No abstract

A selection for yeast mutants resistant to GAL4-VP16-induced toxicity previously identified two genes, ADA2 and ADA3, which may function as adaptors for some transcriptional activation domains and thereby facilitate activation. Here we identify two new genes by the same selection, one of which is identical to GCN5. We show that gcn5 mutants share properties with ada mutants, including slow growth, temperature sensitivity and reduced activation by the VP16 and GCN4 activation domains. Double mutant studies suggest that ADA2 and GCN5 function together in a complex or pathway. Moreover, we demonstrate that GCN5 binds to ADA2 both by the two-hybrid assay in vivo and by co-immunoprecipitation in vitro. This suggests that ADA2 and GCN5 are part of a heteromeric complex that mediates transcriptional activation. Finally, we demonstrate the functional importance of the bromodomain of GCN5, a sequence found in other global transcription factors such as the SWIISNF complex and the TATA binding protein-associated factors. This domain is not required for the interaction between GCN5 and ADA2 and thus may mediate a more general activity of transcription factors.

The analysis of single nucleotide polymorphisms (SNPs) is increasingly utilizedto investigate the genetic causes of complex human diseases. Here we present a high-throughput genotyping platform that uses a one-primer assay to genotype over 10,000 SNPs per individual on a single oligonucleotide array. This approach uses restriction digestion to fractionate the genome, followed by amplification of a specific fractionated subset of the genome. The resulting reduction in genome complexity enables allele-specific hybridization to the array. The selection of SNPs was primarily determined by computer-predicted lengths of restriction fragments containing the SNPs, andwas further driven by strict empirical measurements of accuracy, reproducibility, andaverage call rate, which we estimate to be >9.5%, >99.9%, and>95%, respectively. With average heterozygosity of 0.38 andgenome scan resolution of 0.31 cM, the SNP array is a viable alternative to panels of microsatellites (STRs). As a demonstration of the utility of the genotyping platform in whole-genome scans, we have replicated and refined a linkage region on chromosome 2p for chronic mucocutaneous candidiasis and thyroid disease, previously identified using a panel of microsatellite (STR) markers

Mutations in yeast ADA2, ADA3, and GCN5 weaken the activation potential of a subset of acidic activation domains. In this report, we show that their gene products form a heterotrimeric complex in vitro, with ADA2 as the linchpin holding ADA3 and GCN5 together. Further, activation by LexA-ADA3 fusions in vivo are regulated by the levels of ADA2. Combined with a prior observation that LexA-ADA2 fusions are regulated by the levels of ADA3 (N. Silverman, J. Agapite, and L. Guarente, Proc. Natl. Acad. Sci. USA 91:11665-11668, 1994), this finding suggests that these proteins also form a complex in cells. ADA3 can be separated into two nonoverlapping domains, an amino-terminal domain and a carboxyl-terminal domain, which do not separately complement the slow-growth phenotype or transcriptional defect of a ⌬ada3 strain but together supply full complementation. The carboxyl-terminal domain of ADA3 alone suffices for heterotrimeric complex formation in vitro and activation of LexA-ADA2 in vivo. We present a model depicting the ADA complex as a coactivator in which the ADA3 amino-terminal domain mediates an interaction between activation domains and the ADA complex.In eukaryotes, several factors that are important in the activation of transcription by RNA polymerase II are in large, heteromeric complexes. For example, the yeast SWI2/SNF2, SWI1, SWI3, SNF5, and SNF6 proteins form a large multisubunit complex, which apparently counters repression by chromatin (4,5,7,23). Mutations in SWI2 and SNF5 result in decreased transcription and altered chromatin structure at certain promoters (19). These phenotypes can be suppressed by mutations in histone genes. In another case, the yeast SRB2, SRB4, SRB5, and SRB6 proteins form a holoenzyme complex with RNA polymerase II and certain basal transcription factors (21). The SRB proteins interact with the carboxyl-terminal domain of the largest subunit of RNA polymerase II and are important for both basal and activated transcription in vitro (33). In higher eukaryotes, the TFIID complex is composed of TATA-binding protein (TBP) and TBP-associated factors (TAFs) (9). While TBP with the other basal factors is sufficient for basal transcription, the TAFs are required for activated transcription (12, 13). Thus, the TAFs are proposed to be coactivators or adaptors required to mediate the stimulatory signal from activators to basal factors. There is also evidence that a family of factors interact with TBP in yeast cells (10,25,34).In addition to the TAFs, other factors, such as the yeast ADA2, ADA3, and GCN5 gene products, have been proposed to be coactivators (3,22,24). Mutations in ADA2, ADA3, and GCN5 were isolated in a selection for mutants which confer resistance to toxicity from overexpressed GAL4-VP16. This toxicity is postulated to occur by titration of basal transcription factors away from productive transcription complexes by the strong acidic activation domain of GAL4-VP16 (2). If this titration by GAL4-VP16 requires proteins with coactivator or adaptor function, alterations in thes...

We describe the isolation of a yeast gene, ADA3, mutations in which prevent the toxicity of GAL4-VP16 in vivo. Toxicity was previously proposed to be due to the trapping of general transcription factors required at RNA polymerase II promoters (S. L. Berger, B. Pina, N. Silverman, G. A. Marcus, J. Agapite, J. L. Regier, S. J. Triezenberg, and L. Guarente, Cell 70:251-265, 1992). trans activation by VP16 as well as the acidic activation domain of GCN4 is reduced in the mutant. Other activation domains, such as those of GALA and HAP4, are only slightly affected in the mutant. This spectmm is similar to that observed for mutants with lesions inADA2, a gene proposed to encode a transcriptional adaptor. TheADA3 gene is not absolutely essential for cell growth, but gene disruption mutants grow slowly and are temperature sensitive. Strains doubly disrupted for ada2 and ada3 grow no more slowly than single mutants, providing further evidence that these genes function in the same pathway. Selection of initiation sites by the general transcriptional machinery in vitro is altered in the ada3 mutant, providing a clue that ADA3 could be a novel general transcription factor involved in the response to acidic activators.The activation of transcription in eukaryotes requires transcriptional activators, which are proteins that bind to sites distal from the TATA box, termed enhancers or upstream activation sequences (UASs) (4, 25, 44). These activators contain discrete activation domains that augment transcription initiation in the vicinity of the TATA box (8). The activation domains of one major class of activators are characterized by a high concentration of amino acids with acidic side chains. Acidic activation domains are found in many yeast activators, including GAL4, GCN4, and HAP4 (23,34,40). A protein from the virion of herpes simplex virus, VP16, contains an unusually potent acidic activation domain (54, 62). These acidic activators function in many eukaryotic cells, ranging from yeasts to mammals, indicating that their mechanism of action has been conserved in eukaryotes.The transcription initiation reaction at the TATA box is mediated by general transcription factors, including the TATA box-binding protein (TBP). TBP was purified as a monomer from Saccharomyces cerevisiae (9) but was found as part of a multiprotein complex in higher cells (16,52 However, in the absence of chromatin assembly, robust transcriptional activation can still be observed in vitro, indicating the existence of a chromatin-independent mechanism of activation. In this mechanism, the activator is thought to contact some component of the general transcriptional machinery. Chromatographic studies show that the * Corresponding author. acidic activation domain of VP16 is capable of binding to TBP and also to TFIIB (36,39,60). How these interactions relate to transcriptional activation has not yet been clarified.Other evidence suggests that protein-protein interactions between activation domains and the transcriptional machinery occur via intermediary f...