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

Abstract

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.

Cell Cycle-dependent Expression and Nucleolar Localization of hCAP-H

“…Schmiesing et al (2000) have proposed that H3 phosphorylation is initiated at chromatin sites on which hCAP-E is associated throughout interphase. To establish the timing of association of hCAP-H with relative to the initiation of chromatin condensation, HeLa cells were stained simultaneously with a mouse mAb specific to histone H3 phos- et al, 1997) and with rabbit Ab2575.…”

“…Because the phosphorylation of histone H3 has been associated with the onset of chromosome condensation, Hirano et al (1999) proposed that the association and activation of condensin with mitotic chromosomes may be secondary to the phosphorylation of histone H3. In Drosophila, the phosphorylation of histone H3 by Aurora B kinase is required for recruitment of condensin to chromosomes (Giet and Glover, 2001), and Schmiesing et al (2000) have reported that in human cells, a fraction of the SMC heterodimer is associated with discrete chromosomal foci in interphase and that these foci colocalize with the sites of early histone H3 phosphorylation. However, Kimura and Hirano (2001) have demonstrated that the phosphorylation of H3 at Ser-10 is not sufficient to target the 13S condensin complex to chromosomes in vitro, independently of the phosphorylation status of the non-SMC subunits.…”

“…The composition of the condensin complex is conserved in all organisms studied to date. Very recently, Kimura et al (2001) demonstrated that the homologues of Xenopus condensin subunits, barren-1/ hCAP-H (Cabello et al, 1997), hCAP-C, hCAP-E, CNAP1 (Schmiesing et al, 2000), and hCAP-G form a heteropentamer capable of inducing chromosome condensation in the Xenopus egg extract model. In Saccharomyces cerevisiae the condensin complex also consists of five subunits: Smc4p (XCAP-C), Smc2p (XCAP-E), Brn1p (XCAP-H), Ycs4p (XCAP-D2), and Ycs5p (XCAP-G).…”

“…The composition of the condensin complex is conserved in human cells. Homologues of all five condensin subunits have been identified in the human genome and cDNAs corresponding hCAP-H, hCAP-C, hCAP-E, and the homologue of XCAP-D2, CNAP1, have been cloned (Cabello et al, 1997;Schmiesing et al, 1998;Schmiesing et al, 2000). Schmiesing et al (2000) reported that immunoprecipitation of HeLa cell extracts with CNAP1-specific antibody reveals the association with hCAP-C, hCAP-E, and two unidentified proteins corresponding to the predicted molecular weights of hCAP-H and hCAP-G. Kimura and Hirano (2001) have established the identity of these two proteins by coprecipitation of all five subunits of the 13S holocomplex from HeLa cell extracts…”

Histone Hyperacetylation in Mitosis Prevents Sister Chromatid Separation and Produces Chromosome Segregation Defects

“…H3 phosphorylation at Ser10 is governed by the activity of aurora/Ipl1 kinases (Speliotes et al, 2000;Giet and Glover 2001;Crosio et al, 2002) in competition with the phosphatase activity of the serine/threonine protein phosphatase 1 (Hsu et al, 2000;Murnion et al, 2001). It has been shown that Ser 10-modified histone colo-calizes with the human condensin complex during G2/ prophase (Schmiesing et al, 2000), suggesting that this modification may have a role in destabilizing local chromatin structure to allow access of condensation factors to DNA or in recruiting the condensation machinery on the modified histone amino-terminal tail (Wei et al, 1998;Schmiesing et al, 2000).…”