Interaction analysis of the AAA+ domain of the heterochromatin protein Sir3
Final Report Abstract
Heterochromatin formation at the telomeres and the silent mating-type loci in the yeast Saccharomyces cerevisiae is characterized by the assembly of the Silent Information Regulator (SIR) complex. SIR consists of the histone deacetylase Sir2 and the components Sir3 and Sir4, which are structural components and mediate the interaction of SIR with unmodified chromatin to provide gene silencing. Sir3 carries two nucleosome-binding domains, the N-terminal bromo-adjacent homology domain (BAH) and the AAA+ ATPase-like domain. Mutations in an exposed surface loop of the AAA+ domain abrogate gene silencing and in vitro binding to Sir2/ Sir4. Here, we have identified the C-terminal coiled-coil of Sir4 to interact with this loop of the Sir3 AAA+ domain. We found that a mutation of Sir4-T1314S, which removes a single methyl group, was sufficient to restore silencing at the HM loci to a Sir3 version with a mutation in this loop. Restoration of telomeric silencing required further mutations of Sir4 (E1310V and K1325R). These mutations restored in vitro complex formation between Sir3 and the Sir4 coiled-coil, indicating that the improved affinity between Sir3 and Sir4 is responsible for the restoration of silencing. This conclusion is supported by our analysis of mutations in the AAA+ domain of Sir3 that suppress the silencing and in vitro complex formation defect of mutations in the Sir4 coiled-coil. We have furthermore identified mutations in the core histones that improve silencing, possibly by improving the affinity of the BAH or the AAA+ domain of Sir3 to the nucleosome.
Publications
- (2015) Architektur eines Heterochromatinkomplexes. BIOspektrum 6/2015, S. 597
Ann Ehrenhofer-Murray
(See online at https://doi.org/10.1007/s12268-015-0618-3) - (2017) Variants of the Sir4 Coiled- Coil Domain Improve Binding to Sir3 for Heterochromatin Formation in Saccharomyces cerevisiae. G3 (Bethesda) 7(4): 1117-1126
Samel, Anke, Rudner, Adam and Ann Ehrenhofer-Murray
(See online at https://doi.org/10.1534/g3.116.037739)
