TFIIA is thought to play an important role in transcriptional regulation in higher eukaryotes, but its precise function is unclear. A human cDNA encoding a protein with 45% identity to the small subunit of yeast TFIIA has been isolated. TFIIA activity could be reconstituted by the mixing of recombinant large (~) and small (~/) subunits. TFIIA-depleted HeLa nuclear extracts were used to demonstrate that TFIIA is essential for basal and activated transcription by several distinct classes of activators. Recombinant TFIIA functioned in transcriptional activation whether expressed as a dimer (~+~,) or as a trimer (~+~+y), which closely resembles the native form. Yeast TFIIA also functioned in transcriptional activation, and the human ~, subunit was functionally interchangeable with TOA2, its yeast homolog. Recombinant TFIIA mediated the stimulation of TFIID binding to the TATA region and downstream promoter sequences by the Zta transcriptional activator. Significantly, we found that TFIIA bound directly to Zta in an activation domain-dependent manner. One consequence of the TFIIA-mediated interaction between Zta and TFIID was the formation of a promoter-bound complex resistant to TATA oligonucleotide competition. These results demonstrate that TFIIA is an evolutionarily conserved general factor critical for activator-regulated transcription.

The mechanisms allowing remote enhancers to regulate promoters several kilobase pairs away are unknown but are blocked by the Drosophila suppressor of Hairy-wing protein (Suhw) that binds to gypsy retrovirus insertions between enhancers and promoters. Suhw bound to a gypsy insertion in the cut gene also appears to act interchromosomally to antagonize enhancer-promoter interactions on the homologous chromosome when activity of the Chip gene is reduced. Development of multicellular organisms requires precise temporal and spatial regulation of gene expression. Much of this regulation depends on proteins that bind transcription enhancers. For enhancers separated by a few hundred base pairs from their promoter, DNA looping may be sufficient to allow interactions between basal factors at the promoter and the enhancer-binding proteins. Many complex developmentally regulated genes, however, contain multiple enhancers, which can be many kilobase pairs from the promoter. Enhancers require more than DNA looping to interact with the promoter over such remote distances. For instance, either a UAS or a higher eukaryotic enhancer must be upstream and promoter-proximal to activate transcription in yeast cells, yet both will function downstream of the gene in higher eukaryotic cells (Struhl 1989). This suggests that, in contrast to yeast, higher eukaryotes have factors that facilitate remote enhancer-promoter interactions.The effects of insertions of the gypsy retrovirus on enhancer activity in Drosophila lend support to the enhancer-facilitator hypothesis. Gypsy insertions block enhancer-promoter communication, without inactivating either the enhancer or promoter, when, and only when, they are between the enhancer and promoter (for review, see Dorsett 1996; Geyer 1997). The Suhw protein encoded by suppressor of Hairy-wing [su(Hw)] that binds to specific sequences in gypsy insertions is necessary and sufficient to block enhancers.A common domain in Suhw is required for gypsy insertions to block enhancers in several different genes (Harrison et al. 1993;Kim et al. 1996), suggesting that Suhw blocks all enhancers by the same mechanism. Enhancer blocking is distance independent and reversible (Dorsett 1993), and blocked enhancers remain active because they can activate a second promoter in the other direction (Cai and Levine 1995;Scott and Geyer 1995

Transcriptional adaptor proteins are required for full function of higher eukaryotic acidic activators in the yeast Saccharomyces cerevisiae, suggesting that this pathway of activation is evolutionarily conserved. Consistent with this view, we have identified possible human homologs of yeast ADA2 (yADA2) and yeast GCN5 (yGCN5), components of a putative adaptor complex. While there is overall sequence similarity between the yeast and human proteins, perhaps more significant is conservation of key sequence features with other known adaptors. We show several functional similarities between the human and yeast adaptors. First, as shown for yADA2 and yGCN5, human ADA2 (hADA2) and human GCN5 (hGCN5) interacted in vivo in a yeast twohybrid assay. Moreover, hGCN5 interacted with yADA2 in this assay, suggesting that the human proteins form similar complexes. Second, both yADA2 and hADA2 contain cryptic activation domains. Third, hGCN5 and yGCN5 had similar stabilizing effects on yADA2 in vivo. Furthermore, the region of yADA2 that interacted with yGCN5 mapped to the amino terminus of yADA2, which is highly conserved in hADA2. Most striking is the behavior of the human proteins in human cells. First, GAL4-hADA2 activated transcription in HeLa cells, and second, either hADA2 or hGCN5 augmented GAL4-VP16 activation. These data indicate that the human proteins correspond to functional homologs of the yeast adaptors, suggesting that these cofactors play a key role in transcriptional activation.

Mutations in the suppressor of Hairy-wing [su(Hw)] gene of Drosophila melanogaster can cause female sterility and suppress mutations that are insertions of the gypsy retrotransposon. Gypsy binds the protein (SUHW) encoded by su(Hw), and SUHW prevents enhancers promoter-distal to gypsy from activating gene transcription. SUHW contains 12 zinc fingers flanked by acidic N-and C-terminal domains. We examined the roles of each of the 12 zinc fingers in binding gypsy DNA and classified them into four groups: essential (fingers 6 through 10); beneficial but nonessential (fingers 1, 2, 3, and 11); unimportant (fingers 5 and 12); and inhibitory (finger 4). Because finger 10 is not required for female fertility but is essential for binding gypsy, these results imply that the SUHW-binding sites required for oogenesis differ in sequence from the gypsy-binding sites. We also examined the functions of the N-and C-terminal domains of SUHW by determining the ability of various deletion mutants to support female fertility and to alter expression of gypsy insertion alleles of the yellow, cut, forked, and Ultrabithorax genes. No individual segment of the N-and C-terminal domains of SUHW is absolutely required to alter expression of gypsy insertion alleles. However, the most important domain lies between residues 737 and 880 in the C-terminal domain. This region also contains the residues required for female fertility, and the fertility domain may be congruent with the enhancer-blocking domain. These results imply that SUHW blocks different enhancers and supports oogenesis by the same or closely related molecular mechanisms. Several spontaneous mutations in Drosophila melanogasterare insertions of the gypsy retrotransposon into or next to a gene (42). Expression of the phenotypes of gypsy insertion alleles can be suppressed to wild-type or near wild-type levels by suppressor of Hairy-wing [su(Hw)] mutations (42, 54). Other than suppression of gypsy insertions, female sterility is the only known phenotype associated with su(Hw) mutations (38).su(Hw) encodes a protein (SUHW) with 12 zinc finger motifs (44) that binds to a repeated consensus sequence downstream of the 5Ј long terminal repeat (LTR) of gypsy (11,61). Gypsy and the SUHW protein bound to gypsy can alter gene expression by multiple mechanisms. When gypsy is in a transcribed region of a gene and oriented in the same (parallel) direction, primary gene transcripts can be truncated and processed at the polyadenylation sites in the gypsy LTRs (13). Binding of SUHW to gypsy increases polyadenylation of gene transcripts in the 5Ј LTR (11, 13). Truncation of gene transcripts in gypsy occurs in parallel gypsy insertions in the achaete-scute complex (3) and the forked gene (29). The gypsy LTR poly(A) sites may also explain why parallel gypsy insertions in an intron of the Ultrabithorax gene display stronger phenotypes than antiparallel insertions (47).SUHW also alters gene expression by preventing enhancers and silencers from activating or repressing transcription (1,2,12,19,20,28,3...

The Drosophila protein Chip potentiates activation by several enhancers and is required for embryonic segmentation. Chip and its mammalian homologs interact with and promote dimerization of nuclear LIM proteins. No known Drosophila LIM proteins, however, are required for segmentation, nor for expression of most genes known to be regulated by Chip. Here we show that Chip also interacts with diverse homeodomain proteins using residues distinct from those that interact with LIM proteins, and that Chip potentiates activity of one of these homeodomain proteins in Drosophila embryos and in yeast. These and other observations help explain the roles of Chip in segmentation and suggest a model to explain how Chip potentiates activation by diverse enhancers.