SPECIFICITY FOR THE HAIRY ENHANCER OF SPLIT BASIC HELIX-LOOP-HELIX (BHLH) PROTEINS MAPS OUTSIDE THE BHLH DOMAIN AND SUGGESTS 2 SEPARABLE MODES OF TRANSCRIPTIONAL REPRESSION/

The Hairy/Enhancer of split/Deadpan family of basic helix-loop-helix ( bHLH) proteins function as transcriptional repressors. We have examine d the mechanisms of repression used by the Hairy and E(SPL) proteins b y assaying the antagonism between wild-type or altered Hairy/E(SPL) an d Scute bHLH proteins during sex determination in Drosophila melanogas ter. Domain swapping and mutagenesis of the Hairy and E(SPL) proteins show that three evolutionarily conserved domains are required for thei r function: the bHLH, Orange, and WRPW domains. However, the suppressi on of Scute activity by Hairy does not require the WRPW domain. We sho w that the Orange domain is an important functional domain that confer s specificity among members of the Hairy/E(SPL) family. In addition, w e show that a Xenopus Hairy homology conserves not only Hairy's struct ure but also its biological activity in our assays. We propose that tr anscriptional repression by the Hairy/E(SPL) family of bHLH proteins i nvolves two separable mechanisms: repression of specific transcription al activators, such as Scute, through the bHLH and Orange domains and repression of other activators via interaction of the C-terminal WRPW motif with corepressors, such as the Groucho protein.