Fig. 5.

Immuno-FISH and ChIP analyses of the alphoid^tetO-HAC/NBS1. (A) ChIP analysis of centromeric chromatin in alphoid^tetO-HAC/NBS1 clones 3 and 6. Normal mouse IgG (Top), antibodies against CENP-A, H3K4me2, H3K4me3, and H3K9me3 were used for analysis. The assemblies of these proteins on the alphoid DNA of the original alphoid^tetO-HAC in AB2.2.18.21 cell line (Left), the alphoid^tetO-HAC carrying the human NBS1 gene in NBS1 no. 3 and NBS1 no. 6 cell lines (Right), are shown. The bars show the percentage recovery of the various target DNA loci by immunoprecipitation with each antibody to input DNA. Error bars indicate SD (n = 2 or 3). Analyzed loci are alphoid^tetO (alphoid DNA with tetO motif on the alphoid^tetO-HAC), bsr (the marker gene in the HAC vector sequence), and 3′ and 5′ ends of NBS1. rDNA (5S ribosomal DNA), alphoid^chr.21 (centromeric alphoid DNA of chromosome 21), and sat2 (pericentromeric satellite 2) were used as controls. Immunoprecipitated DNAs were quantified by real-time PCR. (B) Positions of probes for ChIP analysis in the original HAC and in the HAC carrying the NBS1 gene are shown by colored boxes. (C) Immuno-FISH analysis of metaphase chromosome spreads containing the alphoid^tetO-HACs. Cells with the original alphoid^tetO-HAC (AB2.2.18.21) and with alphoid^tetO-HAC carrying the NBS1 gene (clones 3 and 6) were used for analysis. Immunolocalization of the centromeric protein CENP-A on metaphases was performed by indirect immunofluorescence with anti–CENP-A antibody and Alexa 488-conjugated secondary antibody (green). HAC-specific DNA sequence (BAC) was used as a FISH probe to detect the HAC (red). CENP-A and BAC signals on the HACs overlap one another.