Human Chromatid Cohesin Component hRad21 Is Phosphorylated in M Phase and Associated with Metaphase Centromeres

Abstract

Sister chromatids duplicated in S phase are connected with each other during G 2 and M phase until the onset of anaphase. This chromatid cohesion is essential for correct segregation of genetic material to daughter cells. Recently, understanding of the molecular mechanisms governing chromatid cohesion in yeast has been greatly advanced, whereas these processes in mammalian cells remain unclear. We report here biochemical and cytological analyses of human Rad21, a homologue of the yeast cohesin subunit, Scc1p/Mcd1p. hRad21 is a nuclear phosphorylated protein. Its abundance does not change during the cell cycle, and it becomes hyperyphosphorylated in M phase. Most hRad21 is not associated with chromatin when the nuclear envelope breakdown takes place in prophase. However, a detailed analysis of the spread chromosomes indicated that hRad21 remains associated with prometaphase-like chromosomes along their entire lengths. The mitotic chromatin-bound hRad21 becomes dissociated in a highly regulated manner because hRad21 remains specifically at the centromeres but disappears from the arm regions on metaphase-like chromosomes. Interestingly, hRad21 at the metaphase centromeres appears to be present at the inner pairing domain where the two sister chromatids are supposed to be in intimate contact. These results suggest that hRad21 has a critical role in chromatid cohesion in human mitotic cells.Accurate chromosome segregation during mitosis into two daughter cells is one of the requirements for stable genome maintenance. Until recently, the molecular mechanisms regulating chromatid cohesion had not been well understood. However, recent studies, mostly conducted in budding yeast, have outlined the process generally (reviewed in Ref. 1). Concurrent with DNA replication, the sister chromatids become connected along their entire length. Several distinct groups of proteins are involved in establishing and maintaining chromatid cohesion. The most intensively investigated protein complex, budding yeast cohesin, consists of four subunit proteins, Scc1p/ Mcd1p, Scc3p, Smc1p, and Smc3p (2-4), and serves as a physical glue between sister chromatids. Cohesin is phylogenetically conserved. Rad21p in fission yeast and human Rad21 (hRad21) are homologues of Scc1p/Mcd1p (5, 6). Smc1p and Smc3p are found in budding yeast, Xenopus, and mammals (7-9). They are members of the SMC (structural maintenance of chromosome) protein family that is characterized by the presence of coiled-coil domains and ATPase domains (reviewed in Ref. 10). Very recently, two Scc3p homologues, SA1 and SA2, have been found in Xenopus and human cells (11). Interestingly, in Xenopus, two distinct classes of cohesin, termed xcohesin SA1 and x-cohesin SA2 , are present. These two complexes share Xenopus (X)SCC1, XSCC2, and XRAD21 and differ via containing either XSA1 or XSA2 (11). Immunodepletion of Xenopus cohesin from egg extracts led to a failure of chromatid cohesion (8). Therefore, the cohesin complex is likely to be conserved in all eukaryotes, including humans.H...

Cohesin Associates with Spindle Poles in a Mitosis-specific Manner and Functions in Spindle Assembly in Vertebrate Cells

“…Human Rad21 localization at spindle poles was also reported previously (Hoque and Ishikawa, 2001). These results were further confirmed by the localization of the recombinant hSMC1 fused to GFP at mitotic spindle poles in a stable HeLa cell line expressing hSMC1-GFP (Supplemental Figure S1A).…”

“…Only a small amount of cohesin (ϳ5%) remains at the chromosome-pairing domain to mediate sister chromatid cohesion, which is eventually terminated by the cleavage of Rad21 at the onset of anaphase leading to chromosome segregation (Waizenegger et al, 2000;Hoque and Ishikawa, 2001;Gimenez-Abian et al, 2004). However, the functional significance of the abundant cytoplasmic cohesin remained an enigma.…”

“…During G2/prophase, the majority of cohesin dissociates from chromosomes as an intact complex in a Polo-like kinase-reguThis article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E08 -04 -0419) on January 7, 2009. lated manner (Losada et al, 2000;Sumara et al, 2000;Gregson et al, 2001). Only a small amount of cohesin (ϳ5%) remains at the chromosome-pairing domain to mediate sister chromatid cohesion, which is eventually terminated by the cleavage of Rad21 at the onset of anaphase leading to chromosome segregation (Waizenegger et al, 2000;Hoque and Ishikawa, 2001;Gimenez-Abian et al, 2004). However, the functional significance of the abundant cytoplasmic cohesin remained an enigma.…”

Localization of hRad9, hHus1, hRad1, and hRad17 and Caffeine-sensitive DNA Replication at the Alternative Lengthening of Telomeres-associated Promyelocytic Leukemia Body

“…Mitotic spreads of chromosome were prepared by Cytospin as described (36), except cells were swollen in 75 mM KCl. Cells were prepermeabilized prior to fixation with 0.1% Triton X-100 in T buffer (20 mM HEPES-KOH (pH 7.4), 50 mM NaCl, 3 mM MgCl 2 , and 0.3 M sucrose).…”

Molecular insights into mechanisms regulating faithful chromosome separation in female meiosis

“…137,152,153 Kinase phosphorylation plays important roles in this process. 154 The second is the anaphase pathway. The remaining centromeric cohesin is cleaved by separase at the transition from metaphase to anaphase.…”