Fig. 7.
DAXX contributes to the mislocalization of CENP-A and CIN in CHAF1B-depleted cells. (A) Representative images of metaphase chromosome spreads showing the localization of CENP-A at centromeric and non-centromeric regions in HeLaCENP-A–TAP cells transfected with the indicated siRNAs for 72 h. Metaphase chromosome spreads were prepared 72 h post transfection, and cells were immunostained with an antibody against CENP-A and stained with DAPI. Scale bars: 5 µm. (B) Quantification of CENP-A signal intensities at centromeric (left) and non-centromeric (right) regions in metaphase chromosome spreads of HeLaCENP-A–TAP cells transfected with the indicated siRNAs. Each circle represents a spot on a centromeric or non-centromeric region. ‘Cells’ and ‘Chr’ denote the numbers of cells and chromosomes analyzed, respectively. Red lines indicated mean±s.d. for YFP signal intensities across areas measured in the number of cells indicated from three independent experiments. (C) The proportion of cells exhibiting defective chromosome segregation in HeLaCENP-A–TAP cells transfected with the indicated siRNAs. N denotes the number of cells analyzed. Error bars represent s.e.m. from three independent experiments. P-values shown in B,C were calculated using one way-ANOVA with Tukey's post hoc test. (D) Model for CENP-A mislocalization and CIN in CHAF1B-depleted cells. We propose that depletion of CHAF1B promotes DAXX-mediated mislocalization of CENP-A to non-centromeric regions. Support for this model includes the CUT&RUN sequencing data and the suppression of CENP-A mislocalization and CIN phenotypes by depletion of DAXX in CHAF1B-depleted cells.