title: Synthetic lethal screens identify gene silencing processes in yeast and implicate the acetylated amino terminus of Sir3 in recognition of the nucleosome core.
abstract: Dot1 methylates histone H3 lysine 79 (H3K79) on the nucleosome core and is involved in Sir protein-mediated silencing. Previous studies suggested that H3K79 methylation within euchromatin prevents nonspecific binding of the Sir proteins, which in turn facilitates binding of the Sir proteins in unmethylated silent chromatin. However, the mechanism by which the Sir protein binding is influenced by this modification is unclear. We performed genome-wide synthetic genetic array (SGA) analysis and identified interactions of DOT1 with SIR1 and POL32. The synthetic growth defects found by SGA analysis were attributed to the loss of mating type identity caused by a synthetic silencing defect. By using epistasis analysis, DOT1, SIR1, and POL32 could be placed in different pathways of silencing. Dot1 shared its silencing phenotypes with the NatA N-terminal acetyltransferase complex and the conserved N-terminal bromo adjacent homology (BAH) domain of Sir3 (a substrate of NatA). We classified all of these as affecting a common silencing process, and we show that mutations in this process lead to nonspecific binding of Sir3 to chromatin. Our results suggest that the BAH domain of Sir3 binds to histone H3K79 and that acetylation of the BAH domain is required for the binding specificity of Sir3 for nucleosomes unmethylated at H3K79.
authors.affiliation: The Netherlands Cancer Institute, Division of Cellular Biochemistry, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands.
authors.forename: Tibor
authors.initials: T
authors.lastname: van Welsem
authors.forename: Floor
authors.initials: F
authors.lastname: Frederiks
authors.forename: Kitty F
authors.initials: KF
authors.lastname: Verzijlbergen
authors.forename: Alex W
authors.initials: AW
authors.lastname: Faber
authors.forename: Zara W
authors.initials: ZW
authors.lastname: Nelson
authors.forename: David A
authors.initials: DA
authors.lastname: Egan
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Gottschling
authors.forename: Fred
authors.initials: F
authors.lastname: van Leeuwen
mesh_terms: D000107:Acetylation; D002843:Chromatin / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D020868:Gene Silencing; D005804:Genes, Lethal; D011495:Histone-Lysine N-Methyltransferase; D006657:Histones / Q000378:metabolism; D008745:Methylation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D009707:Nucleosomes / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
chemical_list: D002843:Chromatin; D006657:Histones; D009687:Nuclear Proteins; D009707:Nucleosomes; C522758:Pol32 protein, S cerevisiae; C084121:SIR1 protein, S cerevisiae; C084122:SIR3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; C416235:Dot1 protein, S cerevisiae; D011495:Histone-Lysine N-Methyltransferase; D004259:DNA-Directed DNA Polymerase