The LRS and SIN domains: two structurally equivalent but functionally distinct nucleosomal surfaces required for transcriptional silencing.

The nucleosome constitutes the central structure of chromatin and comprises two chains each of histones H2A, H2B, H3, and H4, which are assembled into a histone octamer and around which 147 bp of DNA is wrapped ∼1.7 times (18). Previous studies identified two nucleosomal surfaces, SIN (switch independent) and LRS, which are important for transcriptional repression in Saccharomyces cerevisiae (13, 24, 33). These domains lie at opposite ends of the crescent-shaped, quasisymmetric H3-H4 heterodimer. Histone residues altered in sin mutants or in five of the lrs mutant alleles can be structurally superimposed by a rotation of 180° around a symmetry axis at superhelical location (SHL) ±1.5. Thus, the two “ends” of each (H3-H4) “crescent” are structurally equivalent yet organize different regions of the DNA (Fig. ​(Fig.1).1). Alterations within these two clusters relieve distinct forms of transcriptional repression: the sin mutant alleles partially bypass the need for the SWI/SNF chromatin remodeling complex (13, 24, 33), and lrs mutant alleles lead to a loss of repression of genes placed in transcriptionally silent regions of the genome (e.g., the ribosomal DNA locus or telomeres) (24, 33). FIG. 1. Mapping of Sin and Lrs alterations onto the surface of the yeast nucleosome core particle. (A) Surface representation of the yeast nucleosome core particle (Brookhaven PDB 1ID3) viewed down the DNA superhelix axis (disk surface). Sin alleles map to SHL ... Sin− alterations in histones H3 and H4 were identified in yeast genetic screens for mutations that alleviated the transcriptional defects associated with inactivation of the SWI/SNF chromatin remodeling complex (13, 24, 33). In addition, the expression of Sin− histones leads to derepression of the PHO5 gene in high-phosphate conditions even in wild-type yeast, suggesting that Sin− histones also weaken more general, nucleosome-mediated repression of basal transcription (38). sin mutations within genes encoding histones H3 or H4 lead to single-amino-acid changes that cluster around the nucleosomal dyad axis (SHL ±0.5) (13, 18). Because interactions between DNA and the histone octamer are strongest at the dyad region of the nucleosome, disruption of histone-DNA contacts in this region may be the rate-limiting step in nucleosomal dissociation and sliding (20). X-ray structures of Sin− mononucleosomes have demonstrated that Sin− versions of histone H4 or H3 do not affect the overall structure of the nucleosome but rather disrupt only a small percentage of histone-DNA contacts (22). These subtle changes in histone-DNA interactions provide a likely explanation for why Sin− mononucleosomes dissociate at significantly lower ionic strength and why they have a lower energy barrier towards temperature-induced nucleosome repositioning (“sliding”) (7, 15, 22). In contrast, nucleosomal arrays reconstituted with Sin− versions of histone H4 do not exhibit altered positioning of nucleosomes, nor do Sin− nucleosomes within these arrays show altered accessibility to restriction enzymes (12). Interestingly, these Sin− nucleosomal arrays show defects in formation of condensed, 30-nm-like fibers. This folding defect mimics the loss of condensation due to removal of the histone N-terminal domains (8, 12). These results have led to two models for how Sin− histones might compensate for inactivation of SWI/SNF in vivo: (i) the enhanced thermal mobility of Sin− mononucleosomes makes chromatin remodeling unnecessary, and (ii) the unfolded state of Sin− nucleosomal arrays may mimic the “remodeled state” of chromatin acted on by SWI/SNF. These models may not be mutually exclusive, as the enhanced mobility of Sin− nucleosomes may inhibit chromatin condensation. In contrast to the Sin− alterations, little is known about the biochemistry of Lrs− histones. The lrs mutants were identified in an unbiased yeast genetic screen for mutations that alleviate silencing of an RNA polymerase II (Pol II) reporter gene that had been inserted into the ribosomal DNA locus (24). Additionally, the lrs mutant alleles were found to alleviate transcriptional silencing at telomeres and, to a lesser extent, the silent mating type loci (HM loci). Given the striking structural similarity of the LRS and SIN domains, it is attractive to hypothesize that these two surfaces fulfill similar functions. Here we have compared the in vivo and in vitro phenotypes of Lrs− and Sin− histone alterations. We find that lrs mutants do not exhibit Sin− phenotypes, nor do sin mutants exhibit defects in ribosomal DNA silencing. In contrast to Sin− versions of H4, we find that an Lrs− version of histone H3 does not disrupt formation of condensed, 30-nm-like fibers in vitro. Our genetic and biochemical studies indicate that these two nucleosomal surfaces have distinct functionalities in vitro and in vivo.