EXAMINATION OF MAMMALIAN BASIC HELIX-LOOP-HELIX TRANSCRIPTION FACTORSUSING A YEAST ONE-HYBRID SYSTEM

Basic helix-loop-helix (bHLH) transcription factors play diverse roles in controlling many developmental events, Although a great deal is un derstood about how bHLH factors activate gene transcription via E-box DNA consensus sequences, studies of bHLH factor function in higher euk aryotes often have been hindered by the presence of multiple family me mbers, As a first step in developing a simplified in vivo system to ex amine bHLH factor activities, we examined whether the bHLH muscle regu latory factors MRF4 and MyoD function appropriately in yeast. We show that Gal4-MRF4 fusion proteins, or native MRF4 proteins, activate expr ession of an E-box HIS3 reporter gene whereas MyoD proteins remain ina ctive, Deletion of the MRF4 transcription activation domain (TAD) or p oint mutations that abolish MRF4 DNA interactions inhibit HIS3 express ion, Substitution of the MRF4 TAD with the Gal4 TAD also produces a fu nctional protein, demonstrating that these transcription activation do mains are functionally equivalent in yeast, Replacement of the MRF4 TA D with the related MyoD TAD, however, generates an inactive protein, s uggesting that some specificity exists between bHLH family members, Us ing this experimental system, we also demonstrate that mammalian cDNA libraries can be screened successfully for cDNAs encoding novel bHLH p roteins that interact with E-box targets, Thus, this in vivo yeast sys tem provides a novel approach to facilitate functional studies of bHLH factor regulation.