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First published online September 2, 2003
doi: 10.1242/10.1242/jcs.00697

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Epigenetic assembly of centromeric chromatin at ectopic -satellite sites on human chromosomes

Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan

View larger version (24K): [in a new window]   Fig. 1. Cell lines obtained by transfection of an alphoid YAC constructs into HT1080 cells. (A) Following transfection of 21-I alphoid YAC (7C5hTEL) DNA, the de novo assembly of functional centromeres was observed coincident with MAC formation. MAC formation was not observed for transfection with 21-II alphoid YAC (B13hTEL). 7C5HT1-19 is a subclone of a MAC-containing cell line (7C5HT1). (B) The structure and copy number of the alphoid YAC in the 7C5HT1 and derivative cell lines were determined by Southern blot analysis (Ikeno et al., 1998; Masumoto et al., 1998).

View larger version (37K): [in a new window]   Fig. 5. Effects of TSA treatment on the ectopic alphoid YAC sites. (A) 7C5HT1-19 cells were analyzed after 2 days of TSA treatment or after a further 7 days of culture without TSA, by western blotting using anti-acetylated histone H3 (upper) and H4 (lower) antibodies. (B) 7C5HT1-19 cells were treated with TSA and applied to TAU gel (left and middle) and SDS gel (right) and analyzed by western blotting with anti-acetylated histone H4 (Lys 12) or anti-CENP-A antibody. (C) Proportions of cells containing a minichromosome. Cells were treated with the indicated concentrations of TSA for 2 days and then cultured for 7 days without TSA. More than 50 metaphase cells were analyzed for each sample. Bars indicate s.e.m. (D) ChIP analysis of TSA-treated 7C5HT1-19 cells using anti-acetylated histone H3 antibody. HT1-19 cells were treated with 1 µg/ml of TSA for 2 days and then cultured for 7 days without TSA. Immunoprecipitated DNA was quantitated by real-time PCR using primers indicated in Fig. 4A. The bar chart shows the percentage of recovery by immunoprecipitation (% IP) using normal IgG or anti-acetylated histone H3 antibody. The colors of the columns denote the different DNA sequences as shown in lower panel. Bars indicate s.e.m. (E) Relative transcription levels of the bsr gene in TSA-treated cells were analyzed by real-time RT-PCR. The transcription levels of the ß-actin gene were not significantly changed by the TSA treatment in this analysis, so each level of bsr transcript was normalized to that of the ß-actin transcript. Bars indicate s.e.m.

View larger version (46K): [in a new window]   Fig. 3. Minichromosome re-formation in the cell line 7C5HT1-19. (A) The proportion of cells containing minichromosomes (solid line) or the integrated alphoid YAC site (dashed line). 7C5HT1-19 cells were cultured in selective (square) or nonselective (circle) medium for the number of days indicated. More than 50 metaphase cells were analyzed for each observation. (B) 7C5HT1-19 metaphase chromosomes after 80 days in culture in either nonselective (left) or selective (middle and right) medium. FISH probes specific to the whole of human chromosome 16 (green) and to YAC arm sequences (red) were used. Arrowheads indicate alphoid YAC DNA. Scale bar: 2 µm. (C) FISH signal intensities with the chromosome 16 probe on re-formed minichromosomes relative to that of the host chromosome 16. The diamond indicates the average intensity for the short arm of chromosome 16. Bars indicate maximum and minimum values. (D) Assembly of CENPs on re-formed minichromosomes. Indirect immunofluorescence and simultaneous staining by FISH was performed to detect CENPs (green) and the YAC arm (red). Arrowheads indicate minichromosomes. Scale bar: 2 µm. (E) Stability of re-formed minichromosomes in 7C5HT1-19 cells after 60 days in nonselective medium. (F) RT-PCR analysis of bsr transcripts. (Upper) Portions of RT-PCR products amplified with primers specific to the bsr gene and the human ß-actin gene (1, 1/2, 1/4 and 1/10, 1/20, 1/40, respectively) were electrophoresed in agarose gel. (lower) Ratios of transcriptional products of bsr to those of the 7C5HT1-19 cell line at day 0 quantitated by real-time RT-PCR. Both analyses showed the same transcription levels of bsr. The relative copy number of bsr genes was quantitated by real-time PCR.

View larger version (43K): [in a new window]   Fig. 2. Assembly and disassembly of CENPs on an integrated alphoid YAC. Metaphase chromosomes from the cell lines 7C5HT1-19 (A-F) and B13HT1 (M-O) were analyzed by FISH with the 21-I probe (red in A,B) or the YAC arm probe (red in C-F,M-O), in combination with immunofluorescence using antibodies against CENPs (green in A-F,M-O). Scale bars: 2 µm. Chromosomes were counterstained with DAPI (blue). Arrowheads indicate the integration sites of the alphoid YAC. (G-L) Plots of the intensity profile of immunofluorescence signals (black) for anti-CENP antibodies and FISH signals (red) along the dotted line on chromosome 16 shown in A-F. Intensities were measured between the positions marked by the star and the asterisk, as shown in merged images of A-F. (P) The intensity of CENP-B immunofluorescence at each alphoid YAC integration site relative to that of the resident centromere on chromosome 16 was plotted. The red dotted line indicates the lower limit of CENP-B signal that is above background level. (Q) The proportion of cells containing the CENPs signals on the alphoid YAC DNA sites. Bars indicate s.e.m. (standard error of mean).

View larger version (25K): [in a new window]   Fig. 4. ChIP analysis of CENP-A and CENP-B on introduced alphoid YAC DNA. (A) Positions of PCR primer pairs on alphoid YAC DNA are indicated. (B) Standard curves of real-time PCR using the primer pairs indicated in A and sequentially diluted input DNAs. All the primer pairs showed a concentration-dependent amplification of the target sequences. (C-F) Fixed and sonicated chromatin from the cell lines indicated was immunoprecipitated with mouse normal IgG, anti-CENP-A antibody, and anti-CENP-B antibody. The immunoprecipitated DNA was quantitated by real-time PCR using the primers indicated in A. (C) The bar charts show the percentage recovery (% IP) of 5S ribosomal DNA fragment by immunoprecipitation using the indicated antibodies. (D-F) The bar charts show the relative enrichment, calculated by dividing the % IP of each DNA region by that of the 5S ribosomal DNA fragment immunoprecipitated by normal mouse IgG (D), anti-CENP-A antibody (E) and anti-CENP-B antibody (F). ChIP analysis was performed four times and the average of the results is presented. The colors of the columns denote the different DNA sequences as shown in A. Bars indicate s.e.m.

View larger version (47K): [in a new window]   Fig. 6. De novo assembly of CENPs at the integrated 10 kb alphoid YAC DNA site without chromosome rearrangement. (A-G) Metaphase chromosomes from the del.22HT1-3 cell line, untreated (A,C,E) or treated (B,D,F,G) with 1000 ng/ml of TSA, analyzed by immunofluorescence using antibodies against CENPs (green), and FISH using the YAC arm probe (red). The chromosomes were counterstained with DAPI (blue). Arrowheads indicate the integration sites of the 10 kb alphoid YAC. Scale bars: 2 µm. (H-M) Intensity plot of CENP signals (black) and YAC signals (red) at the 10 kb alphoid YAC site on the host chromosome DNA. (N) The plots of relative intensity of CENP-A (upper), -B (middle), -C (lower) signals at each alphoid YAC integration site on the host centromeres in the del.22HT1-3 cell line untreated (square) or treated (diamond) with 1000 ng/ml of TSA. Red dotted lines indicate the lower limit of signals distinguishable from background levels.

View larger version (28K): [in a new window]   Fig. 7. Schematic representation of suppressed centromeres and functional centromere chromatin, and hypothetical models for the mechanism of assembly of centromere components on alphoid YAC. (A) On an artificial chromosome with a functional centromere/kinetochore structure, CENP-A and CENP-B assemble onto alphoid arrays in the introduced alphoid YAC DNA but not on YAC arms. Bsr genes on the YAC arms are transcribed, and histone H3 in chromatin on the genes is acetylated. (B) On the ectopic alphoid YAC loci, disassembly of centromere proteins from the insert alphoid arrays coincides with the reduction of the transcriptional activity from the bsr gene on the YAC arms and with the hypoacetylated level of histone H3 of the gene. (C) Increased gene expression on the YAC arms and hyperacetylation of histones by TSA causes structural change in chromatin at the integrated alphoid YAC DNA. Centromeric proteins can re-assemble at such regions and active centromeres re-form. Selection pressure might induce this process gradually during long-term culture.