Regulatory Specificity of R2R3 MYB Transcription Factors

The Control of Branched Flavonoid Pathways in Maize as a Case Study

Much progress has been made in elucidating the mechanisms of transcriptional regulation in eukaryotes. It is becoming clear that the regulatory specificity of transcription factors (TFs) is not given only by their interaction with conserved DNA-sequence motifs in the promoters of the genes that they control, but also by the interactions with other factors, which may or may not bind the target promoters. This strategy, also known as combinatorial transcriptional control, has been best studied in plants in studies focusing on the regulation of flavonoid biosynthesis in maize.

Flavonoid biosynthetic pathway showing the red phlobaphese pigments charachteristic

Figure 1: Flavonoid biosynthetic pathway showing the red phlobaphene pigments characteristic of maize pericarp and cob glumes expressing the P1 gene and the purple anthocyanins present in many flowering plants. The names of the maize (top) and Arabidopsis (bottom) genes encoding the enzymes of the pathway are indicated

There are three main reasons why studying transcriptional regulation in this system is ideal: 1) It provides a very well described plant biosynthetic pathway for which all the structural genes have been cloned, thus providing an array of co-regulated target genes for study; 2) the products of this pathway are pigments that serve as excellent visual markers not essential for the viability of the plant; 3) the extensive genetic studies of this pathway have yielded a rich collection of mutants that make studies in vivo possible.

The anthocyanin branch of the flavonoid pathway is regulated by the R2R3-MYB domain protein C1, which can only activate its target genes in the presence of the bHLH cofactor R (Fig. 1). We have established that R is critical in determining the specificity of C1, and that it has a dual role: it is an essential co-activator of C1 and it also enhances transcriptional activation by directly binding or by recruiting a second binding factor (indicated as RIF in Fig. 2) to the ARE, a cis element present in several anthocyanin biosynthetic genes. The identification of these two activities of R was assisted by using a mutant of P1, an R2R3-MYB regulator of the phlobaphene pigments (Fig. 1), which is capable of interacting with R (P1*, Fig. 2). The P1* protein controls a1 (Fig. 1), but not other anthocyanin specific genes, when in the absence of R. In the presence of R, however, it can activate the anthocyanin biosynthetic pathway.

The lab is now targeting R as a main player in the regulation of anthocyanin biosynthesis. We identified in yeast two-hybrid screens several proteins that interact with R.  Indeed, R contains multiple protein-protein interaction domains, and all the available evidence suggests that R plays a role as a scaffold for protein-protein interactions on the promoters of the genes it controls.

The MIR region (MYB-interacting region, amino acids 1-251) of R interacts with the MYB domain protein C1. The acidic region of R (amino acids 252-415) heterodimerizes with PAC1, a WD repeat protein, which has been shown to be involved in the regulation of anthocyanin biosynthesis. In addition, R contains a dimerization domain at its C-terminus (amino acids 525-610) which has structural similarity to an ACT domain, and which is necessary for its regulatory function.

Model to explain the regulatory specificity of C1 and P1

Figure 2: Model to explain the regulatory specificity of C1 and P1. P1* corresponds to a P1 mutant capable of interacting with R.

The role of the bHLH domain of R in transcriptional regulation remians elusive. We have shown that the bHLH domain of R is necessary for the activation of endogenous genes in maize BMS cells but it is dispensable for the activation of transiently introduced genes. This finding suggests an involvement of R in chromatin functions. Indeed, we recently identified in yeast two-hybrid screens a novel AGENET containing, EMSY-like protein RIF1 (R Interacting Factor1), which specifically interacts with the bHLH domain of R. This protein is required for endogenous flavonoid gene expression but not for promoter-reporter gene expression and it is part of the regulatory complex on at least one of the flavonoid gene promoters. Current experiments aim to understand the function of this protein in more detail and how transcriptional regulation and chromatin remodeling are linked.

In addition to RIF1, we identified two novel maize bHLH proteins which interact with the bHLH domain of R. We are currently characterizing these proteins and investigating their involvement in the regulatory function of R.

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