RESUMO
We have developed a highly specific antibody set for acetylation sites in yeast histones H4 (K5, K8, K12, and K16); H3 (K9, K14, K18, K23, and K27); H2A (K7); and H2B (K11 and K16). Since ELISA does not assure antibody specificity in chromatin immunoprecipitation, we have employed additional screens against the respective histone mutations. We now show that telomeric and silent mating locus heterochromatin is hypoacetylated at all histone sites. At the INO1 promoter, RPD3 is required for strongly deacetylating all sites except H4 K16, ESA1 for acetylating H2A, H2B, and H4 sites except H4 K16, and GCN5 for acetylating H2B and H3 sites except H3 K14. These data uncover the in vivo usage of acetylation sites in heterochromatin and euchromatin.
Assuntos
Anticorpos Antifúngicos/metabolismo , Proteínas de Ciclo Celular , Eucromatina/metabolismo , Heterocromatina/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae , Leveduras/metabolismo , Acetilação , Acetiltransferases/metabolismo , Animais , Anticorpos Antifúngicos/imunologia , Ensaio de Imunoadsorção Enzimática , Eucromatina/imunologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Heterocromatina/imunologia , Histona Acetiltransferases , Peptídeos e Proteínas de Sinalização Intracelular , Mutação , Testes de Precipitina/métodos , Regiões Promotoras Genéticas/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Leveduras/genética , Leveduras/imunologiaRESUMO
Histone deacetylases such as human HDAC1 and yeast RPD3 are trichostatin A (TSA)-sensitive enzymes that are members of large, multiprotein complexes. These contain specialized subunits that help target the catalytic protein to histones at the appropriate DNA regulatory element, where the enzyme represses transcription. To date, no deacetylase catalytic subunits have been shown to have intrinsic activity, suggesting that noncatalytic subunits of the deacetylase complex are required for their enzymatic function. In this paper we describe a novel yeast histone deacetylase HOS3 that is relatively insensitive to the histone deacetylase inhibitor TSA, forms a homodimer when expressed ectopically both in yeast and Escherichia coli, and has intrinsic activity when produced in the bacterium. Most HOS3 protein can be found associated with a larger complex in partially purified yeast nuclear extracts, arguing that the HOS3 homodimer may be dissociated from a very large nuclear structure during purification. We also demonstrate, using a combination of mass spectrometry, tandem mass spectrometry, and proteolytic digestion, that recombinant HOS3 has a distinct specificity in vitro for histone H4 sites K5 and K8, H3 sites K14 and K23, H2A site K7, and H2B site K11. We propose that while factors that interact with HOS3 may sequester the catalytic subunit at specific cellular sites, they are not required for HOS3 histone deacetylase activity.
