ADR1 Activation Domains Contact the Histone Acetyltransferase GCN5 and the Core Transcriptional Factor TFIIB

Abstract

The yeast transcriptional activator ADR1, which is required for ADH2 and peroxisomal gene expression, contains four separable and partially redundant activation domains (TADs). Mutations in ADA2 or GCN5, encoding components of the ADA coactivator complex involved in histone acetylation, severely reduced LexA-ADR1-TAD activation of a LexA-lacZ reporter gene. Similarly, the ability of the wild-type ADR1 gene to activate an ADH2-driven promoter was compromised in strains deleted for ADA2 or GCN5. In contrast, defects in other general transcription cofactors such as CCR4, CAF1/POP2, and SNF/SWI displayed much less or no effect on LexA-ADR1-TAD activation. Using an in vitro protein binding assay, ADA2 and GCN5 were found to specifically contact individual ADR1 TADs. ADA2 could bind TAD II, and GCN5 physically interacted with all four TADs. Both TADs I and IV were also shown to make specific contacts to the C-terminal segment of TFIIB. In contrast, no significant binding to TBP was observed. TAD IV deletion analysis indicated that its ability to bind GCN5 and TFIIB was directly correlated with its ability to activate transcription in vivo. ADR1 TADs appear to make several contacts, which may help explain both their partial redundancy and their varying requirements at different promoters. The contact to and dependence on GCN5, a histone acetyltransferase, suggests that rearrangement of nucleosomes may be one important means by which ADR1 activates transcription.In Saccharomyces cerevisiae, the transcriptional activator ADR1 is required for expression of the glucose-repressible alcohol dehydrogenase gene (ADH2) under nonfermentative conditions (1). It also regulates genes required in glycerol metabolism (2, 3) and peroxisome function and biogenesis (4, 5). ADR1 is a zinc finger, DNA-binding protein that is 153 kDa in size (6, 7). Its regulation of ADH2 under nonfermentative growth conditions occurs by binding to UAS1, a palindromic site, located 110 bp 1 upstream of the ADH2 TATAA sequence The presence of four transactivation regions suggests that ADR1 may make multiple protein contacts to transcriptional cofactors and/or core transcriptional components. The observation that TADs II and III are functionally redundant (9) suggests that some of these contacts may be made to the same protein.There are a number of potential targets for ADR1 activation domains. Core transcriptional components including TBP, TFIIB, TFIIF, TFIIE, and TAFs have been implicated in mammalians system as being direct contacts for transcriptional activators (12). In yeast, the GAL4 activation domain has been shown to bind TBP but not TFIIB in vitro (13). In addition to these core transcriptional factors, other cofactors or coactivators may mediate the action of activators. The ADA2 complex is one such coactivator complex. These proteins have been shown to bind activators like VP16 and GCN4 (14, 15) and to be required for maximal transcriptional activity of several yeast activators (16). However, some yeast activators like HAP4 and GAL4 (16,17) are ...

The yeast Ada complex mediates the ligand-dependent activation function AF-2 of retinoid X and estrogen receptors

“…Interestingly, ada5 and ada1 mutants exhibit more severe phenotypic defects and a broader spectrum of tran- scriptionally affected genes than the ada2 and gcn5 mutants (Marcus et al 1996;Horiuchi et al 1997), suggesting that like Ada3, Ada1 and Ada5 possess additional functions that are independent of Ada2 and Gcn5 (see below). Several subunits of the Ada complexes have been shown to interact directly in vitro with the ADs of several activator proteins: Interactions have been reported between Ada2 and either VP16 (Silverman et al 1994), Gcn4 (Barlev et al 1995), Adr1 (Chiang et al 1996), or GR ( 1) (Henriksson et al 1997), between Gcn5 and Adr1 (Chiang et al 1996), and between Ada5 and VP16 (Marcus et al 1996). We show here for the first time that Ada3 can also be the direct target for a transcriptional activator.…”

Adr1 and Cat8 synergistically activate the glucose-regulated alcohol dehydrogenase gene ADH2 of the yeast Saccharomyces cerevisiae

“…Synergistic derepression of ADH2 by Cat8 and Adr1 could be the result of recruiting a common coactivator\ mediator of transcriptional activation. Possible candidates may be Ada2 and the histone acetyltransferase Gcn5, as well as basal transcription factors TFIIB and components of TFIID (TBP, Taf90 and Taf130\145) for which direct contacts to activation domains within Adr1 have been demonstrated (Chiang et al, 1996 ;Komarnitsky et al, 1998).…”

“…Mutagenesis of individual positions within UAS1 allowed derivation of the sequence TTGGRGA or its reverse complement as the preferred Adr1-binding site (Cheng et al, 1994). In addition to its DNA-binding domain, Adr1 also contains four transcription activation domains (TADI-IV) which contact several coactivators and basal transcription factors such as Ada2, Gcn5 and TFIIB as well as subunits of TFIID (Cook et al, 1994 ;Chiang et al, 1996 ;Komarnitsky et al, 1998). Although several regulators of UAS1\Adr1-dependent gene expression have been described [the Cat1\Snf1\ Ccr1 protein kinase (Ciriacy, 1977 ;Denis, 1987), cAMP-dependent protein kinases Tpk1, 2, 3 (Cherry et al, 1989) and the protein phosphatase complex The synthetic DNA fragment ADH2-UAS2 was inserted into the promoter test plasmid pJS401 (∆UAS-ICL1-lacZ URA3 2µ), which contains the basal ICL1 promoter but is devoid of all upstream regulatory elements.…”

Adenovirus E1A requires the yeast SAGA histone acetyltransferase complex and associates with SAGA components Gcn5 and Tra1

“…Transcriptional co-activator proteins are required for E1A function Adr1 activates transcription of several genes in yeast through direct interaction with components of the TFIID complex and transcriptional co-activators including Ada2 and the HAT Gcn5 (Chiang et al, 1996;Komarnitsky et al, 1998). Gcn5 serves as a coactivator of transcription by acetylation of speci®c lysine residues in the amino-terminal tails of nucleosomal histones, which is associated with transcriptional activation (Kuo et al, 1996(Kuo et al, , 1998.…”