Identification of the residues in the Myb domain of maize C1 that specify the interaction with the bHLH cofactor R

Abstract

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 basic helix–loop–helix domain of maize R links transcriptional regulation and histone modifications by recruitment of an EMSY-related factor

“…4). Such a model had been predicted from extensive transient expression experiments and mutational analyses of the A1 promoter (9,11,19,28), but a direct in vivo tethering of C1 to the A1 promoter was never shown before. Most significant from a mechanistic perspective, however, our results support a model in which C1 is primarily responsible for specifying the promoters to which R needs to be recruited, while R furnishes a docking platform for the recruitment of additional factors to the complex, including RIF1, as shown in this study.…”

“…Microprojectile bombardment of maize BMS suspension cells and transient expression assays for luciferase and ␤-glucuronidase (GUS) were performed as previously described (9). For transient expression assays of firefly luciferase and Renilla luciferase (Renilla), the DualLuciferase Reporter Assay System (Promega) was used.…”

“…R/B belong to the group IIIf of plant bHLH factors (12), a subfamily that is shared with similar anthocyanin regulators in various plants as well as with the Arabidopsis GL3/EGL3 regulators of epidermal cell patterning (13). All of these factors function by interacting with R2R3-MYB proteins, recognizing particular signature motifs in the corresponding MYB DNA-binding domains (9,14). In addition, members of this group of bHLH proteins contain a conserved ACT-like domain at the C termini, which participates in homodimer formation (15).…”

“…They participate in the transcriptional regulation of the anthocyanin pathway genes through the cooperation with the R2R3-MYB transcription factor C1 or its paralog, PL1 (7). C1 and R/B physically interact through the MYB domain of C1 and the N-terminal region of R (which does not contain the bHLH motif) (8,9), and C1 makes direct DNA contacts with specific cis-regulatory elements, which, in the case of the A1 gene (encoding dihydroflavonol reductase, DFR), correspond to the high-and low-affinity P1 binding sites ( ha PBS and la PBS, respectively) (10,11). R/B belong to the group IIIf of plant bHLH factors (12), a subfamily that is shared with similar anthocyanin regulators in various plants as well as with the Arabidopsis GL3/EGL3 regulators of epidermal cell patterning (13).…”

Jasmonic acid control of GLABRA3 links inducible defense and trichome patterning inArabidopsis

“…Our results, however, suggest that JA signaling activates the function of GL1 protein in an indirect manner. Several R2R3-Myb proteins, including GL1, share a conserved motif within their Myb domains that physically interacts with R/B-like bHLH proteins (Grotewold et al, 2000;Zimmermann et al, 2004). The gl1-S92F mutant, in which the Myb domain of GL1 is disrupted (Ser92Phe substitution), did not induce any trichomes in response to wounding or MeJA treatment ( Fig.…”