A potential role for human cohesin in mitotic spindle aster assembly.

The cohesin multiprotein complex containing SMC1, SMC3, Scc3 (SA), and Scc1 (Rad21) is required for sister chromatid cohesion in eukaryotes. Although metazoan cohesin associates with chromosomes and was shown to function in the establishment of sister chromatid cohesion during interphase, the majority of cohesin was found to be off chromosomes and reside in the cytoplasm in metaphase. Despite its dissociation from chromosomes, however, microinjection of an antibody against human SMC1 led to disorganization of the metaphase plate and cell cycle arrest, indicating that human cohesin still plays an important role in metaphase. To address the mitotic function of human cohesin, the subcellular localization of cohesin components was reexamined in human cells. Interestingly, we found that cohesin localizes to the spindle poles during mitosis and interacts with NuMA, a spindle pole-associated factor required for mitotic spindle organization. The interaction with NuMA persists during interphase. Similar to NuMA, a significant amount of cohesin was found to associate with the nuclear matrix. Furthermore, in the absence of cohesin, mitotic spindle asters failed to form in vitro. Our results raise the intriguing possibility that in addition to its well demonstrated function in sister chromatid cohesion, cohesin may be involved in spindle assembly during mitosis.

Specification of kinetochore-forming chromatin by the histone H3 variant CENP-A.

The mechanisms that specify precisely where mammalian kinetochores form within arrays of centromeric heterochromatin remain largely unknown. Localization of CENP-A exclusively beneath kinetochore plates suggests that this distinctive histone might direct kinetochore formation by altering the structure of heterochromatin within a sub-region of the centromere. To test this hypothesis, we experimentally mistargeted CENP-A to non-centromeric regions of chromatin and determined whether other centromere-kinetochore components were recruited. CENP-A-containing non-centromeric chromatin assembles a subset of centromere-kinetochore components, including CENP-C, hSMC1, and HZwint-1 by a mechanism that requires the unique CENP-A N-terminal tail. The sequence-specific DNA-binding protein CENP-B and the microtubule-associated proteins CENP-E and HZW10 were not recruited, and neocentromeric activity was not detected. Experimental mistargeting of CENP-A to inactive centromeres or to acentric double-minute chromosomes was also not sufficient to assemble complete kinetochore activity. The recruitment of centromere-kinetochore proteins to chromatin appears to be a unique function of CENP-A, as the mistargeting of other components was not sufficient for assembly of the same complex. Our results indicate at least two distinct steps in kinetochore assembly: (1) precise targeting of CENP-A, which is sufficient to assemble components of a centromere-prekinetochore scaffold; and (2) targeting of kinetochore microtubule-associated proteins by an additional mechanism present only at active centromeres.

A human condensin complex containing hCAP-C-hCAP-E and CNAP1, a homolog of Xenopus XCAP-D2, colocalizes with phosphorylated histone H3 during the early stage of mitotic chromosome condensation.

Structural maintenance of chromosomes (SMC) family proteins play critical roles in structural changes of chromosomes. Previously, we identified two human SMC family proteins, hCAP-C and hCAP-E, which form a heterodimeric complex (hCAP-C-hCAP-E) in the cell. Based on the sequence conservation and mitotic chromosome localization, hCAP-C-hCAP-E was determined to be the human ortholog of the Xenopus SMC complex, XCAP-C-XCAP-E. XCAP-C-XCAP-E is a component of the multiprotein complex termed condensin, required for mitotic chromosome condensation in vitro. However, presence of such a complex has not been demonstrated in mammalian cells. Coimmunoprecipitation of the endogenous hCAP-C-hCAP-E complex from HeLa extracts identified a 155-kDa protein interacting with hCAP-C-hCAP-E, termed condensation-related SMC-associated protein 1 (CNAP1). CNAP1 associates with mitotic chromosomes and is homologous to Xenopus condensin component XCAP-D2, indicating the presence of a condensin complex in human cells. Chromosome association of human condensin is mitosis specific, and the majority of condensin dissociates from chromosomes and is sequestered in the cytoplasm throughout interphase. However, a subpopulation of the complex was found to remain on chromosomes as foci in the interphase nucleus. During late G(2)/early prophase, the larger nuclear condensin foci colocalize with phosphorylated histone H3 clusters on partially condensed regions of chromosomes. These results suggest that mitosis-specific function of human condensin may be regulated by cell cycle-specific subcellular localization of the complex, and the nuclear condensin that associates with interphase chromosomes is involved in the reinitiation of mitotic chromosome condensation in conjunction with phosphorylation of histone H3.

Identification of two distinct human SMC protein complexes involved in mitotic chromosome dynamics.

The structural maintenance of chromosomes (SMC) family member proteins previously were shown to play a critical role in mitotic chromosome condensation and segregation in yeast and Xenopus. Other family members were demonstrated to be required for DNA repair in yeast and mammals. Although several different SMC proteins were identified in different organisms, little is known about the SMC proteins in humans. Here, we report the identification of four human SMC proteins that form two distinct heterodimeric complexes in the cell, the human chromosome-associated protein (hCAP)-C and hCAP-E protein complex (hCAP-C/hCAP-E), and the human SMC1 (hSMC1) and hSMC3 protein complex (hSMC1/hSMC3). The hCAP-C/hCAP-E complex is the human ortholog of the Xenopus chromosome-associated protein (XCAP)-C/XCAP-E complex required for mitotic chromosome condensation. We found that a second complex, hSMC1/hSMC3, is required for metaphase progression in mitotic cells. Punctate vs. diffuse distribution patterns of the hCAP-C/hCAP-E and hSMC1/hSMC3 complexes in the interphase nucleus indicate independent behaviors of the two complexes during the cell cycle. These results suggest that two distinct classes of SMC protein complexes are involved in different aspects of mitotic chromosome organization in human cells.