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.     

Abundance of the RSC nucleosome-remodeling complex is important for the cells to tolerate DNA damage in Saccharomyces cerevisiae

The essential Nps1p/Sth1p is a catalytic subunit of the nucleosome-remodeling complex, RSC, of Saccharomyces cerevisiae that can alter nucleosome structure by using the energy of ATP hydrolysis. Besides the ATPase domain, Nps1p harbors the bromodomain, of which the function(s) have not yet been defined. We have isolated a temperature-sensitive mutant allele of NPS1, nps1-13, which has amino acid substitutions within the bromodomain. This mutation perturbed the interaction between the RSC components and enhanced the sensitivity of the cells to several DNA-damaging treatments at the permissive temperature. Reduced expression of NPS1 also caused DNA damage sensitivity. These results suggest the importance of the Nps1p bromodomain in RSC integrity and a model in which high amounts of RSC would be required for the cells to overcome DNA damage.     

Sth1p,a Saccharomyces cerevisiae Snf2p/Swi2p homolog,is an essential ATPase in RSC and differs from Snf/Swi in its interactions with histones and chromatin-associated proteins.

The essential Sth1p is the protein most closely related to the conserved Snf2p/Swi2p in Saccharomyces cerevisiae. Sth1p purified from yeast has a DNA-stimulated ATPase activity required for its function in vivo. The finding that Sth1p is a component of a multiprotein complex capable of ATP-dependent remodeling of the structure of chromatin (RSC) in vitro, suggests that it provides RSC with ATP hydrolysis activity. Three sth1 temperature-sensitive mutations map to the highly conserved ATPase/helicase domain and have cell cycle and non-cell cycle phenotypes, suggesting multiple essential roles for Sth1p. The Sth1p bromodomain is required for wild-type function; deletion mutants lacking portions of this region are thermosensitive and arrest with highly elongated buds and 2C DNA content, indicating perturbation of a unique function. The pleiotropic growth defects of sth1-ts mutants imply a requirement for Sth1p in a general cellular process that affects several metabolic pathways. Significantly, an sth1-ts allele is synthetically sick or lethal with previously identified mutations in histones and chromatin assembly genes that suppress snf/swi, suggesting that RSC interacts differently with chromatin than Snf/Swi. These results provide a framework for understanding the ATP-dependent RSC function in modeling chromatin and its connection to the cell cycle.     

The yeast RSC chromatin-remodeling complex is required for kinetochore function in chromosome segregation

The accurate segregation of chromosomes requires the kinetochore, a complex protein machine that assembles onto centromeric DNA to mediate attachment of replicated sister chromatids to the mitotic spindle apparatus. This study reveals an important role for the yeast RSC ATP-dependent chromatin-remodeling complex at the kinetochore in chromosome transmission. Mutations in genes encoding two core subunits of RSC, the ATPase Sth1p and the Snf5p homolog Sfh1p, interact genetically with mutations in genes encoding kinetochore proteins and with a mutation in centromeric DNA. RSC also interacts genetically and physically with the histone and histone variant components of centromeric chromatin. Importantly, RSC is localized to centromeric and centromere-proximal chromosomal regions, and its association with these loci is dependent on Sth1p. Both sth1 and sfh1 mutants exhibit altered centromeric and centromere-proximal chromatin structure and increased missegregation of authentic chromosomes. Finally, RSC is not required for centromeric deposition of the histone H3 variant Cse4p, suggesting that RSC plays a role in reconfiguring centromeric and flanking nucleosomes following Cse4p recruitment for proper chromosome transmission.     

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.     

Chromatin remodeling through directional DNA translocation from an internal nucleosomal site

The RSC chromatin remodeler contains Sth1, an ATP-dependent DNA translocase. On DNA substrates, RSC/Sth1 tracks along one strand of the duplex with a 3' --> 5' polarity and a tracking requirement of one base, properties that may enable directional DNA translocation on nucleosomes. The binding of RSC or Sth1 elicits a DNase I-hypersensitive site approximately two DNA turns from the nucleosomal dyad, and the binding of Sth1 requires intact DNA at this location. Results with various nucleosome substrates suggest that RSC/Sth1 remains at a fixed position on the histone octamer and that Sth1 conducts directional DNA translocation from a location about two turns from the nucleosomal dyad, drawing in DNA from one side of the nucleosome and pumping it toward the other. These studies suggest that nucleosome mobilization involves directional DNA translocation initiating from a fixed internal site on the nucleosome.     

RSC mobilizes nucleosomes to improve accessibility of repair machinery to the damaged chromatin

Repair of DNA double-strand breaks (DSBs) protects cells and organisms, as well as their genome integrity. Since DSB repair occurs in the context of chromatin, chromatin must be modified to prevent it from inhibiting DSB repair. Evidence supports the role of histone modifications and ATP-dependent chromatin remodeling in repair and signaling of chromosome DSBs. The key questions are, then, what the nature of chromatin altered by DSBs is and how remodeling of chromatin facilitates DSB repair. Here we report a chromatin alteration caused by a single HO endonuclease-generated DSB at the Saccharomyces cerevisiae MAT locus. The break induces rapid nucleosome migration to form histone-free DNA of a few hundred base pairs immediately adjacent to the break. The DSB-induced nucleosome repositioning appears independent of end processing, since it still occurs when the 5'-to-3' degradation of the DNA end is markedly reduced. The tetracycline-controlled depletion of Sth1, the ATPase of RSC, or deletion of RSC2 severely reduces chromatin remodeling and loading of Mre11 and Yku proteins at the DSB. Depletion of Sth1 also reduces phosphorylation of H2A, processing, and joining of DSBs. We propose that RSC-mediated chromatin remodeling at the DSB prepares chromatin to allow repair machinery to access the break and is vital for efficient DSB repair.     

Specialized RSC: Substrate Specificities for a Conserved Chromatin Remodeler

The remodel the structure of chromatin (RSC) nucleosome remodeling complex is a conserved chromatin regulator with roles in chromatin organization, especially over nucleosome depleted regions therefore functioning in gene expression. Recent reports in Saccharomyces cerevisiae have identified specificities in RSC activity toward certain types of nucleosomes. RSC has now been shown to preferentially evict nucleosomes containing the histone variant H2A.Z in vitro. Furthermore, biochemical activities of distinct RSC complexes has been found to differ when their nucleosome substrate is partially unraveled. Mammalian BAF complexes, the homologs of yeast RSC and SWI/SNF complexes, are also linked to nucleosomes with H2A.Z, but this relationship may be complex and extent of conservation remains to be determined. The interplay of remodelers with specific nucleosome substrates and regulation of remodeler outcomes by nucleosome composition are tantalizing questions given the wave of structural data emerging for RSC and other SWI/SNF family remodelers.     

RSC Nucleosome-remodeling complex plays prominent roles in transcriptional regulation throughout budding yeast gametogenesis

RSC is a nucleosome-remodeling complex of Saccharomyces cerevisiae essential for growth that can alter histone-DNA interaction by using the energy of ATP hydrolysis. Nps1p/Sth1p is an ATPase subunit of RSC. A mutation in the conserved ATPase domain of Nps1p causes a sporulation defect with decreased expression of early meiotic genes, especially IME2. This defect is partially suppressed by the overexpression of either IME1 or IME2. A homozygous diploid of a novel temperature-sensitive nps1 mutation, nps1-13, harboring amino acid substitutions within the bromodomain, was unable to sporulate. Overexpression of IME, IME2, or both of these genes allowed the completion of meiosis I and meiosis II in nps1-13 but not the formation of mature asci. In nps1-13 carrying YEpIME1, the expression of a group of sporulation-specific genes, which express at the middle stages of sporulation and are required for spore-wall formation, notably diminished, and several late sporulation genes expressed at the early stages of sporulation. These results suggest that Nps1p/RSC plays important roles during the spore development process by controlling gene expression for initiating both meiosis and spore morphogenesis, and ensures proper expression timing of late meiotic genes.