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Regulatory Switch Enforced by Basic Helix-Loop-Helix and ACT-Domain

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Regulatory Switch Enforced by Basic Helix-Loop-Helix and ACT-Domain Regulatory switch enforced by basic helix-loop-helix PNAS PLUS and ACT-domain mediated dimerizations of the maize transcription factor R Que Konga,1, Sitakanta Pattanaika,b,1, Antje Fellerc,1, Joshua R. Werkmana, Chenglin Chaid, Yongqin Wangd, Erich Grotewoldc,d,2, and Ling Yuana,b,2 aDepartment of Plant and Soil Sciences and bKentucky Tobacco Research and Development Center, University of Kentucky, Lexington, KY 40546; and cMolecular, Cellular and Developmental Biology Program and dDepartment of Molecular Genetics and Center for Applied Plant Sciences, Ohio State University, Columbus, OH 43210 Edited by Peter H. Quail, University of California, Berkeley, CA, and approved May 21, 2012 (received for review April 10, 2012) The maize R2R3-MYB regulator C1 cooperates with the basic helix– the formation of MAX–MAD and MAX–MNT heterodimers loop–helix (bHLH) factor R to activate the expression of anthocya- results in transcriptional repression through the recruitment of nin biosynthetic genes coordinately. As is the case for other bHLH histone deacetylase complexes (5). factors, R harbors several protein–protein interaction domains. In plants, bHLH factors constitute one of the largest families Here we show that not the classical but rather a briefly extended of regulatory proteins (1). The Arabidopsis genome encodes for R bHLH region forms homodimers that bind canonical G-box DNA ∼162 bHLH proteins, about 10% of all of the known transcrip- motifs. This bHLH DNA-binding activity is abolished if the C-termi- tion factors in this plant (6–8). Maize R was the first plant bHLH nal ACT (aspartokinase, chorismate, and TyrA) domain is licensed to transcription factor described (9). R belongs to a small gene homodimerize. Then the bHLH remains in the monomeric form, family, which includes B, and R/B specify anthocyanin pigmen- allowing it to interact with R-interacting factor 1 (RIF1). In this con- tation in different plant tissues (10). They participate in the figuration, the R–RIF1 complex is recruited to the promoters of transcriptional regulation of anthocyanin pathway genes through a subset of anthocyanin biosynthetic genes, such as A1, through the cooperation with the R2R3–MYB transcription factor C1 or the interaction with its MYB partner C1. If, however, the ACT do- its paralog PL1 (11). C1 and R/B interact physically through the main remains monomeric, the bHLH region dimerizes and binds to MYB domain of C1 and the N-terminal region of R (12, 13), and G-boxes present in several anthocyanin genes, such as Bz1. Our C1 is responsible for tethering R through high-affinity P1-binding results provide a mechanism by which a dimerization domain in sites (haPBS) and low-affinity PBS (laPBS) cis-regulatory elements a bHLH factor behaves as a switch that permits distinct configura- to flavonoid biosynthetic gene promoters, such as A1 (14, 15). R/B tions of a regulatory complex to be tethered to different promoters. belong to the bHLH group IIIf (7), a subfamily that is shared Such a combinatorial gene regulatory framework provides one with similar anthocyanin regulators in various plants as well as mechanism by which genes lacking obviously conserved cis-regula- with the Arabidopsis GL3/EGL3 regulators of epidermal cell tory elements are regulated coordinately. patterning (16). All these factors function by interacting with R2R3–MYB proteins, recognizing particular signature motifs in gene regulation | promoter switch the corresponding MYB DNA-binding domains (13, 17). In addition, members of this group of bHLH proteins contain a he evolution of multicellular organisms was accompanied by conserved aspartokinase, chorismate mutase, TyrA (ACT)-like Tan increase in the complexity of gene-regulatory mechanisms, domain at the C termini, which participates in homodimer for- reflected in the expansion of transcription factor families and in mation (18). The importance of the bHLH motif in the regula- the intricacy of the interactions between regulatory proteins and tion of anthocyanin accumulation was revealed recently by the cis-regulatory elements in what is known today as “combinatorial identification of R-interacting factor1 (RIF1), a maize nuclear transcriptional control.” A premise of combinatorial control is protein with homology to breast cancer gene 2 (BRCA2)-inter- that different arrangements of a discrete number of regulatory acting EMSY factor. RIF1 specifically interacts with the bHLH proteins can be used to regulate a much larger number of genes. motif of R and links R function with promoter-specific histone Therefore, understanding how interactions between different functions (19). Surprisingly, however, thus far the canonical regulatory proteins impact their ability to deploy the expression function of the bHLH region of R as a DNA-binding homo- or of specific gene sets is of fundamental biological importance. heterodimerization domain has not been demonstrated. The basic helix–loop–helix (bHLH) family of transcription Here, we show that, for the bHLH region of R to dimerize, it factors is among the largest in multicellular organisms (1). The must be extended at least eight residues to include a couple of hallmark of the family is a bHLH domain, which consists of two evenly spaced leucine residues in what appears to be a short LZ functionally distinct regions. Generally, the basic region of the bHLH domain directly contacts DNA harboring an E-box se- quence (CANNTG), and the HLH region provides the potential Author contributions: E.G. and L.Y. designed research; Q.K., S.P., A.F., J.R.W., C.C., and for homo- and heterodimerization. In addition to the HLH Y.W. performed research; Q.K., S.P., A.F., J.R.W., C.C., Y.W., E.G., and L.Y. analyzed data; motif, bHLH factors often contain additional protein–protein and E.G. and L.Y. wrote the paper. PLANT BIOLOGY interaction domains. For example, members of the MYC family The authors declare no conflict of interest. of mammalian cell proliferation regulators, such as MAD or This article is a PNAS Direct Submission. MNT, contain a leucine-zipper (LZ) region that contributes to Freely available online through the PNAS open access option. the selective interaction with MAX, another bHLH-LZ protein 1Q.K., S.P., and A.F. contributed equally to this work. (2). MAX can form homo- or heterodimers with several related 2To whom correspondence may be addressed. E-mail: [email protected] or lyuan3@ proteins, including MAD (3) and MNT (4). Providing a textbook uky.edu. example of combinatorial transcriptional control, MYC–MAX See Author Summary on page 11918 (volume 109, number 30). – – and MAX MAX complexes bind E-boxes, but only the MYC This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. MAX heterodimer activates cell-proliferation genes. In contrast, 1073/pnas.1205513109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1205513109 PNAS | Published online July 9, 2012 | E2091–E2097 Downloaded by guest on September 24, 2021 – motif. When this briefly extended bHLH dimerizes, it binds R411 478 to dimerize in plant cells was confirmed by the activation DNA, and we show by Systemic Evolution of Ligands by Expo- of a luciferase reporter construct harboring GAL4-binding sites – – nential Enrichment (SELEX) that the R DNA-binding prefer- by coexpression of the pAD-R411 478 and pBD-R411 478 constructs – ence is to the G-box (CACGTG), a special type of E-box. in tobacco protoplasts but not by pAD-R411 478 (Fig. 1D). pBD- – However, if the bHLH region also includes the ACT domain, the R411 478 alone also resulted in the activation of the reporter, – R DNA-binding activity is lost, and the monomeric bHLH do- suggesting that pBD-R411 478 can interact with an endogenous main interacts with RIF1. We show that this configuration of the transactivator. Taken together, these results provide conclusive C1 R–RIF1 complex underlies the control of the expression of evidence that the briefly extended bHLH from R (residues 411– the A1 gene, in which recruitment to the A1 promoter is medi- 478p Fig. 1A) can mediate robust homodimer formation. ated primarily by the C1 DNA-binding activity. Mutations that We next investigated whether the ability of R bHLH to form abolish ACT-mediated dimerization restore the ability of R to homodimers conferred proteins carrying this domain the ca- dimerize through the bHLH and to recognize G-boxes. We pacity to bind DNA. Binding of purified recombinant N6His- – further show that the topology of the complex responsible for the R411 478 was tested by EMSA on a biotin-labeled, double- regulation of another anthocyanin biosynthetic gene, Bz1, pri- stranded 20-bp oligonucleotide (Fig. 2A, WT probe) harboring marily involves interactions of R with DNA, and in this case C1- an E-box with the core motif CACGTG (a G-box). As a control, mediated DNA interactions play a secondary role. In this con- EMSA also was performed on a mutant probe (Fig. 2A,M figuration, RIF1 is not part of the regulatory complex, as shown 411–478 probe) lacking a functional G-box. Binding of N6His-R to by ChIP experiments. Our results provide a mechanism by which the WT probe, but not to the M probe, was observed (Fig. 2A, – a dimerization domain in a bHLH factor behaves as a switch that N His-R411 478, compare lanes 5 and 6). Increasing concen- fi 6 permits different con gurations of regulatory complexes to be trations of the unlabeled WT probe, but not of the M probe, tethered to different promoters, helping explain the elusive 411–478 cis competed with the binding of N6His-R to the WT probe problem of how genes lacking conserved -regulatory elements (Fig. 2A,lanes7–11). Interestingly, and suggesting that the in- can be coordinately regulated. teraction with DNA further stabilizes homodimer formation, 411–524 N6His-R also showed robust and specific DNA-binding ac- Results and Discussion 411–478 A 411–524 fl tivity similar to that of N6His-R (Fig.
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