The Drosophila homeodomain protein Even-skipped (Eve) has previously been shown to function as a sequence-specific transcriptional repressor, and in vitro and in vivo experiments have shown that the protein can actively block basal transcription. However, the mechanism of repression is not known. Here, we present evidence establishing a direct interaction between Eve and the TATA-binding protein (TBP). Using cotransfection assays with minimal basal promoters whose activity can be enhanced by coexpression of TBP, we found that Eve could efficiently block, or squelch, this enhancement. Squelching did not require Eve DNA-binding sites on the reporter plasmids but was dependent on the presence of the Eve repression domain. Further support for an in vivo interaction between the Eve repression domain and TBP was derived from a two-hybridtype assay with transfected cells. Evidence that Eve and TBP interact directly was provided by in vitro binding assays, which revealed a specific protein-protein interaction that required an intact Eve repression domain and the conserved C terminus of TBP. The Eve homeodomain was also required for these associations, suggesting that it may function in protein-protein interactions. We also show that a previously characterized artificial repression region behaves in a manner similar to that of the Eve repression domain, including its ability to squelch TBP-enhanced expression in vivo and to bind TBP specifically in vitro. Our results suggest a model for transcriptional repression that involves an interaction between Eve and TBP.It is clear by now that control of gene expression in eukaryotes involves repression as well as activation of transcription. A significant number of proteins that are capable of functioning as transcriptional repressors in various assays have been identified, and many of them are known to play key roles in a variety of important cellular and developmental processes. These include, for example, the homeodomain protein ␣2, which functions with other proteins to control cell type in Saccharomyces cerevisiae (19,21); the homeodomain proteins Even-skipped (Eve) and Engrailed (En), which are involved in pattern formation during early Drosophila embryogenesis (13, 17); and in mammals, the Zn 2ϩ finger-containing v-erbA oncoprotein, or thyroid hormone receptor (8), and the WT1 Wilms tumor gene product (31). These proteins all share the property that they are sequence-specific DNA binding proteins capable of recognizing binding sites in target genes and repressing transcription.There are a number of ways in which transcriptional repressors can function, and even those whose action involves sequence-specific DNA binding can employ distinct mechanisms (for reviews, see references 26 and 39). Perhaps the simplest involves competition for DNA-binding sites, whereby the repressor interferes with binding of either an activator or a basal transcription factor, by virtue of adjacent or overlapping binding sites. A second mechanism, called quenching, involves simultaneous DNA bindi...

TATA binding protein (TBP) is a critical transcription factor involved in transcription by all three RNA polymerases (RNAPs). Studies using in vitro systems and yeast have shown that the C-terminal core domain (CTD) of TBP is necessary and sufficient for many TBP functions, but the significance of the N-terminal domain (NTD) of TBP is still obscure. Here, using transient expression assays in Drosophila Schneider cells, we show that the NTD of Drosophila TBP (dTBP) strongly activates transcription when fused to the GAL4 DNA binding domain (DBD). Strikingly, the activity of the NTD is completely repressed in the context of full-length dTBP. In contrast to the much weaker activation obtained by either full-length dTBP or the dTBP CTD fused to the GAL4 DBD, activation by the NTD is dependent on the presence of GAL4 binding sites and is susceptible to the effects of a dominant negative TFIIB mutant, TFIIB∆C202, a property observed previously with certain authentic activation domains. Activation by the NTD, but not full-length dTBP or the CTD, seems to be mediated by the action of a strong activation domain, likely a glutamine-rich region. In conclusion, the dTBP NTD can behave as a very strong activator that is masked in the full-length protein, suggesting possible roles for the dTBP NTD in RNAP II-mediated transcription. TATA binding protein Drosophila Activation domain N-terminal domain