Different Mechanisms Participate in the R-dependent Activity of the R2R3 MYB Transcription Factor C1

Abstract

The R2R3 MYB transcription factor C1 requires the basic helix-loop-helix factor R as an essential co-activator for the transcription of maize anthocyanin genes. In contrast, the R2R3 MYB protein P1 activates a subset of the C1-regulated genes independently of R. Substitution of six amino acids in P1 with the C1 amino acids results in P1*, whose activity on C1-regulated and P1-regulated genes is R-dependent or R-enhanced, respectively. We have used P1* in combination with various promoters to uncover two mechanisms for R function. On synthetic promoters that contain only C1/P1 binding sites, R is an essential co-activator of C1. This function of R is unlikely to simply be the result of an increase in the C1 DNA-binding affinity, since transcriptional activity of a C1 mutant that binds DNA at a higher affinity, comparable with P1, remains R-dependent. The differential transcriptional activity of C1 fusions with the yeast Gal4 DNA-binding domain in yeast and maize cells suggests that part of the function of R is to relieve C1 from a plant-specific inhibitor. A second function of R requires cis-regulatory elements in addition to the C1/P1 DNAbinding sites for R-enhanced transcription of a1. We hypothesize that R functions in this mode by binding or recruiting additional factors to the anthocyanin regulatory element conserved in the promoters of several anthocyanin genes. Together, these findings suggest a model in which combinatorial interactions with co-activators enable R2R3 MYB factors with very similar DNA binding preferences to discriminate between target genes in vivo.Flowering plants express a large number of proteins containing the conserved R2R3 MYB DNA-binding domain. About 125 R2R3 Myb genes are present in the Arabidopsis genome (1), and many more are predicted to be expressed in maize and related monocots (2, 3). Similar to other transcription factor families, the R2R3 MYB factors show exquisite regulatory specificity in vivo, while recognizing very similar DNA sequences in vitro (4 -9). Thus, mechanisms other than discrimination between similar DNA-binding sites are at play in the control of specific sets of target genes by each R2R3 MYB transcription factor in vivo.The regulation of flavonoid biosynthetic gene expression by the cooperation of R2R3 MYB and basic helix-loop-helix (bHLH)1 transcription factors provides one of the best described examples of combinatorial gene regulation in plants (10,11). Anthocyanin accumulation in maize is controlled by two classes of regulatory proteins that act in concert: C1 or PL1, two closely related R2R3 MYB domain proteins (12), and R or B, which are members of the R/B family of bHLH domain proteins (13). Extensive genetic and molecular studies have shown that the C1 or Pl1 genes require a member of the bHLH-containing R or B gene family to activate transcription of the anthocyanin biosynthetic genes (10). The C1-and R/Bencoded proteins physically interact, and this interaction is mediated by the MYB domain of C1 and the N-terminal region of B (14) or R (15).The m...

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

“…1B), no recruitment of R to DNA was observed (Fig. 4, R-GFP), as expected based on current models on how C1 and R function (28). However, when p35SC1 (C1) was cotransformed, a very significant recruitment of R-GFP to DNA was observed, yet only to the P region of the A1 promoter (Fig.…”

“…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.…”

The Arabidopsis Transcription Factor MYB12 Is a Flavonol-Specific Regulator of Phenylpropanoid Biosynthesis

“…Recent studies of the protein-protein interaction specificities of the R2R3-MYB protein family revealed specific conserved amino acid signatures to be the structural basis for interaction of R2R3-MYB and R/B-like BHLH proteins (Grotewold et al, 2000;Hernandez et al, 2004;Zimmermann et al, 2004). None of the identified amino acid motifs was found to be present in the MYB12 or ZmP amino acid sequences.…”

Caught Red-Handed:RcEncodes a Basic Helix-Loop-Helix Protein Conditioning Red Pericarp in Rice

“…These genes have different tissue specificities and exhibit no activation domains or DNA binding activity alone (Goff et al, 1992). Recent experiments in maize suggest that R functions in part to free C1 from interaction with a repressor protein as well as to recognize R-specific anthocyanin promoter elements (Hernandez et al, 2004). Maize also contains a bHLH protein, INTENSIFIER1 (In1), whose dominant allele acts as a negative regulator of pigmentation (Burr et al, 1996).…”