The maize Myb transcription factor C1 depends on the basic helix-loop-helix (bHLH) proteins R or B for regulatory function, but the closely related Myb protein P does not. We have used the similarity between the Myb domains of C1 and P to identify residues that specify the interaction between the Myb domain of C1 and the N-terminal region of R. Substitution of four predicted solvent-exposed residues in the first helix of the second Myb repeat of P with corresponding residues from C1 is sufficient to confer on P the ability to physically interact with R. However, two additional Myb domain amino acid changes are needed to make the P regulatory activity partially dependent on R in maize cells. Interestingly, when P is altered so that it interacts with R, it can activate the Bz1 promoter, normally regulated by C1 ؉ R but not by P. Together, these findings demonstrate that the change of a few amino acids within highly similar Myb domains can mediate differential interactions with a transcriptional coregulator that plays a central role in the regulatory specificity of C1, and that Myb domains play important roles in combinatorial transcriptional regulation. Combinatorial interactions between transcription factors are of central importance to regulation of gene expression in eukaryotes. These interactions can either modulate transcription factor activity or contribute to the biological specificity of factors with very similar DNA-interaction motifs. Elucidation of the mechanisms by which proteins with very similar DNA-binding domains achieve regulatory specificity remains a fundamental question in biology today.Proteins containing the Myb-homologous DNA-binding domain are widespread in eukaryotes (reviewed in refs. 1 and 2). The vertebrate c-myb gene plays an essential regulatory role in the proliferation and differentiation of hematopoietic cells. Besides c-myb, at least two other myb-related genes (A-myb and B-myb) are present in vertebrates (3). The products of these genes have Myb domains, each consisting of three head-to-tail Myb motifs (R1, R2, and R3). Oncogenic versions of c-myb, such as v-myb, contain only R2 and R3, as do hundreds of plant Myb-domain proteins (4). Myb domains formed by the R2 and R3 Myb motifs bind DNA. Each Myb motif contains three ␣-helices, and the third helix of each Myb motif makes sequencespecific DNA contacts. The second and third helices of each Myb motif form a helix-turn-helix structure when bound to DNA, similar to motifs found in the repressor and in homeo domains (5). In addition to their well-established roles in DNA binding, Myb domains are also emerging as important protein-protein interaction motifs. These Myb domain-mediated proteinprotein interactions play key roles in the biological specificity of the corresponding factors (6-13). However, the mechanisms by which protein-protein interactions contribute to the regulatory specificity of Myb domain proteins are poorly understood.In f lowering plants, several hundred genes containing the conserved Myb DNA-binding domain have b...

The maize basic-helix-loop-helix (bHLH) factor R belongs to a group of proteins with important functions in the regulation of metabolism and development through the cooperation with R2R3-MYB transcription factors. Here we show that in addition to the bHLH and the R2R3-MYB-interacting domains, R contains a dimerization region located C-terminal to the bHLH motif. This protein-protein interaction domain is important for the regulation of anthocyanin pigment biosynthesis by contributing to the recruitment of the C1 R2R3-MYB factor to the C1 binding sites present in the promoters of flavonoid biosynthetic genes. The R dimerization region bares structural similarity to the ACT domain present in several metabolic enzymes. Protein fold recognition analyses resulted in the identification of similar ACT-like domains in several other plant bHLH proteins. We show that at least one of these related motifs is capable of mediating homodimer formation. These findings underscore the function of R as a docking site for multiple protein-protein interactions and provide evidence for the presence of a novel dimerization domain in multiple plant bHLH proteins.Proteins containing the basic-helix-loop-helix (bHLH) 3 domain compose one of the largest transcription factor families in plants (1-4). The bHLH signature that defines the family is constituted by an N-terminal ϳ16-amino acid-long basic ␣-helix that binds DNA to the canonical E-box (CANNTG) (5) and a C-terminal helix-loop-helix (HLH) domain involved in homo-and/or heterodimerization (6). Some factors, however, such as the Id myogenic regulator, lack the basic region and function as inhibitors by forming heterodimers that cannot bind DNA (7). It is common for bHLH proteins to contain additional protein-protein interaction domains that contribute in unique ways to their regulatory function (8). For example, the Myc proto-oncoprotein forms heterodimers with Max through the respective bHLH and adjacent basic leucine zipper (bZip) domains (9). In contrast to Myc, which cannot homodimerize, Max can form homo-or heterodimers with several related proteins, including Mad1 and Mnt (10). The bHLH region of Myc mediates the interaction with Miz-1, a POZ transcription factor that permits Myc to bind and repress promoters lacking the CACGTG E-box (10, 11). Plant bHLH proteins are also characterized by the presence of several conserved domains in addition to the bHLH motif. For example, the analysis of the 133 Arabidopsis bHLH factors uncovered 40 or more domains present in three or more proteins (3). By and large the function of these domains is not known. Among the few for which functions have been identified are the N-terminally located small APB domain present in several phytochrome-interacting factors (12) and the region that mediates the interaction with R2R3-MYB factors (13, 14) central to providing R2R3-MYB transcriptional regulators with very similar DNA binding preferences with the ability to control distinct sets of target genes in vivo (15).The bHLH/R2R3-MYB cooperation is best e...

JunD is a versatile AP-1 transcription factor that can activate or repress a diverse collection of target genes. Precise control of junD expression and JunD proteinprotein interactions modulate tumor angiogenesis, cellular differentiation, proliferation and apoptosis. Molecular and clinical knowledge of two decades has revealed that precise JunD activity is elaborated by interrelated layers of constitutive transcriptional control, complex post-transcriptional regulation and a collection of post-translational modifications and protein-protein interactions. The stakes are high, as inappropriate JunD activity contributes to neoplastic, metabolic and viral diseases. This article deconvolutes multiple layers of control that safeguard junD gene expression and functional activity. The activity of JunD in transcriptional activation and repression is integrated into a regulatory network by which JunD exerts a pivotal role in cellular growth control. Our discussion of the JunD regulatory network integrates important open issues and posits new therapeutic targets for the neoplastic, metabolic and viral diseases associated with JunD/AP-1 expression.