Fig. 4. CENP-C is mistargeted with CENP-A during interphase. To determine if CENP-C is recruited to non-centromeric regions of chromatin to which CENP-A has been mistargeted, CENP-A was overexpressed and co-localized with other CENPs recognized by human autoimmune sera. A stable CHO cell line was used that expresses an HA-tagged human CENP-A cDNA from a CMV-based tetracycline-regulated promoter. (A) SH-CREST antiserum, which primarily recognizes CENPs-A, -B, and -C, stains exclusively centromeres in uninduced control interphase CHO cells. A low level of anti-HA antibody background staining is observed. DNA was counterstained with DAPI. (B) SH-CREST was found to localize throughout the interphase chromatin of CHO cells that were induced to express the HA-tagged CENP-A transgene. CENP-A-(HA) was detected using anti-HA antibody. (C) To determine if the non-centromeric staining of CREST observed following CENP-A overexpression was in part due to the mistargeting of factors other than CENP-A recognized by the serum, CENP-A was overexpressed in the CHO cell line, and then eluted from fixed cell preparations by salt and DNase treatment prior to immunostaining. Exogenous centromeric and non-centromeric CENP-A was significantly reduced in extracted preparations when compared with unextracted (B), as detected by immunofluorescence using anti-HA antibody. Speckles of CENP-A remained at non-centromeric sites in the extracted preparations, perhaps corresponding to matrix-associated regions of the genome. Importantly, centromeric and non-centromeric SH-CREST staining remained following the elution of CENP-A, indicating the presence of CENP-B and/or -C at the ectopic sites. Bars, 10 µm.
