Basic helix-loop-helix (bHLH) transcription factors play diverse roles in 
controlling many developmental events. Although a great deal is understood about 
how bHLH factors activate gene transcription via E-box DNA consensus sequences, 
studies of bHLH factor function in higher eukaryotes often have been hindered by 
the presence of multiple family members. As a first step in developing a 
simplified in vivo system to examine bHLH factor activities, we examined whether 
the bHLH muscle regulatory factors MRF4 and MyoD function appropriately in 
yeast. We show that Gal4-MRF4 fusion proteins, or native MRF4 proteins, activate 
expression of an E-box HIS3 reporter gene whereas MyoD proteins remain inactive. 
Deletion of the MRF4 transcription activation domain (TAD) or point mutations 
that abolish MRF4 DNA interactions inhibit HIS3 expression. Substitution of the 
MRF4 TAD with the Gal4 TAD also produces a functional protein, demonstrating 
that these transcription activation domains are functionally equivalent in 
yeast. Replacement of the MRF4 TAD with the related MyoD TAD, however, generates 
an inactive protein, suggesting that some specificity exists between bHLH family 
members. Using this experimental system, we also demonstrate that mammalian cDNA 
libraries can be screened successfully for cDNAs encoding novel bHLH proteins 
that interact with E-box targets. Thus, this in vivo yeast system provides a 
novel approach to facilitate functional studies of bHLH factor regulation.