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.
TenHarmsel
A
A
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511.
Austin
R J
RJ
Savenelli
N
N
Biggin
M D
MD
D000818:Animals; D001426:Bacterial Proteins; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004330:Drosophila / Q000235:genetics* / Q000378:metabolism; D029721:Drosophila Proteins; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005786:Gene Expression Regulation; D005801:Genes, Homeobox; D018398:Homeodomain Proteins; D008961:Models, Structural; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D014157:Transcription Factors; D014158:Transcription, Genetic; D014162:Transfection
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D029968:Escherichia coli Proteins; C474481:EspD protein, E coli; D018398:Homeodomain Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; C052837:eve protein, Drosophila; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase