Mark D. Biggin

A COMPARISON OF IN VIVO AND IN VITRO DNA-BINDING SPECIFICITIES SUGGESTS A NEW MODEL FOR HOMEOPROTEIN DNA BINDING IN DROSOPHILA EMBRYOS

Little is known about the range of DNA sequences bound by transcription factors in vivo. Using a sensitive UV cross‐linking technique, we show that three classes of homeoprotein bind at significant levels to the majority of genes in Drosophila embryos. The three classes bind with specificities different from each other; however, their levels of binding on any single DNA fragment differ by no more than 5‐ to 10‐fold. On actively transcribed genes, there is a good correlation between the in vivo DNA‐binding specificity of each class and its in vitro DNA‐binding specificity. In contrast, no such correlation is seen on inactive or weakly transcribed genes. These genes are bound poorly in vivo, even though they contain many high affinity homeoprotein‐binding sites. Based on these results, we suggest how the in vivo pattern of homeoprotein DNA binding is determined.

Transcription factors and the control of Drosophila development

Drosophila is a uniquely advantageous system for carrying out both biochemical and genetic analyses of proteins that regulate spatial and temporal patterns of transcription. Here we discuss what is known about the mechanisms of action and biological functions of transcription factors that act on genes controlling Drosophila embryogenesis.

Cooperative binding at a distance by even-skipped protein correlates with repression and suggests a mechanism of silencing.

In this study, we examined how the Drosophila developmental control gene even-skipped (eve) represses transcription. Tissue culture cells were used to show that eve contains domains which inhibit transcriptional activators present at the Ultrabithorax (Ubx) proximal promoter when bound up to 1.5 kb away from these activators. Different portions of eve were fused to a heterologous DNA binding domain to show that three adjacent regions of eve contribute to silencing. There appear to be two mechanisms by which eve protein represses transcription. In this study, we used in vitro transcription and DNA binding experiments to provide evidence for one of these mechanisms. Repression in vitro correlates with binding of eve protein to two low-affinity sites in the Ubx proximal promoter. Occupancy of these low-affinity sites is dependent upon cooperative binding of other eve molecules to a separate high-affinity site. Some of these sites are separated by over 150 bp of DNA, and the data suggest that this intervening DNA is bent to form a looped structure similar to those caused by prokaryotic repressors. One of the low-affinity sites overlaps an activator element bound by the zeste transcription factor. Binding of eve protein is shown to exclude binding by zeste protein. These data suggest a mechanism for silencing whereby a repressor protein would be targeted to DNA by a high-affinity element, which itself does not overlap activator elements. Cooperative binding of further repressor molecules to distant low-affinity sites, and competition with activators bound at these sites lead to repression at a distance.

A domain of the even-skipped protein represses transcription by preventing TFIID binding to a promoter: repression by cooperative blocking.

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.

Bending DNA can repress a eukaryotic basal promoter and inhibit TFIID binding.

Previous studies indicated that repression by eve involves cooperative DNA binding and leads to the formation of a DNA loop which encompasses the DNA sequences normally bound by the RNA polymerase II general transcription factors. To test the general principle of whether bending of a basal promoter sequence can contribute directly to repression of transcription, a minicircle template of 245 bp was used. In a purified transcription system, transcription from the minicircular DNA is greatly reduced compared with that from the identical DNA fragment in linear form. Transcription is also reduced when the minicircle contains a single-stranded nick, indicating that transcription is reduced because of DNA bending, rather than any constraint on supercoiling. We show that the reduced transcription from the minicircle in these experiments is not due to a reduced rate of elongation by RNA polymerase II. Rather, repression occurs, at least in part, because binding of the general transcription factor TFIID to the minicircle is strongly inhibited compared with binding to the linear DNA. We suggest that bending DNA may be a mechanism by which eukaryotic transcription may be regulated, by modulating the activity of the general transcription factors.