Swapping the Gene-Specific and Regional Silencing Specificities of the Hst1 and Sir2 Histone Deacetylases

Abstract

Sir2 and Hst1 are NAD؉ -dependent histone deacetylases of budding yeast that are related by strong sequence similarity. Nevertheless, the two proteins promote two mechanistically distinct forms of gene repression. Hst1 interacts with Rfm1 and Sum1 to repress the transcription of specific middle-sporulation genes. Sir2 interacts with Sir3 and Sir4 to silence genes contained within the silent-mating-type loci and telomere chromosomal regions. To identify the determinants of gene-specific versus regional repression, we created a series of Hst1::Sir2 hybrids. Our analysis yielded two dual-specificity chimeras that were able to perform both regional and gene-specific repression. Regional silencing by the chimeras required Sir3 and Sir4, whereas gene-specific repression required Rfm1 and Sum1. Our findings demonstrate that the nonconserved Nterminal region and two amino acids within the enzymatic core domain account for cofactor specificity and proper targeting of these proteins. These results suggest that the differences in the silencing and repression functions of Sir2 and Hst1 may not be due to differences in enzymatic activities of the proteins but rather may be the result of distinct cofactor specificities.The Sirtuin family of proteins is a large class of NAD ϩ -dependent deacetylases with a highly conserved enzymatic core domain (9). These enzymes are found in all kingdoms of life, and many organisms contain multiple Sirtuin family members (13). Although Sirtuin homologs have NAD ϩ -dependent deacetylase activities, their cellular functions differ as a result of their subcellular localization and specific protein-protein interactions (15,32). For example, although human SIRT2 deacetylates histone H4 during mitosis, it has also been found in the cytoplasm and appears to function as a tubulin deacetylase required for exit from mitosis (11,30,32,46). The SIRT1 protein deacetylates and activates acetyl-coenzyme A synthetase, as well as regulating transcription factors, such as p53, FOXO, and human immunodeficiency virus Tat (16,23,31,45,47,48). Determining how these proteins form complexes with their cofactors and recognize their targets is therefore key to understanding how these proteins function in the cell.The yeast Saccharomyces cerevisiae has five members of the Sirtuin family, HST1 to HST4 and SIR2, the founding member of the family (3, 10). Similar to the homologs in higher eukaryotes, the yeast proteins vary in their cellular localizations and perhaps substrate specificities (32,40). Sir2 functions as a NAD ϩ -dependent histone deacetylase and is involved in modifying chromatin structure and functioning as a regional transcriptional silencer (19,21,43). Silencing in yeast, like heterochromatin in higher eukaryotes, renders large regions of the chromosome transcriptionally inactive in a non-gene-specific manner. These regions include the silenced mating type loci, the subtelomeric domains, and the ribosomal DNA (rDNA) locus (39). The histone tails within these regions are hypoacetylated, and the DNA is gene...

The Duplicated Deacetylases Sir2 and Hst1 Subfunctionalized by Acquiring Complementary Inactivating Mutations

“…However, an alternative possibility is that the N-and C-terminal portions both contribute to both interactions by folding together to form an interaction surface. The first possibility is consistent with the previous finding that a chimeric protein containing the N terminus of ScSir2 and the C terminus of ScHst1 (S-H) interacts with both the Sir and Sum1 complexes (17,30). Yet, these results do not exclude the second possibility, in which either the N or the C terminus might be sufficient to allow the protein to interact with the Sir or Sum1 complex, as long as the remainder of the domain is provided by the paralog.…”

“…2), has high homology with ScSir2, suggesting that small differences between these deacetylases determine specificity. Indeed, replacing two amino acids in the zinc-binding module of ScSir2 with the amino acids from ScHst1, N378Q and L379I, enables ScSir2 to interact with ScRfm1 and repress a promoter containing a heterologous ScSum1 binding site (30).…”

“…It was previously concluded that the ancestral deacetylase that underwent duplication had both Sir2-like and Hst1-like functions (17,18), indicating that subfunctionalization occurred. In addition, distinct regions of ScSir2 and ScHst1 were found to confer specificities for their respective complexes (17,30). However, it was not known whether these specificity-determining regions were ancestral, in which case inactivating mutations would lead to VOL.…”

“…Although ScSir2 and ScHst1 have different functions, they can partially substitute for one another (3,17). Moreover, a chimeric protein composed of the N terminus of ScSir2 and the C terminus of ScHst1 complements both sir2⌬ and hst1⌬ mutations in S. cerevisiae and interacts with ScSir4 and ScSum1 (17,30). Therefore, different portions of the proteins confer specificity for the Sir or Sum1 complex.…”

Reinventing Heterochromatin in Budding Yeasts: Sir2 and the Origin Recognition Complex Take Center Stage

“…Consistent with this idea, KlSir2 complements an hst1⌬ deletion in S. cerevisiae (56) and partially suppresses a sir2⌬ mating defect (23). Studies of chimeric ScSir2-Hst1 molecules indicate that distinct regions of these deacetylases enable them to associate with the SIR or SUM1 complexes (41,56,80), and these interaction domains are conserved in KlSir2 (41). The most parsimonious model is that the ancestral Sir2 also utilized these interaction domains and that after duplication the paralogs acquired complementary inactivating mutations that reduced their affinities for one of the two complexes.…”