The Transcriptional Repressor Even-skipped Interacts Directly with TATA-Binding Protein

Abstract

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...

Phosphorylation of the ASF/SF2 RS domain affects both protein-protein and protein-RNA interactions and is necessary for splicing.

“…The GST-ASF derivatives, GST-ClkK and GST-ClkR proteins, were expressed in and purified from E. coli essentially as de scribed (Um et al 1995). E. coli cells (IMIOI; 100 ml) trans formed with the appropriate plasmids were induced with 1 mM IPTG at 15°C overnight.…”

Tuning cell differentiation patterns and single cell dynamics by regulating proteins’ functionalities in a toggle switch

“…First, we assume that k rep and k unrep have equal values for both the functional and non-functional dimers in the case where non-functional dimers can bind to DNA but do not repress transcription. This occurs, e.g., in the example described of the repressor Even-skipped (Eve) where phosphorylation of the repressor domain by glycogen-synthase kinase-3 allows transcription without affecting Eve's DNA binding ability [48]. It is further noted that the domain where the repressor binds to DNA can be upstream to the elongation sequence and thus, a non-functional dimer bound to DNA does not necessarily affect transcription (e.g.…”

“…Eve is an example of such situations since, as described, has two separable domains and the reduction of its ability to repress transcription does not affect Eve's DNA binding ability [48].…”

“…A well characterized example demonstrating these principles of regulation of partial functionality of a gene regulatory protein is the repressor Even-skipped (Eve) [48]. Eve is a sequence-specific homeodomain repressor that is involved in pattern formation during early embryogenesis [46,48].…”

“…Eve is a sequence-specific homeodomain repressor that is involved in pattern formation during early embryogenesis [46,48]. It is comprised of two separable domains: a DNA-binding domain and a repressor domain, which directly interacts with the TATA-binding protein (TBP) [48].…”