A Domain of the even-skipped Protein Represses Transcription by Preventing TFIID Binding to a Promoter: Repression by Cooperative Blocking

Abstract

We examined the mechanism by which the C-terminal 236 amino acids of the even-skipped protein (region CD) repress transcription. A fusion protein, CDGB, was created that contains region CD fused to the glucocorticoid receptor DNA binding domain. This protein repressed transcription in an in vitro system containing purified fractions of the RNA polymerase II general transcription factors, and repression was dependent upon the presence of high-affinity glucocorticoid receptor binding sites in the promoter. Repression by CDGB was prevented when the promoter DNA was preincubated with TFIID or TBP, whereas preincubation of the template DNA with CDGB prevented TFIID binding. Together, these results strongly imply that CDGB represses transcription by inhibiting TFIID binding, and further experiments suggested a mechanism by which this may occur. Region CD can mediate cooperative interactions between repressor molecules such that molecules bound at the glucocorticoid receptor binding sites stabilize binding of additional CDGB molecules to low-affinity binding sites throughout the basal promoter. Binding to some of these low-affinity sites was shown to contribute to repression. Further experiments suggested that the full-length eve protein also represses transcription by the same mechanism. We speculate that occupancy of secondary sites within the basal promoter by CDGB or the eve protein inhibits subsequent TFIID binding to repress transcription, a mechanism we term cooperative blocking.

Drosophila Mediator Complex Is Broadly Utilized by Diverse Gene-Specific Transcription Factors at Different Types of Core Promoters

“…5B and D). Previous reports show that the repression domain of Even-skipped directly targets TBP (1,38). We also confirmed that the TFIID complex in the nuclear extract specifically interacts with Even-skipped under the same conditions in which Even-skipped failed to interact with dMediator ( Fig.…”

The Human Cut Homeodomain Protein Can Repress Gene Expression by Two Distinct Mechanisms: Active Repression and Competition for Binding Site Occupancy

“…In support of this mode of action, the unliganded thyroid hormone receptor was found to prevent assembly of a functional preinitiation complex (24), and the Dr-1 and Eve proteins were shown to interact, in vivo and in vitro, with the TATA-binding protein (65,68). That the TATA-binding protein can be a target for repression was also demonstrated by in vitro transcription studies using purified fractions of the RNA polymerase II general transcription factors (5,68). Thus, active repression domains appear to inhibit the formation of a functional preinitiation complex, but the exact step that is inhibited, DNA binding or protein-protein interactions, is still a matter of debate (5,14,65).…”

The Adenovirus E1A Repression Domain Disrupts the Interaction between the TATA Binding Protein and the TATA Box in a Manner Reversible by TFIIB

“…In addition, MOT1 prevents TBP binding to the TATA element in an ATP-dependent way (3,4). Recently, the Drosophila homeodomain protein Even-skipped (Eve) was reported to inhibit transcription by interaction with TBP and to prevent TFIID binding to the TATA box (5,53). However, neither topoisomerase I, MOT1, nor Eve interferes with TFIIB-TBP interaction.…”