Confirmation of functional heterozygotes for CENP-A location. (A) ChIP analysis of diploid lines FIBL1, FIBL2, and FIBL4. In FIBL1, CENP-A was enriched at both D17Z1 and D17Z1-B, indicating functional heterozygosity. CENP-A was enriched at D17Z1 in FIBL2, suggesting functional homozygosity. In FIBL4, CENP-A appeared more enriched on D17Z1, but immunostaining of FIBL4 revealed tetraploidy for HSA17. Immunostaining of FIBL3, the diploid version of FIBL4, showed one HSA17 with CENP-A at D17Z1 and the other with CENP-A at D17Z1-B (Fig. 3C). (B) Homologs (H) of FIBL3 were separated into mouse–human somatic cell hybrids. H1 and H2 were molecularly distinguished from one another using PCR for a polymorphic 17q locus. (C) CENP-A location was confirmed by ChIP on each HSA17 homolog. CENP-A was maintained stably at D17Z1 on H1 and at D17Z1-B on H2. Data represent three independent experiments. CENP-A enrichment at the target site is reported as percent of input shown relative to the CENP-A–negative site mouse 5s rDNA. Error bars represent SEM.