High-affinity binding sites for the Deformed protein are required for the function of an autoregulatory enhancer of the Deformed gene.
The homeotic selector gene Deformed (Dfd) is required to specify the identity of head segments during Drosophila development. Previous experiments have shown that for the Dfd segmental identity function to operate in epidermal cells, the Dfd gene must be persistently expressed. One mechanism that provides persistent embryonic expression of Dfd is an autoregulatory circuit. Here, we show that the control of this autoregulatory circuit is likely to be directly mediated by the binding of Dfd protein to an upstream enhancer in Dfd locus DNA. In a 25-kb region around the Dfd transcription unit, restriction fragments with the highest binding affinity for Dfd protein map within the limits of the upstream autoregulatory element at approximately -5 kb. A minimal autoregulatory element, within a 920-bp segment of upstream DNA, has four moderate- to high-affinity binding sites for Dfd protein, with the two highest affinity sites sharing an ATCATTA consensus sequence. Site-specific mutagenesis of these four sites results in an element that has low affinity for Dfd protein when assayed in vitro and is nonfunctional when assayed in embryos.
Regulski
M
M
Department of Biology, Yale University, New Haven, Connecticut 06511.
Dessain
S
S
McGinnis
N
N
McGinnis
W
W
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004330:Drosophila / Q000196:embryology / Q000235:genetics* / Q000254:growth & development; D029721:Drosophila Proteins; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D005796:Genes; D018398:Homeodomain Proteins; D008969:Molecular Sequence Data; D015183:Restriction Mapping
D004268:DNA-Binding Proteins; C066676:Dfd protein, Drosophila; D029721:Drosophila Proteins; D018398:Homeodomain Proteins