lane M shows a 100-bp DNA ladder. (C and D) PCR-primer extension analyses of the MNase digests with CENP-B box primers (left and middle panels) or mid primers (right panels). Left and middle panels, DNA (1 μg) from each of the digests shown in B was subjected to PCR-primer extension reactions using ³²P-labeled CENP-B box l-primer (C) or r-primer (D), and the samples were electrophoresed for 4 h (left panels) or 6 h (middle panels). Lanes 1 to 8 correspond to the samples shown in lanes 1 to 8 of B. Lanes 9 to 12 show sequence ladders as position markers. Lane M is a ³²P-labeled MspI digest of pBR322. The distance in bases from the CENP-B primer is indicated at the left. The area of the CENP-B box is marked with a shaded box, and hypersensitive bands are marked with asterisks (*). Right panels, higher-resolution analysis of the CENP-B box regions using mid l-primer (C) or r-primer (D). Lane 1 corresponds to lane 4 of B, and lanes 2 and 3 to lanes 6 and 7 of B. Lanes 4 to 7 show sequence ladders as position markers. The CENP-B box is indicated by a bracket at the right. The distance in bases from the CENP-B box is indicated at the right. (E) Mapping of MNase-hypersensitive sites around the CENP-B box and x-box regions. The DNA sequence obtained by direct sequencing of HeLa nuclei using the CENP-B box l-primer is shown (C, left, lanes 9 to 12) (61). The nucleotide position is indicated at the top as the distance in bases from the 5′ terminus of CENP-B box r-primer. Numbering around the CENP-B box region (left half of the figure) is counted from the adjacent CENP-B box. Position 342 corresponds to position 0, which is the start site for mapping around the x-box region (right half of the figure). Hypersensitive sites in the l-strand are indicated by upward-pointing arrowheads, and those in the r-strand by downward-pointing arrowheads. The upward-pointing open arrowheads with brackets at the left side of the CENP-B box indicate approximate loci (within brackets) for hypersensitive sites, because the DNA sequence of this region could not be read by direct sequencing. The CENP-B box is indicated by an open box (positions 1 to 17 or 343 to 359) and the corresponding area in the adjacent α-satellite monomer that has no affinity to CENP-B is shown as an open box (box) (positions 172 to 189). The mid-primer and x-box areas are indicated by brackets. R is A or G; Y is T or C; W is A or T; and S is G or C. (F) Mapping of 5′ termini of the α-satellite DNA fragments recovered by CHIP using anti-CENP-A antibody. 5′ termini of each strand from 94 clones of α-satellite DNA fragments (Fig. 3) were mapped on the α-satellite dimer repeat. The length of each vertical bar represents the frequency of the 5′ terminus mapping to each position on the sequence. The distance from the 5′ terminus of the CENP-B box r-strand is shown in bases at the top. Base 0 corresponds to base 342 from the adjacent CENP-B box.

Size distributions of the centromeric chromatin separated by glycerol gradient sedimentation were examined by CHIP using anti-CENP-A or anti-CENP-C antibodies. HeLa nuclei (6 × 10⁸, 3 ml) were digested with MNase for 15 min at 60 U/ml, and the solubilized chromatin fractions were centrifuged through a 5 to 20% glycerol density gradient. Aliquots of 2 ml were fractionated from the bottom. DNA was extracted from 10 μl of each fraction by phenol after proteinase K digestion and electrophoresed through a 1% agarose gel (top). Each 1.5-ml aliquot was subjected to CHIP analysis using anti-CENP-A (middle) or anti-CENP-C (bottom) antibodies. Distributions of CENP-A, CENP-B, and CENP-C were detected by Western blotting using ACA serum (AK) after SDS-7.5% (for CENP-B and CENP-C) and 12.5% (for CENP-A) PAGE. Marker proteins, catalase (240 kDa) and thyroglobulin (670 kDa) were centrifuged in parallel, and their positions are indicated at the top.

Quantitative analysis of CENP-A, -B, and -C in CENP-A/B/C chromatin. The four MNase digests shown in Fig. 1B were immunoprecipitated with anti-CENP-A antibodies (lanes 1 to 4; 12-μl samples of αCA). The supernatants were subsequently precipitated with ACA serum (lanes 5 to 8; 12-μl samples of ACA) to isolate and detect the residual CENPs. The precipitated proteins were resolved by SDS-12.5% PAGE and immunoblotted using ACA serum. Because of overloading of CENP-A in the αCA samples (lanes 2 to 4), 1.5-μl samples were also immunoblotted and are shown below. The diffuse bands between CENP-A and CENP-B in both samples were IgGs released from the antibody-Sepharose beads. Lane M shows a CENP-A/B/C marker mixture containing CENP-A (61 fmol/μl), CENP-B (23 fmol/μl), and CENP-C (25 fmol/μl), A/B/C molar ratio = 2.7:1:1.

Proposed model for the distribution of CENPs. (A) The A/B/C chromatin complex (CENP-A nucleosome/CENP-B/CENP-C complex; red) occupies 50% of the αI-type array, and the normal nucleosome/CENP-B complex (green) occupies the remaining 50%. (B) The distribution of CENP-A, -B, and -C in S- and G₂-phases as seen by light microscopy. Red represents the prekinetochore, which contains the A/B/C chromatin complex. Green represents the centromeric heterochromatin, which consists of the normal nucleosome/CENP-B complex. (C) The figure represents the results of electron microscopy observations and the present results in M-phase. The CENP-A/B/C chromatin complex (red) constitutes the inner kinetochore domain, and the CENP-B/normal nucleosome complex (green) constitutes the pairing domain.