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The attachment of sister kinetochores to microtubules from opposite spindle poles is essential for faithful chromosome segregation. Kinetochore assembly requires centromere‐specific nucleosomes containing the histone H3 variant CenH3. However, the functional roles of the canonical histones (H2A, H2B, H3, and H4) in chromosome segregation remain elusive. Using a library of histone point mutants in Saccharomyces cerevisiae , 24 histone residues that conferred sensitivity to the microtubule‐depolymerizing drugs thiabendazole (TBZ) and benomyl were identified. Twenty‐three of these mutations were clustered at three spatially separated nucleosomal regions designated TBS‐I, ‐II, and ‐III ( T BZ/ b enomyl‐ s ensitive regions I–III). Elevation of mono‐polar attachment induced by prior nocodazole treatment was observed in H2A‐I112A (TBS‐I), H2A‐E57A (TBS‐II), and H4‐L97A (TBS‐III) cells. Severe impairment of the centromere localization of Sgo1, a key modulator of chromosome bi‐orientation, occurred in H2A‐I112A and H2A‐E57A cells. In addition, the pericentromeric localization of Htz1, the histone H2A variant, was impaired in H4‐L97A cells. These results suggest that the spatially separated nucleosomal regions, TBS‐I and ‐II, are necessary for Sgo1‐mediated chromosome bi‐orientation and that TBS‐III is required for Htz1 function.

The chromosomal passenger complex consisting of Borealin, INCENP, Survivin, and Aurora B follows a dynamic pattern of localization to perform its role as a regulator of chromosome alignment, aspects of the spindle assembly checkpoint, and cytokinesis. Post-translational modifications of chromosomal passenger proteins play an important role in regulating the localization and function of the complex. Borealin displays a slower electrophoretic mobility during mitosis as a result of phosphorylation. Here we show that phosphorylation at S219 is responsible for this mobility shift. An S219A mutant of Borealin that cannot be phosphorylated at this site displays a defect in centromere localization that is evident in cells arrested in mitosis with nocodazole. Further, the S219A form of Borealin is unable to efficiently rescue mitotic defects that occur upon knock-down of the endogenous protein. These defects are correlated with a reduction in the intensity of Mad2 staining at kinetochores in cells expressing the S219A form of Borealin. These results highlight an important role for phosphorylation of Borealin at S219 in the proper progression through mitosis.

The centromere is a chromatin structure essential for correct segregation of sister chromatids, and defects in this region often lead to aneuploidy and cancer. We have previously reported purification of the interphase centromere complex (ICEN) from HeLa cells, and have demonstrated the presence of 40 proteins (ICEN1-40), along with CENP-A, -B, -C, -H and hMis6, by proteomic analysis. Here we report analysis of seven ICEN components with unknown function. Centromere localization of EGFP-tagged ICEN22, 24, 32, 33, 36, 37 and 39 was observed in transformant cells. Depletion of each of these proteins by short RNA interference produced abnormal metaphase cells carrying misaligned chromosomes and also produced cells containing aneuploid chromosomes, implying that these ICEN proteins take part in kinetochore functions. Interestingly, in the ICEN22, 32, 33, 37 or 39 siRNA-transfected cells, CENP-H and hMis6 signals disappeared from all the centromeres in abnormal mitotic cells containing misaligned chromosomes. These results suggest that the seven components of the ICEN complex are predominantly localized at the centromeres and are required for kinetochore function perhaps through or not through loading of CENP-H and hMis6 onto the centromere.

Background The pericentromeric heterochromatin is an important element for the regulation of gene silencing. Its spatial distribution during interphase appears to be cell-type specific. This study analyzes three-dimensional (3D) centromere distribution patterns during cellular differentiation along the neutrophil pathway. Methods Differentiation of the promyelocytic leukemia cell line NB4 was induced by retinoic acid. Centromeres in interphase nuclei were visualized by immunofluorescence staining of centromere-associated proteins with CREST serum. 3D images of nuclei were obtained by confocal microscopy. Automated methods for the segmentation of point-like objects in 3D images were implemented to detect the position of centromeres. Features of centromere localization patterns were determined by constructing the minimal spanning tree of the centromere distribution. Results In differentiated NB4 cells, the number of centromere conglomerates (chromocenters) was decreased and the distance between chromocenters was increased as compared with untreated controls. The nuclear volume did not differ between the two groups. Conclusions The measured rearrangement of centromeres indicates a progressive clustering of heterochromatin and a global remodeling of interphase chromosome territories during differentiation of NB4 cells. The developed methods for the analysis of 3D centromere distribution patterns provide the opportunity for a fast and objective analysis of heterochromatin remodeling. Cytometry 47:217–225, 2002. © 2002 Wiley-Liss, Inc.