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• W2107455091 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 DNA-binding 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. 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 DNA-binding 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 (1Stracke R. Werber M. Weisshaar B. Curr. Opin. Plant Biol. 2001; 4: 447-456Crossref PubMed Scopus (1500) Google Scholar), and many more are predicted to be expressed in maize and related monocots (2Rabinowicz P.D. Braun E.L. Wolfe A.D. Bowen B. Grotewold E. Genetics. 1999; 153: 427-444PubMed Google Scholar, 3Jiang C. Gu J. Chopra S. Gu X. Peterson T. Gene (Amst.). 2004; 326: 13-22Crossref PubMed Scopus (104) Google Scholar). Similar to other transcription factor families, the R2R3 MYB factors show exquisite regulatory specificity in vivo, while recognizing very similar DNA sequences in vitro (4Sainz M.B. Grotewold E. Chandler V.L. Plant Cell. 1997; 9: 611-625PubMed Google Scholar, 5Sablowski R.W.M. Moyano E. Culianez-Macia F.A. Schuch W. Martin C. Bevan M. EMBO J. 1994; 13: 128-137Crossref PubMed Scopus (275) Google Scholar, 6Grotewold E. Drummond B. Bowen B. Peterson T. Cell. 1994; 76: 543-553Abstract Full Text PDF PubMed Scopus (533) Google Scholar, 7Romero I. Fuertes A. Benito M.J. Malpica J.M. Leyva A. Paz-Ares J. Plant J. 1998; 14: 273-284Crossref PubMed Scopus (244) Google Scholar, 8Urao T. Yamaguchi-Shinozaki K. Urao S. Shinozaki K. Plant Cell. 1993; 5: 1529-1539Crossref PubMed Scopus (453) Google Scholar, 9Suzuki A. Wu C.Y. Washida H. Takaiwa F. Plant Cell Phys. 1998; 39: 555-559Crossref PubMed Scopus (48) Google Scholar). 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) 1The abbreviations used are: bHLH, basic helix-loop-helix; ARE, anthocyanin regulatory element; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. transcription factors provides one of the best described examples of combinatorial gene regulation in plants (10Mol J. Grotewold E. Koes R. Trends Plant Sci. 1998; 3: 212-217Abstract Full Text Full Text PDF Scopus (671) Google Scholar, 11Irani N.G. Hernandez J.M. Grotewold E. Rec. Adv. Phytochem. 2003; 38: 59-78Crossref Scopus (29) Google Scholar). 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 (12Cone K.C. Cocciolone S.M. Burr F.A. Burr B. Plant Cell. 1993; 5: 1795-1805Crossref PubMed Scopus (212) Google Scholar), and R or B, which are members of the R/B family of bHLH domain proteins (13Ludwig S.E. Wessler S.R. Cell. 1990; 62: 849-851Abstract Full Text PDF PubMed Scopus (142) Google Scholar). 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 (10Mol J. Grotewold E. Koes R. Trends Plant Sci. 1998; 3: 212-217Abstract Full Text Full Text PDF Scopus (671) Google Scholar). The C1- and R/B-encoded proteins physically interact, and this interaction is mediated by the MYB domain of C1 and the N-terminal region of B (14Goff S.A. Cone K.C. Chandler V.L. Genes Dev. 1992; 6: 864-875Crossref PubMed Scopus (332) Google Scholar) or R (15Grotewold E. Sainz M.B. Tagliani L. Hernandez J.M. Bowen B. Chandler V.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13579-13584Crossref PubMed Scopus (266) Google Scholar). The maize P1 gene, encoding another R2R3 MYB transcription factor, controls the accumulation of 3-deoxyflavonoids and red phlobaphene pigments by activating a subset of the anthocyanin biosynthetic genes controlled by C1 and R, without a need for a known bHLH partner. P1 and C1 activate the expression of some common genes in the flavonoid pathway such as a1, and they interact with different affinities to the same cis-acting regulatory elements in the a1 gene promoter (4Sainz M.B. Grotewold E. Chandler V.L. Plant Cell. 1997; 9: 611-625PubMed Google Scholar, 6Grotewold E. Drummond B. Bowen B. Peterson T. Cell. 1994; 76: 543-553Abstract Full Text PDF PubMed Scopus (533) Google Scholar). The a1 promoter has a modular structure in which the proximal high affinity P1 binding sites (haPBS) and the distal low affinity P1-binding sites (laPBS) are separated by the anthocyanin regulatory element (ARE) (16Lesnick M.L. Chandler V.L. Plant Physiol. 1998; 117: 437-445Crossref PubMed Scopus (63) Google Scholar). In transient expression experiments, these three elements contribute to the regulation of a1 by P1 or by C1 + R (4Sainz M.B. Grotewold E. Chandler V.L. Plant Cell. 1997; 9: 611-625PubMed Google Scholar, 6Grotewold E. Drummond B. Bowen B. Peterson T. Cell. 1994; 76: 543-553Abstract Full Text PDF PubMed Scopus (533) Google Scholar, 17Tuerck J.A. Fromm M.E. Plant Cell. 1994; 6: 1655-1663PubMed Google Scholar). In addition, transposon insertions and mutations in the ARE differentially affect the in vivo regulation of a1 by P1 or C1 + R (18Pooma W. Gersos C. Grotewold E. Genetics. 2002; 161: 793-801PubMed Google Scholar). Unlike P1, C1 activates the transcription of the a2, bz1, and bz2 genes, which are specific for the anthocyanin branch of the pathway (4Sainz M.B. Grotewold E. Chandler V.L. Plant Cell. 1997; 9: 611-625PubMed Google Scholar, 6Grotewold E. Drummond B. Bowen B. Peterson T. Cell. 1994; 76: 543-553Abstract Full Text PDF PubMed Scopus (533) Google Scholar, 16Lesnick M.L. Chandler V.L. Plant Physiol. 1998; 117: 437-445Crossref PubMed Scopus (63) Google Scholar). The a2, bz1, and bz2 gene promoters are also modular (16Lesnick M.L. Chandler V.L. Plant Physiol. 1998; 117: 437-445Crossref PubMed Scopus (63) Google Scholar, 19.Lesnick, M. L. (1997) Analysis of the cis-acting Sequences Required for C1/B Activation of the Maize Anthocyanin Biosynthetic Pathway. Ph.D. thesis, pp. 32-53, University of Oregon, Eugene, ORGoogle Scholar), containing ARE and C1-binding sites. Although the MYB domains of P1 and C1 are over 70% identical (20Grotewold E. Athma P. Peterson T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4587-4591Crossref PubMed Scopus (204) Google Scholar) and they recognize very similar DNA sequences (4Sainz M.B. Grotewold E. Chandler V.L. Plant Cell. 1997; 9: 611-625PubMed Google Scholar), only C1 has an absolute requirement for the bHLH factor R to activate transcription of the anthocyanin biosynthetic genes (R-dependent transcription). In contrast, P1 controls gene expression independently of R (R-independent transcription). The substitution of six residues in the MYB domain of P1 with the corresponding residues from C1 generates the P1* protein (Fig. 1A), which, unlike P1, is able to physically interact with R (15Grotewold E. Sainz M.B. Tagliani L. Hernandez J.M. Bowen B. Chandler V.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13579-13584Crossref PubMed Scopus (266) Google Scholar). Similar to P1, P1* activates a1 but not bz1 in the absence of R, indicating that the DNA-binding properties of P1* have not been altered. Interestingly, however, in the presence of R, P1* mediates a robust activation of the bz1 promoter (15Grotewold E. Sainz M.B. Tagliani L. Hernandez J.M. Bowen B. Chandler V.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13579-13584Crossref PubMed Scopus (266) Google Scholar). The cooperation between bHLH and R2R3 MYB transcription factors is not limited to the regulation of flavonoid biosynthesis by C1- and R-like proteins. The GL1 R2R3 MYB protein interacts with the GL3 and EGL3 bHLH factors to regulate the accumulation of trichomes in Arabidopsis (21Payne T. Zhang F. Lloyd A.M. Genetics. 2000; 156: 1349-1362PubMed Google Scholar, 22Zhang F. Gonzalez A. Zhao M. Payne C.T. Lloyd A. Development. 2003; 130: 4859-4869Crossref PubMed Scopus (621) Google Scholar). Similarly, the bHLH rd22BP1 and R2R3 MYB AtMYB2 Arabidopsis proteins cooperate for drought- and abscisic acid-regulated gene expression (23Abe H. Yamaguchi-Shinozaki K. Urao T. Iwasaki T. Hosokawa D. Shinozaki K. Plant Cell. 1997; 9: 1859-1868PubMed Google Scholar). Whereas the Arabidopsis genome contains more than 120 genes encoding bHLH proteins (24Atchley W.R. Fitch W.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5172-5176Crossref PubMed Scopus (501) Google Scholar, 25Buck M.J. Atchley W.R. J. Mol. Evol. 2003; 56: 742-750Crossref PubMed Scopus (103) Google Scholar, 26Heim M.A. Jakoby M. Werber M. Martin C. Weisshaar B. Bailey P.C. Mol. Biol. Evol. 2003; 20: 735-747Crossref PubMed Scopus (733) Google Scholar, 27Toledo-Ortiz G. Huq E. Quail P.H. Plant Cell. 2003; 15: 1749-1770Crossref PubMed Scopus (921) Google Scholar), the factors that cooperate with R2R3 MYB proteins belong to a small subgroup of bHLH proteins that share a common motif in their N termini. 2E. L. Braun and E. Grotewold, unpublished observations. This motif corresponds to the region in R that interacts with C1 (14Goff S.A. Cone K.C. Chandler V.L. Genes Dev. 1992; 6: 864-875Crossref PubMed Scopus (332) Google Scholar, 15Grotewold E. Sainz M.B. Tagliani L. Hernandez J.M. Bowen B. Chandler V.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13579-13584Crossref PubMed Scopus (266) Google Scholar). These findings suggest a general mechanism of cooperation between R2R3 MYB proteins and bHLH factors in transcriptional regulation. How this cooperation contributes to the regulatory specificity of R2R3 MYB proteins is the subject of this study. Herein, we have investigated the cooperation between C1 and R for the regulation of flavonoid biosynthetic genes. Using P1*, we uncovered two components for this synergy. One component, manifested by the R-enhanced activity of C1 and P1* on the a1 promoter requires, in addition to the high affinity P1-binding sites, cis-regulatory sequences within the ARE. The second component is manifested by the R-dependent activity of C1 on promoters containing only the haPBS. We generated a mutant of C1 (C1SH) that binds DNA with a higher affinity than C1 and comparable to P1. Using C1SH, we demonstrate that the R-dependent activity of C1 is not solely due to the intrinsic low DNA-binding affinity of C1, since the C1SH transcriptional activity continues to be R-dependent. The differential activity in yeast and maize cells of chimeras of C1 with the yeast Gal4 DNA-binding domain suggests that part of the function of R is to relieve C1 from an inhibitor. Together, our findings uncover two distinct and separable mechanisms by which R cooperates with C1 for transcriptional activity. In addition, our results provide a model to explain how R2R3 MYB factors with related DNA binding preferences achieve regulatory specificity through combinatorial interactions with accessory factors. Plasmids Used in Yeast Experiments—The P1 and C1 cDNAs (20Grotewold E. Athma P. Peterson T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4587-4591Crossref PubMed Scopus (204) Google Scholar, 28Paz-Ares J. Ghosal D. Weinland U. Peterson P.A. Saedler H. EMBO J. 1987; 6: 3553-3558Crossref PubMed Scopus (682) Google Scholar) were cloned under the control of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter in the YEplac112 vector (29Gietz R.D. Sugino A. Gene (Amst.). 1988; 74: 527-534Crossref PubMed Scopus (2521) Google Scholar). A control vector was also constructed with the GAPDH promoter with no downstream gene. The fusions of the MYB domains of P1 and C1 to the Gal4 activation domain (Gal4AD) were constructed by synthesizing the MYB domains and the Gal4 activation domain by PCR and then ligating them at an engineered NotI site. The fusion was then cloned under the GAPDH promoter in YEplac112. The yeast expression constructs corresponding to the N-terminal region of R fused to the Gal4 DNA-binding domain (Gal4DBD-R1–252) was the previously described fusion (15Grotewold E. Sainz M.B. Tagliani L. Hernandez J.M. Bowen B. Chandler V.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13579-13584Crossref PubMed Scopus (266) Google Scholar), but with the yeast selection marker changed from TRP1 to LEU2 by replacing the Gal4AD with the Gal4DBD-R1–252 fusion in pADGAL4 (Stratagene). The yeast strain used for the two-hybrid experiments was PJ69.4a, Mat a trp1–901 leu2–3, ura3–52 his3–200 gal 4D gal 80D LYS2::GAL1-HIS3 GAL2-ADE2 met::GAL7-lacz (30James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar). For the yeast one-hybrid experiments, a strain (YEG102) was constructed by transforming strain AY926 (31Doseff A.I. Arndt K.T. Genetics. 1995; 141: 857-871Crossref PubMed Google Scholar) with a PvuII-linearized pTH plasmid (32Flick J.S. Johnston M. Mol. Cell. Biol. 1990; 10: 4757-4769Crossref PubMed Scopus (125) Google Scholar) in which the double-stranded oligonucleotide primer APB1X2 (5′-GATCGT GTA CCT ACC AAC CTT AAA CGT GTA CCT ACC AAC CTT AAA C-3′), containing two copies of the haPBS, was cloned upstream of a minimal GAL1 yeast promoter driving the expression of the HIS3 gene. Transformations were done using the method described on the World Wide Web at tto.trends.com. Plasmids Used in Transient Expression Experiments—As previously described, all P1 and C1 plant expression vectors include the CaMV 35S promoter, the TMV Ω′ leader, the first intron of maize Adh1-S in the 5′-untranslated region, and the potato proteinase II (pinII) termination signal. Plasmids described previously include p35SP1, p35SC1, p35SR, p35SP1* (corresponding to p35SPI77L,K80R,A83R,T84L,S94G,H95R (15Grotewold E. Sainz M.B. Tagliani L. Hernandez J.M. Bowen B. Chandler V.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13579-13584Crossref PubMed Scopus (266) Google Scholar)); pBz1Luc, containing 2.3 kb of the bz1 promoter and intron upstream of luciferase; and pA1Luc, containing 1.4 kb of the a1 promoter and the Adh1-S intron (4Sainz M.B. Grotewold E. Chandler V.L. Plant Cell. 1997; 9: 611-625PubMed Google Scholar, 6Grotewold E. Drummond B. Bowen B. Peterson T. Cell. 1994; 76: 543-553Abstract Full Text PDF PubMed Scopus (533) Google Scholar, 15Grotewold E. Sainz M.B. Tagliani L. Hernandez J.M. Bowen B. Chandler V.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13579-13584Crossref PubMed Scopus (266) Google Scholar). The p35SC1SH construct was generated by PCR mutagenesis of p35SC1 using the QuikChange® XL kit (Stratagene). p35SBAR (33Grotewold E. Chamberlain M. St. Claire G. Swenson J. Siame B.A. Butler L.G. Snook M. Bowen B. Plant Cell. 1998; 10: 721-740Crossref PubMed Scopus (283) Google Scholar) was used for normalizing the concentration of the 35S promoter delivered in each bombardment. The p35SGal4DBD-C1C-term construct was previously described (36Goff S.A. Cone K.C. Fromm M.E. Genes Dev. 1991; 5: 298-309Crossref PubMed Scopus (124) Google Scholar). The pBz2Luc construct was also previously described (34Bodeau J.P. Walbot V. Mol. Gen. Genet. 1992; 233: 379-387Crossref PubMed Scopus (57) Google Scholar) and was kindly provided by Dr. Bodeau. The pA2Luc construct was obtained from Dr. Lesnick (16Lesnick M.L. Chandler V.L. Plant Physiol. 1998; 117: 437-445Crossref PubMed Scopus (63) Google Scholar). The construct containing three copies of the haPBS present in the a1 gene upstream of the luciferase reporter (p(haPBS)3Luc) corresponds to the 3×APB1–35S construct previously described (6Grotewold E. Drummond B. Bowen B. Peterson T. Cell. 1994; 76: 543-553Abstract Full Text PDF PubMed Scopus (533) Google Scholar). The pGal4BSLuc reporter construct consists of four CGGAGTACTGTCCTCCGAG motifs in tandem upstream of a minimal CaMV 35S promoter. The pA1315LLuc construct is identical to pA1Luc, except for the 6-bp insertion present in the a1315L allele, left as a consequence of the excision of Spm from the a1-m2–7991A::Spm-s allele (18Pooma W. Gersos C. Grotewold E. Genetics. 2002; 161: 793-801PubMed Google Scholar). pUbiGUS (15Grotewold E. Sainz M.B. Tagliani L. Hernandez J.M. Bowen B. Chandler V.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13579-13584Crossref PubMed Scopus (266) Google Scholar) was used to normalize the efficiency of each bombardment. Microprojectile Bombardment and Gene Expression Experiments— Bombardment conditions of maize black Mexican sweet (BMS) suspension cells and transient expression assays for luciferase and GUS were performed essentially as previously described (15Grotewold E. Sainz M.B. Tagliani L. Hernandez J.M. Bowen B. Chandler V.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13579-13584Crossref PubMed Scopus (266) Google Scholar). For each micro-projectile preparation, the mass of DNA was adjusted to 10 μg with p35SBAR (33Grotewold E. Chamberlain M. St. Claire G. Swenson J. Siame B.A. Butler L.G. Snook M. Bowen B. Plant Cell. 1998; 10: 721-740Crossref PubMed Scopus (283) Google Scholar) to equalize the amount of 35S promoter in each bombardment. One μg of each of the regulators and 3 μg of reporter plasmid were used in each bombardment. To normalize luciferase activity to GUS activity, 3 μg of pUbiGUS was included in every bombardment. Each treatment was done in triplicate, and entire experiments were repeated at least twice. The assays for luciferase and GUS, and the normalization of the data were done as described (6Grotewold E. Drummond B. Bowen B. Peterson T. Cell. 1994; 76: 543-553Abstract Full Text PDF PubMed Scopus (533) Google Scholar). Data are expressed as the ratio of arbitrary luciferase light units to arbitrary GUS light units. -Fold activation is calculated as the ratio between the Luc/GUS units of the reporter construct with transcriptional activator divided by the Luc/GUS ratio without the activator. Luciferase for a typical bombardment with just pA1Luc (no activator) gives between 1,000 and 5,000 units, and GUS (from pUbiGUS) gives between 150,000 and 700,000 units. Expression and Purification of Proteins Expressed in Bacteria—The plasmids for the expression of the MYB domains of P1, C1, and C1SH in Escherichia coli were obtained by cloning into the pTYB2 vector (New England Biolabs). The MYB domains (residues 1–119) were synthesized by PCR, adding an NdeI restriction site at the N terminus and an XhoI restriction site at the C terminus, which adds a Cys residue at the N terminus of the protein splicing element intein from Saccharomyces cerevisiae. For expression, E. coli BL21 (DE3) PlyS cells bearing the corresponding plasmids were grown, induced, and purified essentially as described (35Williams C.E. Grotewold E. J. Biol. Chem. 1997; 272: 563-571Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) with the following modifications. After induction of a 1-liter culture with 1 mm isopropyl-1-thio-β-d-galactopyranoside, the cells were harvested by centrifugation and stored at -80 °C until further use. The cells were resuspended in 40 ml of resuspension buffer (20 mm Tris, pH 8, 300 mm NaCl, 1 mm EDTA, 0.1% Triton X-100, and 1 mm phenylmethylsulfonyl fluoride) and passed twice through a French press. The cell lysate was centrifuged at 14,000 × g for 20 min; the supernatant was filtered through two layers of Miracloth (Calbiochem). The chitin beads (New England Biolabs) were equilibrated in CB1 (20 mm Tris, pH 8, 300 mm NaCl, and 1 mm EDTA) and resuspended to give a 50% slurry. Ten ml of chitin bead slurry was added to the cell lysate supernatant and incubated for 2.5 h with rocking at 4 °C. The beads were gently pelleted by centrifugation, and the pellet was then resuspended with 5 ml of CB2 (20 mm Tris, pH 8, 500 mm NaCl, 1 mm EDTA, and 0.1% Triton X-100), loaded onto a column, and washed twice with the same buffer. The column was washed three additional times with CB3 (20 mm Tris, pH 8, 1 m NaCl, 1 mm EDTA, and 0.1% Triton X-100). The column was incubated in CB3 plus 50 mm dithiothreitol overnight in order to allow the self-cleavage of the intein and therefore allow elution of the target protein. The elution was then dialyzed against A-0 buffer (10 mm Tris, pH 7.5, 50 mm NaCl, 1 mm dithiothreitol, 1 mm EDTA, 5% glycerol) at 4 °C and stored at -80 °C until further use. Each wash and elution fraction was collected and analyzed by SDS-PAGE, followed by staining with Coomassie Brillant Blue R-250. Generation of C1 Monoclonal Antibodies and Western Analyses— Monoclonal antibodies were generated using affinity-purified N10His-C1 protein by the Antibody Facility at Cold Spring Harbor Laboratory. For Western analyses, PJ69.4a yeast cultures were grown in 1.5 ml of SC-Leu-Trp medium at 30 °C to an A600 of 0.7. Cells were harvested by centrifugation at 14,000 rpm and washed once with 1.5 ml of distilled water. The pellet was then resuspended in 100 μl of SDS loading buffer (0.06 m Tris-HCl, pH 6.8, 10% glycerol, 2% (w/v) SDS, 5% β-mercaptoethanol, 0.0025% (w/v) bromphenol blue) and heated to 95 °C for 5 min. The suspension was then centrifuged at 14,000 rpm, and 15 μl of extract was loaded and analyzed by 12% SDS-PAGE. The gels were transferred to polyvinylidene difluoride membrane by electrophoresis using the Bio-Rad Mini Trans-Blot transfer cell in 39 mm glycine, 48 mm Tris-HCl, and 20% methanol. The transfer was done at 4 °C and 100 V for 1 h. Membranes were stained with Ponceau Red to verify transfer and then blocked in 1% nonfat dried milk dissolved in 1× TBS (10 mm Tris-HCl, pH 8.0, 150 mm NaCl) with 0.05% Tween 20 overnight at 4 °C. Following blocking, the membranes were incubated with the primary antibodies diluted in 1% nonfat dried milk dissolved in 1× TBS with 0.05% Tween 20 for 2 h at room temperature. The C1 monoclonal antibody C1 40/1–249 was used at 1:150 dilution. For normalization, the blot was probed with an antibody that recognizes the yeast DED1p protein at a dilution of 1:4000. After washing the blots three times for 10 min in 1× TBS, blots were incubated in anti-rabbit IgG, horseradish peroxidase secondary antibody (Amersham Biosciences) for DED1p and anti-mouse IgG, horseradish peroxidase (Amersham Biosciences) for C1 40/1–249 diluted 1:2000 in 1% nonfat dried milk dissolved in 1× TBS with 0.05% Tween 20 for 2 h at room temperature. The blots were then rinsed again with 1× TBS as before, and then they were visualized by using the Amersham ECL kit. Electrophoretic Mobility Shift Assays—End labeling of synthetic oligonucleotide probes (APB01, APB05) was carried out using T4 polynucleotide kinase (Invitrogen) in the presence of a 2 m excess of [γ-32P]ATP (>8,000 Ci/mmol; ICN). The labeled oligonucleotides were then annealed to equal amounts of complementary oligonucleotides (APB10, APB50) by heating to 95 °C and slowly cooling to room temperature. The oligonucleotide pairs APB01-APB10 and APB05-APB50 were annealed to generate APB1 and APB5, respectively. The oligonucleotides were then precipitated on glass filters for quantification in a scintillation counter. The probes used correspond to the following: for APB1, APB10 (5′-GATCCGGGTCAGTGTACCTACCAACCTTAAACAC-3′) and APB01 (5′-GATCGTGTTTAAGGTTGGTAGGTACACTGACCCG-3′); for APB5, APB50 (5′-GATCCGGGTCAGTGTACCCGATCGTCTTAAACAC-3′) and APB05 (5′-GATCGTGTTTAAGACGATCGGGTACACTGACCCG-3′). DNA binding assays were performed on ice for 30 min in a 25-μl total volume in A-0 buffer with 0.8 μg poly(dI/dC) and 1 mm dithiothreitol (unless otherwise indicated). After incubation on ice, ∼10,000 cpm of end-labeled oligonucleotide probe was added and incubated on ice for an additional 30 min. Protein-DNA complexes were resolved on an 8% polyacrylamide gel (80:1, acrylamide/bis-acrylamide) with 0.25× Tris borate-EDTA running buffer at 415 V for 55 min at 4 °C. After electrophoresis, gels were dried onto Whatman paper and subjected to autoradiography at -70 °C overnight, using x-ray film or a Kodak phosphor imager for 2 h and quantified using a Bio-Rad imaging system. The apparent dissociation constants were estimated by carrying out the DNA binding assays as described above with a fixed amount of protein (35 ng) and in the presence of varying amounts of cold double-stranded oligonucleotide (ranging from 0 to 5.240 nmol). After incubation on ice, ∼10,000 cpm of end-labeled oligonucleotide probe was added and incubated on ice for an additional 30 min. Quantification of the free and bound APB1 oligonucleotide was estimated from the radioactivity present in each of the corresponding bands and calibrated against known standards. To compare our studies with others (4Sainz M.B. Grotewold E. Chandler V.L. Plant Cell. 1997; 9: 611-625PubMed Google Scholar, 35Williams C.E. Grotewold E. J. Biol. Chem. 1997; 272: 563-571Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), data were assumed to fit to a linear relationship, although comparable RMS values are obtained using a hyperbolic fit (not shown). It should be noted that the APB1 probe is formed by two overlapping binding sites (6Grotewold E. Drummond B. Bowen B. Peterson T. Cell. 1994; 76: 543-553Abstract Full Text PDF PubMed Scopus (533) Google Scholar), and whereas only one protein can bind to APB1 at a time, thus ruling out a cooperative interaction (not shown), our studies cannot rule out the possibility that each binding site is recognized with different affinities by the different proteins. The ARE Is Required for R-enhanced Activity—Previously, we showed that transferring the interaction with R from C1 to P1 resulted in the P1* protein with novel regulatory activities (15Grotewold E. Sainz M.B. Tagliani L. Hernandez J.M. Bowen B. Chandler V.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13579-13584Crossref PubMed Scopus (266) Google Scholar). In the absence of R, P1* activated the a1 promoter (pA1Luc; Fig. 1B) just like P1 (Fig. 2A) (R-independent transcription; Table I) but not the bz1 promoter (Fig. 2B). In the presence of R, P1* activated bz1, similar to C1 (R-dependent transcription; Table I) (Fig. 2B), and induced anthocyanin accumulation (not shown). P1* displayed a 2–3-fold enhanced activity on the pA1Luc in the presence of R (Fig. 2A, compare P1* and P1* + R). To ascertain whether this R-enhanced activity required cis-elements other than the MYB binding sites, we tested whether P1* displays R-enhanced activity on a promoter containing three copies of the haPBS upstream of a minimal CaMV 35S promoter (p(haPBS)3Luc; Fig. 1B). This synthetic promoter is activated by P1 and C1 + R but not by C1 alone (Fig. 2C). On this promoter, however, the activity of P1* is not enhanced by R (Fig. 2C, compare P1* and P1* + R). These results suggest that other cis-regulatory elements in the a1 promoter participate in the R-enhanced transcription of a1 by P1*.Table IParticipation of R in the regulation of the maize flavonoid biosynthetic genes a1 and bz1Regulatora1bz1P1R-independentNo activationC1R-dependentR-dependentP1*R-enhancedR-dependent" @default.
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