CENP-A is phosphorylated by Aurora B kinase and plays an unexpected role in completion of cytokinesis.

Aurora B is a mitotic protein kinase that phosphorylates histone H3, behaves as a chromosomal passenger protein, and functions in cytokinesis. We investigated a role for Aurora B with respect to human centromere protein A (CENP-A), a centromeric histone H3 homologue. Aurora B concentrates at centromeres in early G2, associates with histone H3 and centromeres at the times when histone H3 and CENP-A are phosphorylated, and phosphorylates histone H3 and CENP-A in vitro at a similar target serine residue. Dominant negative phosphorylation site mutants of CENP-A result in a delay at the terminal stage of cytokinesis (cell separation). The only molecular defects detected in analysis of 22 chromosomal, spindle, and regulatory proteins were disruptions in localization of inner centromere protein (INCENP), Aurora B, and a putative partner phosphatase, PP1gamma1. Our data support a model where CENP-A phosphorylation is involved in regulating Aurora B, INCENP, and PP1gamma1 targeting within the cell. These experiments identify an unexpected role for the kinetochore in regulation of cytokinesis.

Chromatin assembly at kinetochores is uncoupled from DNA replication.

The specification of metazoan centromeres does not depend strictly on centromeric DNA sequences, but also requires epigenetic factors. The mechanistic basis for establishing a centromeric "state" on the DNA remains unclear. In this work, we have directly examined replication timing of the prekinetochore domain of human chromosomes. Kinetochores were labeled by expression of epitope-tagged CENP-A, which stably marks prekinetochore domains in human cells. By immunoprecipitating CENP-A mononucleosomes from synchronized cells pulsed with [(3)H]thymidine we demonstrate that CENP-A-associated DNA is replicated in mid-to-late S phase. Cytological analysis of DNA replication further demonstrated that centromeres replicate asynchronously in parallel with numerous other genomic regions. In contrast, quantitative Western blot analysis demonstrates that CENP-A protein synthesis occurs later, in G2. Quantitative fluorescence microscopy and transient transfection in the presence of aphidicolin, an inhibitor of DNA replication, show that CENP-A can assemble into centromeres in the absence of DNA replication. Thus, unlike most genomic chromatin, histone synthesis and assembly are uncoupled from DNA replication at the kinetochore. Uncoupling DNA replication from CENP-A synthesis suggests that regulated chromatin assembly or remodeling could play a role in epigenetic centromere propagation.

CENP-A associated complex satellite DNA in the kinetochore of the Indian muntjac.

The centromere/kinetochore complex is a chromosomal assembly that mediates chromosome motility and mitotic regulation by interacting with microtubules of the mitotic spindle apparatus. Centromere protein A (CENP-A) is a histone H3 homolog that is concentrated in the chromatin of the inner kinetochore plate of human chromosomes. To identify DNA sequences associated with the inner kinetochore plate, we used anticentromere autoantibodies to immunoprecipitate CENP-A associated chromatin selectively from Indian muntjac fibroblasts. DNA was cloned from immunoprecipitated CENP-A- associated chromatin and characterized by DNA sequence and hybridization analyses. A novel centromeric satellite DNA sequence was identified and shown by fluorescence in situ hybridization analysis to be present at all centromeres of the Indian muntjac. This satellite DNA constitutes a 972 bp monomer repeat and shows partial homology with satellite II DNA of the white-tailed deer. Southern blot analysis of muntjac genomic DNA suggests that this satellite DNA is present in repetitive tandem arrays and contains complex internal arrangements. In conjunction with previous work showing the association of CENP-A with human alpha-satellite DNA, we conclude that the mammalian inner kinetochore plate contains a unique form of chromatin that contains CENP-A in association with complex satellite DNA.

Using time-lapse confocal microscopy for analysis of centromere dynamics in human cells.

Using these methods we have shown that the alpha-satellite domain of the human centromere behaves as an elastic element, stretching in response to spindle forces applied during prometaphase and metaphase (Shelby et al., 1996). These data complement previous observations of centromere stretching during mitosis (e.g., Skibbens et al., 1993) by demonstrating a specific molecular compartment within the centromere, the satellite heterochromatin domain, that supports this strain. Centromere stretching reports on the net force applied across the centromere during mitosis and the availability of a fluorescence-based assay system in human cells provides a robust assay system to complement the elegant DIC-based methods that have been perfected using marsupial and newt cell cultures (Cassimeris et al., 1990; Skibbens et al., 1993, 1995; Rieder et al., 1994). Current applications of this method are directed toward examining the relationship between centromere tension and microtubule dynamics using pharmacological approaches and the behavior of kinetochore-associated regulatory proteins, such as Mad2 and Bub1 (Li and Benezra, 1996; Chen et al., 1996; Taylor and McKeon, 1997), as a function of centromere distortion. In addition, GFP-labeled centromeres can be observed during interphase, providing a novel window into chromosome organization within the nucleus. Our observations show that centromeres distribute into the newly forming nucleus at telophase by what is apparently a uniform isometric expansion, with little evidence for directed motion of individual centromeres contributing to the formation of the G1 nucleus. During interphase, centromeres show very little movement in general, behaving as though embedded in a rigid matrix. Sustained movements of individual centromeres or groups of centromeres are occasionally observed, however, suggesting that chromosome position is subject to change during interphase (Shelby et al., 1996). These experiments complement those described by Belmont and colleagues, who have developed a method to mark specific chromosomal sites with GFP for analysis in vivo (see Chapter 13; Robinett et al., 1996; Straight et al., 1996). These new GFP-based techniques for direct observation of defined DNA sequence domains in vivo carry the logic of in situ hybridization analysis into living cells and, coupled with new methods for observing global chromatin architecture as well as functional nuclear protein domains (Huang et al., 1997; Misteli et al., 1997), promise significant progress toward understanding the dynamic organization of the genome within the living nucleus.

Assembly of CENP-A into centromeric chromatin requires a cooperative array of nucleosomal DNA contact sites.

We investigated the requirements for targeting the centromeric histone H3 homologue CENP-A for assembly at centromeres in human cells by transfection of epitope-tagged CENP-A derivatives into HeLa cells. Centromeric targeting is driven solely by the conserved histone fold domain of CENP-A. Using the crystal structure of histone H3 as a guide, a series of CENP-A/histone H3 chimeras was constructed to test the role of discrete structural elements of the histone fold domain. Three elements were identified that are necessary for efficient targeting to centromeres. Two correspond to contact sites between histone H3 and nucleosomal DNA. The third maps to a homotypic H3-H3 interaction site important for assembly of the (H3/H4)2 heterotetramer. Immunoprecipitation confirms that CENP-A self-associates in vivo. In addition, targeting requires that CENP-A expression is uncoupled from histone H3 synthesis during S phase. CENP-A mRNA accumulates later in the cell cycle than histone H3, peaking in G2. Isolation of the gene for human CENP-A revealed a regulatory motif in the promoter region that directs the late S/G2 expression of other cell cycle-dependent transcripts such as cdc2, cdc25C, and cyclin A. Our data suggest a mechanism for molecular recognition of centromeric DNA at the nucleosomal level mediated by a cooperative series of differentiated CENP-A-DNA contact sites arrayed across the surface of a CENP-A nucleosome and a distinctive assembly pathway occurring late in the cell cycle.

Dynamic elastic behavior of alpha-satellite DNA domains visualized in situ in living human cells.

We have constructed a fluorescent alpha-satellite DNA-binding protein to explore the motile and mechanical properties of human centromeres. A fusion protein consisting of human CENP-B coupled to the green fluorescent protein (GFP) of A. victoria specifically targets to centromeres when expressed in human cells. Morphometric analysis revealed that the alpha-satellite DNA domain bound by CENPB-GFP becomes elongated in mitosis in a microtubule-dependent fashion. Time lapse confocal microscopy in live mitotic cells revealed apparent elastic deformations of the central domain of the centromere that occurred during metaphase chromosome oscillations. These observations demonstrate that the interior region of the centromere behaves as an elastic element that could play a role in the mechanoregulatory mechanisms recently identified at centromeres. Fluorescent labeling of centromeres revealed that they disperse throughout the nucleus in a nearly isometric expansion during chromosome decondensation in telophase and early G1. During interphase, centromeres were primarily stationary, although motility of individual or small groups of centromeres was occasionally observed at very slow rates of 7-10 microns/h.