Reconstitution of yeast silent chromatin: multiple contact sites and O-AADPR binding load SIR complexes onto nucleosomes in vitro.
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.
Martino
Fabrizio
F
Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland.
Kueng
Stephanie
S
Robinson
Philip
P
Tsai-Pflugfelder
Monika
M
van Leeuwen
Fred
F
Ziegler
Mathias
M
Cubizolles
Fabien
F
Cockell
Moira M
MM
Rhodes
Daniela
D
Gasser
Susan M
SM
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
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