| 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 we first compared the LRS domain of the nucleosome with a structurally similar nucleosomal domain, termed the SIN domain, which is required for transcriptional repression at genes regulated by the SWI/SNF chromatin remodeling complex. We show that alterations in the LRS domain do not result in Sin- phenotypes, nor does disruption of the SIN domain lead to loss of ribosomal DNA silencing. Furthermore, whereas disruption of the SIN domain eliminates intramolecular folding of nucleosomal arrays in vitro, alterations in the LRS domain have no effect on chromatin folding in vitro. In contrast to these dissimilarities, we find that the SIN and LRS domains are both required for recruitment of Sir2p and Sir4p to telomeric and silent mating type loci, suggesting that both surfaces can contribute to heterochromatin formation. Our study predicts that while the SIN surface may have structural defects that limit its ability to repress certain types of transcription, the LRS surface is most likely a docking surface for a silencing factor. To identify more precisely 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 Sir3p. We developed a new technique termed TaGAM to identify the dominant mutations as alleles of SIR3. 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. 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. |
