No abstract

Cohesin complex acts in the formation and maintenance of sister chromatid cohesion during and after S phase. Budding yeast Scc1p/Mcd1p, an essential subunit, is cleaved and dissociates from chromosomes in anaphase, leading to sister chromatid separation. Most cohesin in higher eukaryotes, in contrast, is dissociated from chromosomes well before anaphase. The universal role of cohesin during anaphase thus remains to be determined. We report here initial characterization of four putative cohesin subunits, Psm1, Psm3, Rad21, and Psc3, in fission yeast. They are essential for sister chromatid cohesion. Immunoprecipitation demonstrates stable complex formation of Rad21 with Psm1 and Psm3 but not with Psc3. Chromatin immunoprecipitation shows that cohesin subunits are enriched in broad centromere regions and that the level of centromereassociated Rad21 did not change from metaphase to anaphase, very different from budding yeast. In contrast, Rad21 containing similar cleavage sites to those of Scc1p/Mcd1p is cleaved specifically in anaphase. This cleavage is essential, although the amount of cleaved product is very small (<5%). Mis4, another sister chromatid cohesion protein, plays an essential role for loading Rad21 on chromatin. A simple model is presented to explain the specific behavior of fission yeast cohesin and why only a tiny fraction of Rad21 is sufficient to be cleaved for normal anaphase.

The condensin complex in frog extracts, containing two SMC (structural maintenance of chromosomes) and three non-SMC subunits, promotes mitotic chromosome condensation, and its supercoiling activity increases during mitosis by Cdc2 phosphorylation. Here, we report that fission yeast has the same five-member condensin complex, each of which is essential for mitotic condensation. The condensin complex was purified and the subunits were identified by microsequencing. Cnd1, Cnd2, and Cnd3, three non-SMC subunits showing a high degree of sequence conservation to frog subunits, are essential for viability, and their gene disruption leads to a phenotype indistinguishable from that observed in cut3-477 and cut14-208, known mutations in SMC4 and SMC2-like subunits. Condensin subunits tagged with GFP were observed to alter dramatically their localization during the cell cycle, enriched in the nucleus during mitosis, but cytoplasmic during other stages. This stage-specific alteration in localization requires mitosis-specific phosphorylation of the T19 Cdc2 site in Cut3. The T19 site is phosphorylated in vitro by Cdc2 kinase and shows the maximal phosphorylation in metaphase in vivo. Its alanine substitution mutant fails to suppress the temperature-sensitive phenotype of cut3-477, and shows deficiency in condensation, probably because Cut3 T19A remains cytoplasmic. Therefore, direct Cdc2 phosphorylation of fission yeast condensin may facilitate its nuclear accumulation during mitosis.

The CT element of the c-myc gene is required for promoter P1 usage and can drive expression of a heterologous promoter. Both double strand (Sp1) and single strand (hnRNP K) CT-binding proteins have been implicated as mediators of CT action. Although significant levels of CT activity persisted following Sp1 immunodepletion, EGTA totally abolished transactivation, thus implicating another metal requiring factor in CT element activity. As hnRNP K binds to one strand of the CT element, but has no metal requirement, the opposite (purine-rich strand) was examined as a target for a metal-dependent protein. A zinc-requiring purine strand binding activity was identified as cellular nucleic acid binding protein (CNBP), a protein previously implicated in the regulation of sterol responsive genes. Two forms of CNBP differed in their relative binding to the CT- or sterol-response elements. CNBP was shown to be a bona fide regulator of the CT element by cotransfection of a CNBP expression vector that stimulated expression of a CT-driven but not an AP1-dependent reporter. These data suggest that hnRNP K and CNBP bind to opposite strands and co-regulate the CT element.