Scm3 is essential to recruit the histone h3 variant cse4 to centromeres and to maintain a functional kinetochore

The kinetochore is a complex multiprotein structure located at centromeres that is essential for proper chromosome segregation. Budding-yeast Cse4 is an essential evolutionarily conserved histone H3 variant recruited to the centromere by an unknown mechanism. We have identified Scm3, an inner kinetochore protein that immunopurifies with Cse4. Scm3 is essential for viability and localizes to all centromeres. Construction of a conditional SCM3 allele reveals that depletion results in metaphase arrest, with duplicated spindle poles, short spindles, and unequal DNA distribution. The metaphase arrest is mediated by the mitotic spindle checkpoint being dependent on Mad1 and the Aurora kinase B homolog Ipl1. Scm3 interacts with both Ndc10 and Cse4 and is essential to establish centromeric chromatin after DNA replication. In addition, Scm3 is required to maintain kinetochore function throughout the cell cycle. We propose a model in which Ndc10/Scm3 binds to centromeric DNA, which is in turn essential for targeting Cse4 to centromeres.     

The Swi2/Snf2 bromodomain is required for the displacement of SAGA and the octamer transfer of SAGA-acetylated nucleosomes

The SWI/SNF and SAGA chromatin-modifying complexes contain bromodomains that help anchor these complexes to acetylated promoter nucleosomes. To study the importance of bromodomains in these complexes, we have compared the chromatin-remodeling and octamer-transfer activity of the SWI/SNF complex to a mutant complex that lacks the Swi2/Snf2 bromodomain. Here we show that the SWI/SNF complex can remodel or transfer SAGA-acetylated nucleosomes more efficiently than the Swi2/Snf2 bromodomain-deleted complex. These results demonstrate that the Swi2/Snf2 bromodomain is important for the remodeling as well as for the octamer-transfer activity of the complex on H3-acetylated nucleosomes. Moreover, we show that, although the wild-type SWI/SNF complex displaces SAGA that is bound to acetylated nucleosomes, the bromodomain mutant SWI/SNF complex is less efficient in SAGA displacement. Thus, the Swi2/Snf2 bromodomain is required for the full functional activity of SWI/SNF on acetylated nucleosomes and is important for the displacement of SAGA from acetylated promoter nucleosomes.     

The interactions of yeast SWI/SNF and RSC with the nucleosome before and after chromatin remodeling

Interactions of the yeast chromatin-remodeling complexes SWI/SNF and RSC with nucleosomes were probed using site-specific DNA photoaffinity labeling. 5 S rDNA was engineered with photoreactive nucleotides incorporated at different sites in DNA to scan for the subunits of SWI/SNF in close proximity to DNA when SWI/SNF is bound to the 5 S nucleosome or to the free 5 S rDNA. The Swi2/Snf2 and Snf6 subunits of SWI/SNF were efficiently cross-linked at several positions in the nucleosome, whereas only Snf6 was efficiently cross-linked when SWI/SNF was bound to free DNA. DNA photoaffinity labeling of RSC showed that the Rsc4 subunit is in close proximity to nucleosomal DNA and not when RSC is bound to free DNA. After remodeling, the Swi2/Snf2 and Rsc4 subunits are no longer detected near the nucleosomal DNA and are evidently displaced from the surface of the nucleosome, indicating significant changes in SWI/SNF and RSC contacts with DNA after remodeling.     

A mutation in NPS1/STH1, an essential gene encoding a component of a novel chromatin-remodeling complex RSC, alters the chromatin structure of Saccharomyces cerevisiae centromeres.

The NPS1/STH1 gene encodes a nuclear protein essential for the progression of G2/M phase in Saccharomyces cerevisiae . Nps1p shares homology to Snf2/Swi2p, a subunit of a protein complex known as the SNF/SWI complex. Recently, Nps1p was found to be a component of a protein complex termed RSC (3) essential for mitotic growth, whereas its function is unknown. We isolated a temperature-sensitive mutant allele of NPS1 , nps1-105, and found that the mutation increases the sensitivity to thiabendazole (TBZ). At the restrictive temperature, nps1-105 arrested at the G2/M phase in MAD1-dependent manner and missegregated the mini-chromosome with higher frequency than the wild type cells. The nuclease digestion of the chromatin of the mutant cells revealed that the mutation causes the alteration of the chromatin structure around centromeres at the restrictive temperature. The results suggested that, in the nps1-105 mutant, impaired chromatin structure surrounding centromeres may lead to an impairment of kinetochore function and the cells arrest at G2/M phase through the spindle-assembly checkpoint system.     

Nonhistone Scm3 and histones CenH3-H4 assemble the core of centromere-specific nucleosomes

The budding yeast histone H3 variant, Cse4, replaces conventional histone H3 in centromeric chromatin and, together with centromere-specific DNA-binding factors, directs assembly of the kinetochore, a multiprotein complex mediating chromosome segregation. We have identified Scm3, a nonhistone protein that colocalizes with Cse4 and is required for its centromeric association. Bacterially expressed Scm3 binds directly to and reconstitutes a stoichiometric complex with Cse4 and histone H4 but not with conventional histone H3 and H4. A conserved acidic domain of Scm3 is responsible for directing the Cse4-specific interaction. Strikingly, binding of Scm3 can replace histones H2A-H2B from preassembled Cse4-containing histone octamers. This incompatibility between Scm3 and histones H2A-H2B is correlated with diminished in vivo occupancy of histone H2B, H2A, and H2AZ at centromeres. Our findings indicate that nonhistone Scm3 serves to assemble and maintain Cse4-H4 at centromeres and may replace histone H2A-H2B dimers in a centromere-specific nucleosome core.     

The centromere-specific histone variant Cse4p (CENP-A) is essential for functional chromatin architecture at the yeast 2-microm circle partitioning locus and promotes equal plasmid segregation

The centromere protein A homologue Cse4p is required for kinetochore assembly and faithful chromosome segregation in Saccharomyces cerevisiae. It has been regarded as the exquisite hallmark of centromeric chromatin. We demonstrate that Cse4 resides at the partitioning locus STB of the 2-microm plasmid. Cse4p-STB association is absolutely dependent on the plasmid partitioning proteins Rep1p and Rep2p and the integrity of the mitotic spindle. The kinetochore mutation ndc10-1 excludes Cse4p from centromeres without dislodging it from STB. Cse4p-STB association lasts from G1/S through late telophase during the cell cycle. The release of Cse4p from STB chromatin is likely mediated through spindle disassembly. A lack of functional Cse4p disrupts the remodeling of STB chromatin by the RSC2 complex, negates Rep2p binding and cohesin assembly at STB, and causes plasmid missegregation. Poaching of a specific histone variant by the plasmid to mark its partitioning locus with a centromere tag reveals yet another one of the molecular trickeries it performs for achieving chromosome- like fidelity in segregation.     

Direct interaction between Rsc6 and Rsc8/Swh3,two proteins that are conserved in SWI/SNF-related complexes.

The RSC complex of Saccharomyces cerevisiae is closely related to the SWI/SNF complex. Both complexes are involved in remodeling chromatin structure and they share conserved components. The RSC proteins Sth1, Rsc8/Swh3, Sfh1 and Rsc6 are homologs of the SWI/SNF proteins Swi2/Snf2, Swi3, Snf5 and Swp73 respectively. To investigate the RSC complex, we isolated a temperature-sensitive swh3 allele. A screen for multicopy suppressors yielded plasmids carrying the RSC6 and MAK31 loci. RSC6 also suppressed the formamide sensitivity of a strain with a C-terminal truncation of SWH3 . We show that Swh3 and Rsc6 fusion proteins interact in the two-hybrid system and that the swh3-ts mutation impairs this interaction. Finally, bacterially produced Swh3 and Rsc6 fusion proteins interact in vitro , supporting the genetic evidence for direct interaction between Swh3 and Rsc6 in vivo . We have previously shown that Swh3 also interacts with Sth1. These findings, together with the conservation of these proteins in the SWI/SNF complex and in mammalian SWI/SNF-related complexes, strongly suggest that these proteins form a structural core for the complex.