RESUMEN
Chromatin remodelling complexes (CRC) are ATP-dependent molecular machines important for the dynamic organization of nucleosomes along eukaryotic DNA. CRCs SWI/SNF, RSC and INO80 can move positioned nucleosomes in promoter DNA, leading to nucleosome-depleted regions which facilitate access of general transcription factors. This function is strongly supported by transcriptional activators being able to interact with subunits of various CRCs. In this work we show that SWI/SNF subunits Swi1, Swi2, Snf5 and Snf6 can bind to activation domains of Ino2 required for expression of phospholipid biosynthetic genes in yeast. We identify an activator binding domain (ABD) of ATPase Swi2 and show that this ABD is functionally dispensable, presumably because ABDs of other SWI/SNF subunits can compensate for the loss. In contrast, mutational characterization of the ABD of the Swi2-related ATPase Sth1 revealed that some conserved basic and hydrophobic amino acids within this domain are essential for the function of Sth1. While ABDs of Swi2 and Sth1 define separate functional protein domains, mapping of an ABD within ATPase Ino80 showed co-localization with its HSA domain also required for binding actin-related proteins. Comparative interaction studies finally demonstrated that several unrelated activators each exhibit a specific binding pattern with ABDs of Swi2, Sth1 and Ino80.
Asunto(s)
Adenosina Trifosfatasas , Ensamble y Desensamble de Cromatina , Proteínas de Unión al ADN , Unión Proteica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Factores de Transcripción , Activación Transcripcional , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ensamble y Desensamble de Cromatina/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Regulación Fúngica de la Expresión Génica , Dominios Proteicos , Proteínas Nucleares , Proteínas de Ciclo Celular , Factores de Transcripción con Motivo Hélice-Asa-Hélice BásicoRESUMEN
Binding of general transcription factors TFIID and TFIIA to basal promoters is rate-limiting for transcriptional initiation of eukaryotic protein-coding genes. Consequently, activator proteins interacting with subunits of TFIID and/or TFIIA can drastically increase the rate of initiation events. Yeast transcriptional activator Ino2 interacts with several Taf subunits of TFIID, among them the multifunctional Taf1 protein. In contrast to mammalian Taf1, yeast Taf1 lacks bromodomains which are instead encoded by separate proteins Bdf1 and Bdf2. In this work, we show that Bdf1 not only binds to acetylated histone H4 but can also be recruited by Ino2 and unrelated activators such as Gal4, Rap1, Leu3 and Flo8. An activator-binding domain was mapped in the N-terminus of Bdf1. Subunits Toa1 and Toa2 of yeast TFIIA directly contact sequences of basal promoters and TFIID subunit TBP but may also mediate the influence of activators. Indeed, Ino2 efficiently binds to two separate structural domains of Toa1, specifically with its N-terminal four-helix bundle structure required for dimerization with Toa2 and its C-terminal ß-barrel domain contacting TBP and sequences of the TATA element. These findings complete the functional analysis of yeast general transcription factors Bdf1 and Toa1 and identify them as targets of activator proteins.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Proteínas que Contienen Bromodominio , Fosfolípidos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Factor de Transcripción TFIIA , Factores de Transcripción , Fosfolípidos/biosíntesis , Fosfolípidos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , Factor de Transcripción TFIIA/genética , Factor de Transcripción TFIIA/metabolismo , Factor de Transcripción TFIID/genética , Factor de Transcripción TFIID/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas que Contienen Bromodominio/genética , Proteínas que Contienen Bromodominio/metabolismoRESUMEN
The original version of this article unfortunately contained a mistake.
RESUMEN
Expression of phospholipid biosynthetic genes in yeast requires activator protein Ino2 which can bind to the UAS element inositol/choline-responsive element (ICRE) and trigger activation of target genes, using two separate transcriptional activation domains, TAD1 and TAD2. However, it is still unknown which cofactors mediate activation by TADs of Ino2. Here, we show that multiple subunits of basal transcription factor TFIID (TBP-associated factors Taf1, Taf4, Taf6, Taf10 and Taf12) are able to interact in vitro with activation domains of Ino2. Interaction was no longer observed with activation-defective variants of TAD1. We were able to identify two nonoverlapping regions in the N-terminus of Taf1 (aa 1-100 and aa 182-250) each of which could interact with TAD1 of Ino2 as well as with TAD4 of activator Adr1. Specific missense mutations within Taf1 domain aa 182-250 affecting basic and hydrophobic residues prevented interaction with wild-type TAD1 and caused reduced expression of INO1. Using chromatin immunoprecipitation we demonstrated Ino2-dependent recruitment of Taf1 and Taf6 to ICRE-containing promoters INO1 and CHO2. Transcriptional derepression of INO1 was no longer possible with temperature-sensitive taf1 and taf6 mutants cultivated under nonpermissive conditions. This result supports the hypothesis of Taf-dependent expression of structural genes activated by Ino2.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Genes Fúngicos/fisiología , Fosfolípidos/biosíntesis , Dominios y Motivos de Interacción de Proteínas/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factor de Transcripción TFIID/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Colina/metabolismo , ADN de Hongos , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Inositol/metabolismo , Mutagénesis Sitio-Dirigida , Unión Proteica , Subunidades de Proteína/metabolismo , ARN de Hongos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Transcripción GenéticaRESUMEN
It is generally assumed that pathway-specific transcriptional activators recruit pleiotropic coactivators (such as chromatin-modifying complexes or general transcription factors), while specific repressors contact pleiotropic corepressors creating an inaccessible chromatin by the action of histone deacetylases. We have previously shown that the negative regulator Opi1 of yeast phospholipid biosynthesis inhibits transcription by recruiting corepressors Sin3 and Cyc8 in the presence of precursor molecules inositol and choline. To get access to its target genes, Opi1 physically contacts and counteracts DNA-bound activator Ino2. By using chromatin immunoprecipitation, we show that Sin3 and Cyc8 can be detected at Opi1 target promoters INO1 and CHO2 under repressing and derepressing conditions and that corepressor binding is effective even in the absence of Opi1, while Ino2 is absolutely required. Thus, corepressors may be recruited not only by repressors but also by activators such as Ino2. Indeed, we could demonstrate direct interaction of Ino2 with Sin3 and Cyc8. The Opi1 repressor interaction domain within Ino2 is also able to contact Sin3 and Cyc8. Recruitment of corepressors by an activator is not a regulatory exception as we could show that activators Pho4 and Hac1 also contain domains being able to interact with Sin3 and Cyc8.