Reconstitution of heterochromatin-dependent transcriptional gene silencing.
Heterochromatin assembly in budding yeast requires the SIR complex, which contains the NAD-dependent deacetylase Sir2 and the Sir3 and Sir4 proteins. Sir3 binds to nucleosomes containing deacetylated histone H4 lysine 16 (H4K16) and, with Sir4, promotes spreading of Sir2 and deacetylation along the chromatin fiber. Combined action of histone modifying and binding activities is a conserved hallmark of heterochromatin, but the relative contribution of each activity to silencing has remained unclear. Here, we reconstitute SIR-chromatin complexes using purified components and show that the SIR complex efficiently deacetylates chromatin templates and promotes the assembly of altered structures that silence Gal4-VP16-activated transcription. Silencing requires all three Sir proteins, even with fully deacetylated chromatin, and involves the specific association of Sir3 with deacetylated H4K16. These results define a minimal set of components that mediate heterochromatic gene silencing and demonstrate distinct contributions for histone deacetylation and nucleosome binding in the silencing mechanism.
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Aaron
A
Johnson
Geng
G
Li
Timothy W
TW
Sikorski
Stephen
S
Buratowski
Christopher L
CL
Woodcock
Danesh
D
Moazed
D000107:Acetylation; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D042002:Chromatin Assembly and Disassembly; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D020868:Gene Silencing; D006570:Heterochromatin / Q000378:metabolism*; D006655:Histone Deacetylases / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D008239:Lysine; D046912:Multiprotein Complexes; D009154:Mutation; D009243:NAD / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D056490:Nucleosome Assembly Protein 1; D011485:Protein Binding; D012319:RNA Polymerase II / Q000378:metabolism; D012320:RNA Polymerase III / Q000378:metabolism; 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; D056565:Sirtuin 2; D037761:Sirtuins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D006570:Heterochromatin; D006657:Histones; D046912:Multiprotein Complexes; C533556:NAP1 protein, S cerevisiae; D009687:Nuclear Proteins; D056490:Nucleosome Assembly Protein 1; C084122:SIR3 protein, S cerevisiae; C061573:SIR4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; D009243:NAD; D012319:RNA Polymerase II; D012320:RNA Polymerase III; D004262:DNA Restriction Enzymes; C087778:SIR2 protein, S cerevisiae; D056565:Sirtuin 2; D037761:Sirtuins; D006655:Histone Deacetylases; D000251:Adenosine Triphosphatases; C470546:ISW1 protein, S cerevisiae; D008239:Lysine