title: Reconstitution of yeast silent chromatin: multiple contact sites and O-AADPR binding load SIR complexes onto nucleosomes in vitro.
abstract: At yeast telomeres and silent mating-type loci, chromatin assumes a higher-order structure that represses transcription by means of the histone deacetylase Sir2 and structural proteins Sir3 and Sir4. Here, we present a fully reconstituted system to analyze SIR holocomplex binding to nucleosomal arrays. Purified Sir2-3-4 heterotrimers bind chromatin, cooperatively yielding a stable complex of homogeneous molecular weight. Remarkably, Sir2-3-4 also binds naked DNA, reflecting the strong, albeit nonspecific, DNA-binding activity of Sir4. The binding of Sir3 to nucleosomes is sensitive to histone H4 N-terminal tail removal, while that of Sir2-4 is not. Dot1-mediated methylation of histone H3K79 reduces the binding of both Sir3 and Sir2-3-4. Additionally, a byproduct of Sir2-mediated NAD hydrolysis, O-acetyl-ADP-ribose, increases the efficiency with which Sir3 and Sir2-3-4 bind nucleosomes. Thus, in small cumulative steps, each Sir protein, unmodified histone domains, and contacts with DNA contribute to the stability of the silent chromatin complex.
authors.affiliation: Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland.
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Martino
authors.forename: Stephanie
authors.initials: S
authors.lastname: Kueng
authors.forename: Philip
authors.initials: P
authors.lastname: Robinson
authors.forename: Monika
authors.initials: M
authors.lastname: Tsai-Pflugfelder
authors.forename: Fred
authors.initials: F
authors.lastname: van Leeuwen
authors.forename: Mathias
authors.initials: M
authors.lastname: Ziegler
authors.forename: Fabien
authors.initials: F
authors.lastname: Cubizolles
authors.forename: Moira M
authors.initials: MM
authors.lastname: Cockell
authors.forename: Daniela
authors.initials: D
authors.lastname: Rhodes
authors.forename: Susan M
authors.initials: SM
authors.lastname: Gasser
mesh_terms: D001665:Binding Sites; D002843:Chromatin / Q000378:metabolism*; D006655:Histone Deacetylases / Q000302:isolation & purification / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D009707:Nucleosomes / Q000378:metabolism*; D038961:O-Acetyl-ADP-Ribose / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae / Q000302:isolation & purification / Q000378:metabolism*; D056565:Sirtuin 2; D037761:Sirtuins / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D002843:Chromatin; D009707:Nucleosomes; D038961:O-Acetyl-ADP-Ribose; C084122:SIR3 protein, S cerevisiae; C061573:SIR4 protein, S cerevisiae; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; C087778:SIR2 protein, S cerevisiae; D056565:Sirtuin 2; D037761:Sirtuins; D006655:Histone Deacetylases