Target specificities of Drosophila enhancer of split basic helix-loop-helix proteins

Seven Enhancer of split genes in Drosophila melanogaster encode basic-helix -loop-helix transcription factors which are components of the Notch signall ing pathway. They are expressed in response to Notch activation and mediate some effects of the pathway by regulating the expression of target genes. Here we have determined that the optimal DNA binding site for the Enhancer of split proteins is a palindromic 12-bp sequence, 5'-TGGC ACGTG(C/T)(C/T)A -3', which contains an E-box core (CACGTG). This site is recognized by all of the individual Enhancer of split basic helix-loop-helix proteins, consis tent with their ability to regulate similar target genes in vivo. We demons trate that the 3 bp flanking the E-box core are intrinsic to DNA recognitio n by these proteins and that the Enhancer of split and proneural proteins c an compete for binding on specific DNA sequences. Furthermore, the regulati on conferred on a reporter gene in Drosophila by three closely related sequ ences demonstrates that even subtle sequence changes within an E box or fla nking bases have dramatic consequences on the overall repertoire of protein s that can bind in vivo.