Functional analysis of the transcriptional activator encoded by the maize B gene: Evidence for a direct functional interaction between two classes of regulatory proteins

The B, R, C1, and P1 genes regulating the maize anthocyanin pigment biosynthetic pathway encode tissue-specific tianscriptional activators. B and R are functionally duplicate genes that encode proteins with the basic-helix-loop-helix (b-HLH) motif found in Myc proteins. C1 and P1 encode functionally duplicate proteins with homology to the DNA-binding domain of Myb proteins. A member of the b-HLH family (B or R) and a member of the myb family (C1 or P1) are both required for anthocyanin pigmentation. Transient assays in maize and yeast were used to analyze the functional domains of the B protein and its interaction with C1. The results of these studies demonstrate that the b-HLH domain of B and most of its carboxyl terminus can be deleted with only a partial loss of B-protein function. In contrast, relatively small deletions within the B amino-terminal-coding sequence resulted in no tram-activation. Analysis of fusion constructs encoding the DNA-binding domain of yeast GAL4 and portions of B failed to reveal a transcriptional activation domain in the B protein. However, an amino-terminal domain of B was found to recruit a transcriptional activation domain by an interaction with C1. Formation of this complex resulted in the activation of a synthetic promoter containing GAL4 recognition sites, demonstrating that this interaction does not require the normal target promoters for B and C1. B and C1 fusions with yeast GAL4 DNA-binding and transcriptional activation domains were also found to interact when synthesized and assayed in yeast. The domains responsible for this interaction map to a region that contains the Myb homologous repeats of the C1 protein and to the amino terminus of the B protein, which does not contain the b-HLH motif. These studies suggest that the regulation of the maize anthocyanin pigmentation pathway involves a direct interaction between members of two distinct classes of transcriptional activators.