Structural maintenance of chromosomes (SMC) family proteins play critical roles in various nuclear events that require structural changes of chromosomes, including mitotic chromosome organization, DNA recombination and repair, and global transcriptional repression (for reviews, see references 9
, and 18
). The SMC proteins are conserved in eukaryotes as well as in prokaryotes, underscoring their essential roles in the cell. The impairment of SMC function in both prokaryotes and eukaryotes leads to mitotic chromosome segregation defects, suggesting a critical function for SMC family proteins in mitotic chromosome dynamics.
The protein structure of SMC family members is reminiscent of a myosin-like motor protein; it contains conserved head and tail regions with a nucleotide-binding site in the N terminus and a coiled-coil central domain. At least four SMC family proteins are conserved in eukaryotes. For example, the SMC family gene products termed Smc1, Smc2, Smc3, and Smc4 in Saccharomyces cerevisiae
are equivalent to Xenopus
SMC1 (XSMC1), Xenopus
chromosome-associated protein E (XCAP-E), XSMC3, and XCAP-C, and human SMC1 (hSMC1), hCAP-E, hSMC3, and hCAP-C, respectively (9
). XCAP-C and XCAP-E form a heterodimeric complex (XCAP-C–XCAP-E), which is part of the condensin multiprotein complex shown to be required for mitotic chromosome condensation in an in vitro embryonic Xenopus
extract system (11
). The hCAP-E and hCAP-C proteins also form a stable complex (hCAP-C–hCAP-E), which is the human ortholog of XCAP-C–XCAP-E as determined by its amino acid sequence similarity with XCAP-C–XCAP-E and specific localization to mitotic chromosomes (22
). However, the presence of a higher-order complex equivalent to Xenopus
condensin has not been demonstrated in human cells.
The mechanism of SMC-mediated chromosome condensation in the cell is not well understood. The studies using purified Xenopus
condensin complex revealed that the complex utilizes its ATPase activity and introduces writhe in naked supercoiled plasmid DNA (15
). Although this may explain the basic mechanism of condensation, condensation of chromatin fibers in the cell at the correct stage in the cell cycle most likely requires additional highly regulated molecular events. For example, it has been demonstrated that the mitosis-specific phosphorylation of condensin components by Cdc2 kinase is required for the function of Xenopus
condensin in chromosome condensation (14
). The presence of histones on DNA is also an important factor that most likely influences condensin function. Phosphorylation of a specific serine residue in the histone H3 tail is initiated from pericentromeric regions of chromosomes at the end of G2
phase and spreads over the entire chromosome, closely correlating with mitotic chromosome condensation (8
). It was shown recently that this phosphorylation is required for proper condensation and segregation of chromosomes (24
). The role of this phosphorylation at the molecular level is not understood. A possible recruitment of condensation factors, such as the condensin complex, by this modified H3 tail has been suggested. However, no direct evidence of such an interaction has been demonstrated.
In human cells, the hCAP-C–hCAP-E heterodimeric complex is expressed throughout the cell cycle, suggesting the complex is regulated posttranslationally in order to perform its mitosis-specific role (22
). To address the mechanism and regulation of hCAP-C–hCAP-E function, cellular factors that interact with hCAP-C–hCAP-E were purified by coimmunoprecipitation with the endogenous hCAP-C–hCAP-E from HeLa cells. Here we report the identification of the condensation-related SMC-associated protein 1 (CNAP1), which forms a complex with hCAP-C–hCAP-E. CNAP1 was found to be the human homolog of Xenopus
condensin component XCAP-D2 (10
), suggesting the presence of a human condensin complex equivalent to the Xenopus
condensin. To understand the cell cycle-specific regulation of human condensin, comparative immunolocalization analyses of CNAP1 and hCAP-C–hCAP-E and biochemical analysis of the complex at different cell cycle stages were performed using HeLa cells. The results revealed that human condensin is present throughout the cell cycle, but its subcellular localization is cell cycle regulated. The majority of the interphase condensin complex is sequestered in the cytoplasm, while a subpopulation of the complex was found to remain on chromosomes as distinct foci in the interphase nucleus. Biochemical studies revealed that the condensin complex interacts with histone H3 in a DNA-independent manner, suggesting that the chromosome association of condensin is at least partly mediated by the interaction with core histones. Importantly, condensin forms larger foci that colocalize with clusters of phosphorylated histone H3 (phosphor-H3) on locally condensed chromatin during the G2
/M transition. This is the first evidence that demonstrates a direct link between the condensin complex and mitotic phosphorylation of histone H3 and suggests that the interphase nuclear condensin plays a critical role in reinitiation of mitotic chromosome condensation together with phosphor-H3. In this paper, the human condensin complex has been identified and systematically characterized during the cell cycle, providing the basis for understanding SMC-mediated mitotic chromosome condensation in the cell.