EVEN-SKIPPED REPRESSES TRANSCRIPTION BY BINDING TATA-BINDING PROTEIN AND BLOCKING THE TFIID-TATA BOX INTERACTION

The Drosophila homeodomain protein Even-skipped (Eve) is a transcripti onal repressor, and previous studies have suggested that it functions by interfering with the basal transcription machinery. Here we describ e experiments indicating that the mechanism of Eve repression involves a direct interaction with the TATA binding protein (TBP) that blocks binding of TBP-TFIID to the promoter. We first compared Eve activities in in vitro transcription systems reconstituted with either all the g eneral transcription factors or only TBP, TFIIB, TFIIF30, and RNA poly merase II. In each case, equivalent and very efficient levels of repre ssion were observed, indicating that no factors other than those in th e minimal system are required for repression. We then show that Eve ca n function efficiently when its recognition sites are far from the pro moter and that the same regions of Eve required for repression in vivo are necessary and sufficient for in vitro repression. This includes, in addition to an Ala-Pro-rich region, residues within the homeodomain . Using GAL4-Eve fusion proteins, we demonstrate that the homeodomain plays a role in repression in addition to DNA binding, which is to fac ilitate interaction with TBP. Single-round transcription experiments i ndicate that Eve must function prior to TBP binding to the promoter, s uggesting a mechanism whereby Eve represses by competing with the TATA box for TBP binding. Consistent with this, excess TATA box-containing oligonucleotide is shown to specifically and efficiently disrupt the TBP-Eve interaction. Importantly, we show that Eve binds directly to T FIID and that this interaction can also be disrupted by the TATA oligo nucleotide. We conclude that Eve represses transcription via a direct interaction with TBP that blocks TFIID binding to the promoter.