Abstract
The previously identified LRS (Loss of rDNA Silencing) domain of the nucleosome is critically important for silencing at both ribosomal DNA and telomeres. To understand the function of the LRS surface in silencing, we performed an EMS mutagenesis screen to identify suppressors of the H3 A75V LRS allele. We identified dominant and recessive mutations in histones H3, H4, and dominant mutations in the BAH (Bromo Adjacent Homology) domain of SIR3. We further characterized a surface of Sir3p critical for silencing via the LRS surface. We found that all alleles of the SIR3 BAH domain were able to 1) generally suppress the loss of telomeric silencing of LRS alleles, but 2) could not suppress SIN (Swi/Snf Independent) alleles or 3) could not suppress the telomeric silencing defect of H4 tail alleles. Moreover, we noticed a complementary trend in the electrostatic changes resulting from most of the histone mutations that gain or lose silencing and the suppressor alleles isolated in SIR3, and the genes for histones H3 and H4. Mutations in H3 and H4 genes that lose silencing tend to make the LRS surface more electronegative, whereas mutations that increase silencing make it less electronegative. Conversely, suppressors of LRS alleles in either SIR3, histone H3, or H4 also tend to make their respective surfaces less electronegative. Our results provide genetic evidence for recent data suggesting that the Sir3p BAH domain directly binds the LRS domain. Based on these findings, we propose an electrostatic model for how an extensive surface on the Sir3p BAH domain may regulate docking onto the LRS surface.
Highlights
Previous work identified a nucleosome surface named the LRS domain critically important for silencing at all classically defined silent loci in Saccharomyces cerevisiae [1,2]
Three models have been put forth to explain the role of the LRS domain in silencing: (i) these residues are directly required for K79 methylation/Dot1p recognition, (ii) the LRS surface could represent a direct nucleosome-nucleosome interaction surface important for tight packing of nucleosomes and silencing (iii), the surface may represent a site of interaction between the nucleosome and a silencing protein(s) [1]
We hypothesized that the LRS surface is a binding site for a transacting silencing factor, and that this interaction might be electrostatic in nature
Summary
Previous work identified a nucleosome surface named the LRS domain critically important for silencing at all classically defined silent loci in Saccharomyces cerevisiae [1,2]. The relevant residues are located at Super Helical Location (SHL)+/22.5 (equivalent to 4 o’clock on the nucleosome face, with 12 o’clock being the dyad axis) [3] These residues surround histone H3 K79, the site of Dot1p methylation that regulates silencing [4,5]. Bulk nucleosomes are 90% methylated at H3K79 and this modification is widely distributed across the euchromatic yeast genome but markedly depleted at heterochromatic mating-type, ribosomal DNA, and telomeric loci. These results suggest that this residue is important for defining euchromatin, and imply that the absence of such methylation defines a silent chromatin ground state [4,5,6,7]. One model for a condensed chromatin fiber, based on the tetranucleosome structure, suggests that LRS surfaces are solvent-exposed and not involved in intimate nucleosome-nucleosome interactions [10]. (iii) Data presented here and elsewhere [11,12] provide evidence that the LRS surface directly binds the Sir3p BAH domain, and this binding is important for silencing at the telomeres
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