The Plant Cell, Vol. 1, 651-658, July 1990 O 1990 American Society of Plant Physiologists

Fos and Jun Oncogenes Transactivate Chimeric or Native Promoters Containing AP1 /GCN4 Binding Sites in Plant Cells

Pierre Hilson,"Yb Denis de Froidmont,"7b Corinne Lejour," Syu-lchi Hirai," Jean-Marie Jacquemin,'" and Moshe Yaniv'

a Station d'Amelioration des Plantes, 4 rue du Bordia, 5800 Gembloux, Belgium Faculte des Sciences Agronomiques de I'Etat, 2 passage des Deportes, 5800 Gembloux, Belgium Unité des Virus Oncogènes, UA 11 49 du Centre National de Ia Recherche Scientifique, Departement de Biologie

Moléculaire, lnstitut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France

The function of mammalian transcription factors of the leucine zipper class was investigated in leaf-derived protoplasts of tobacco. In transient expression experiments, Fos and Jun strongly activated chimeric promoters composed of the TATA box region of the cauliflower mosaic virus 35s transcript preceded by one to five copies of an APl/GCN4 binding site. Fos and Jun also stimulated a wheat high molecular weight glutenin promoter in which similar binding sites are located more than 500 base pairs from its transcription start site. Both the DNA binding and the transcription activation domains of these proteins were required for proper promoter stimulation by Fos and Jun. Each factor alone was partially active, suggesting that at least the Fos protein can associate with an endogenous plant transcription factor. These observations support the hypothesis that sequences related to APl/GCN4 binding sites could be cis-acting modules involved in the transcriptional regulation of plant genes.

INTRODUCTION

The conservation of transcription mechanisms among mammals, insects, and yeasts has been demonstrated in vivo by the functional interchangeability of promoter ele- ments (Harshman et al., 1988; Jones et al., 1988) and of transactivating factors (Struhl, 1987; Fisher et al., 1988; Kakidani and Ptashne, 1988; Lech et al., 1988; Metzger et al., 1988; Struhl, 1988; Webster et al., 1988; Lambert et al., 1989). In tobacco protoplasts, artificial GAL4-derived proteins were shown to stimulate a chimeric promoter containing GAL4 17-bp binding sites (Ma et al., 1988). Transcription factors of different species showing a com- mon structural organization often bind to closely related elements. For example, similar cis-acting DNA sequences are targets for GCN4, which is the final positive regulator in the general control for amino acid biosynthesis in yeast, and for AP1, which is a mammalian protein fraction con- taining products of the fos and jun multigene families (Vogt et al., 1987; reviewed in Curran and Franza, 1988; Johnson and McKnight, 1989; Mitchell and Tjian, 1989; Struhl, 1989; Vogt and Bos, 1990). Two distinct domains were defined in these factors: the DNA binding domain and the transcription activation domain. The DNA binding domain is bipartite: it contains a leucine zipper region necessary

' To whom correspondence should be addressed.

for the dimerization and a basic region involved in the recognition of DNA (Landschultz et al., 1988). GCN4 or Jun homodimers as well as Fos-Jun heterodimers recog- nize identical DNA sequences (Hirai and Yaniv, 1989; Kouzarides and Ziff, 1989; Sellers and Struhl, 1989). The binding of these factors enhances the transcription of target promoters .through the presumable interaction of their activation domains with elements of the transcription machinery (Lucibello et al., 1988; Hirai et al., 1990).

Recently, severa1 plant genes showing structural simi- larities with these yeast and mammalian transcription fac- tors were identified. The Opaque 2 gene, which probably regulates the transcription of the 22-kD zein genes in maize, encodes a protein that contains a leucine zipper region flanked at its N terminus by a basic region (Hartings et al., 1989). A similar arrangement is also found in cDNAs coding for sequence-specific DNA-binding proteins: TGAla and TGAlb from tobacco (Katagiri et al., 1989) and HBP-1 from wheat (Tabata et al., 1989). All three proteins bind to related sites that share the core sequence TGACG, which is located in the promoters of the cauli- flower mosaic virus (CaMV) 35s transcript and of histone genes. This motif is also part of the CREB/ATF binding site (Hurst and Jones, 1987; Montminy and Bilezikjian, 1987) and resembles the central TGACT motif of the AP1/ GCN4 binding sites.

652 The Plant Cell

An AP1 /GCN4 binding site, the palindromic sequence ATGA(G/C)TCAT, is found in the 3‘ part of the so-called “endosperm box,” a sequence common to promoters of wheat and barley genes (Forde et al., 1985; Colot et al., 1989) coding for gliadins, low molecular weight glutenins, and B hordeins. This sequence is also present twice in the wheat high molecular weight (HM W) glutenin promoters (this paper; Halford et al., 1987). These cereal genes, members of the prolamin storage protein family, have an endosperm-specific, transcriptionally regulated activity (Greene, 1983; Rahman et al., 1984; Bartels and Thomp- son, 1986).

To investigate the relevance of AP1 /GCN4 binding sites in plant promoters and the potential activity of mammalian transcription factors of the leucine zipper class in plant, we undertook transient expression assays in leaf-derived protoplasts of tobacco. We report here that v-fos and c-jun gene products stimulate the transcriptional activity of short chimeric promoters through the AP1 /GCN4 bind- ing sites. These sites also mediate Fos-Jun transactivation of a native wheat HMW glutenin promoter.

RESULTS

Experimental Design of Transactivation Assays

The goal of the experiments was to study the potential action of v-fos and c-jun gene products on promoters in a plant context and the significance of AP1 /GCN4 potential binding sites in plant promoters. For this purpose, leaf- derived protoplasts of tobacco were cotransfected with a reporter plasmid and either plasmids expressing these proteins or a negative control plasmid.

Three different series of plasmids represented in Figure 1 were used in these experiments. First, the reporter plasmids contain the bacterial cat gene fused at its 5’ end to the different promoters tested: (1) short chimeric pro- moters (pMIC plasmids, Figure 1A) and (2) parts of an HMW glutenin promoter (pPGC plasmids, Figure 1 B). Sec- ond, the activator plasmids consist of a correctly oriented open reading frame coding for Fos or Jun proteins placed between the full strong promoter and the polyadenylation sequences from the CaMV 35s transcript (pGY plasmids, Figure 1 C). The same plasmid with no open reading frame was used as negative control (pGY1). Third, the internal control plasmid, used to normalize for transfection efficien- cies, harbors the bacterial gus gene under the control of the full 35s promoter (pBI121, Figure 1 D).

ln practice, the following plasmids were mixed: one reporter plasmid plus one (FOS or JUN) or two (FOS and JUN) activator expression plasmids. In some instances, the gus internal control was added. The Fos-Jun transac- tivations of the different promoters were quantitatively monitored in three independent experiments.

Fos and Jun Transactivate Chimeric Promoters Containing One or Severa1 APl/GCN4 Binding Sites

The activity of Fos and Jun in plant cells was first investi- gated with short promoters specially designed for this study. The chimeric promoters of the pMlC series (Figure IA) consist of a 52-bp fragment overlapping the CaMV 355 TATA box (from position -46 to +6 relative to the transcription start site; Odell et al., 1985) preceded by zero to five concatemerized potential APl binding sites, ATGA(G/C)TCAT, from an HMW glutenin promoter (posi- tion -575 to -559). In this report, we called this sequence the B consensus because its central motif is also located in the B part of the prolamin endosperm box (Kreis et al., 1986). Mouse nuclear extracts enriched for AP1 activity specifically bound the B consensus oligonucleotide in gel retardation assays (data not shown). As illustrated in Fig- ure 2, all the plasmids of this pMlC series gave rise to similar low levels of CAT activity after protoplast transfec- tion in the absence of any activator plasmid (lanes 1 to 6).

Two activator plasmids expressing a hybrid v-fos onco- gene (pGY-FOS; Schuermann et al., 1989) or the mouse c-jun proto-oncogene (pGY-JUN; Ryseck et al., 1988) were added together with the reporter plasmids. These co- transfections resulted in a dramatic stimulation of the chimeric promoters harboring the B consensus [Figure 2; plxB-MIC to p5xB-MIC (lanes 8 to 12) compared with pMlC (lane 7)]. Interestíngly, the stimulation by Fos-Jun increased gradually with the number of binding sites, giving a 13-fold to a 65-fold mean activation for plxB-MIC and p5xB-MIC, respectively.

Because the molecular ratio of the Fos and Jun proteins in the heterodimer is 1 :1, we determined the optimal ratio of pGY-FOS versus pGY-JUN with regard to the transcrip- tion stimulation, assuming that the efficiency of the 35s expression cassette may vary between the different plas- mids. For this purpose, the best inducible plasmid, p5xB- MIC, was cotransfected with different ratios of the Fos and Jun expression plasmids, the total amount of pGY plasmids remaining equal in each sample. In our conditions, the stimulation was optimal with a pGY-F0S:pGY-JUN ratio of 9:l (data not shown).

Figure 3 shows that the cotransfections with pGY-FOS or pGY-JUN alone gave rise to rather good stimulations corresponding for pGY-FOS (lane 4) to 9°/o of the optimum [pGY-FOS(9):pGY-JUN(l)J (lane 1) and a fivefold induction compared with the pGYl control (lane 7), and for pGY- JUN (lane 2) to 37% and an 18-fold activation.

Defined Functional Domains of Fos and Jun Are Required for Proper Transactivation in Plant Protoplasts

To confirm that the enhanced transcriptional activities ob- served are really due to the binding of Fos and Jun proteins

A REPORTER CONSTRUCTS CONTAINING A RECONSTITUTED PROMOTER

AGCIIATTATGAGTCAIAGCA A T A A ~ Z ~ E Z ~ Ã T C G T I ~ ~ _ ~

,

I I t6

-46 CaMV 35s min ima l promoter

8 REPORTER CONSTRUCTS CONTAINING A PART OF THE HMW GLUTENIN PROMOTER

par l ia l -571 endosperm box

c ACTIVATOR CONSTRUCTS

pCY-FDC

358 pro v - f o s 35s le r 35s pro c-10s lock ing 35s l e r (FEJ-FER hybrid) l he ac id ic domoin

p L Y - C D L

355 pro c - l un 355 le r 35s pro c- lun lack ing l he 355 ter leucin z ipper doma in

D G U S INTERNAL CONTROL CONSTRUCl

1'111 121

P NOS le r 358 pro gu=

200 bp LI

Figure 1. Plasmids Used in the in Vivo Transactivation Experiments.

(A) Reporter plasmids containing a chimeric promoter. One to five copies of a double-stranded oligonucleotide called the B consensus and represented in the upper part of the figure were introduced upstream from the CaMV 35s minimal promoter. This oligonucleotide contains an AP1 /GCN4 binding site (ATGAGTCAT underlined sequence) corresponding to the -575/-559 region of an HMW glutenin promoter flanked with Hindlll protruding ends. In p5xB-MIC, the position of the first base pair in each APl/GCN4 motif is given with respect to the transcription Start site in the CaMV 35s promoter region marked by a thin arrow. The thick arrows show the orientation of the B consensus oligonucleotides in the chimeric promoter. (6) Reporter plasmids containing a part of the HMW glutenin promoter. All positions are given with respect to the first ATG of a X genomic clone corresponding to the Glu-1 Dy12 allele of the wheat cultivar Camp Remy. (C) Activator plasmids. These harbor a 35s expression cassette from vvhich fos (dark-shadowed boxes) or jun (light-shadowed boxes) mRNA is transcribed. The abbreviations BR, LZ, and AD stand for Basic Region, Leucine Zipper, and Acidic Domain, respectively. (D) GUS interna1 control plasmid.

654 The Plant Cell

activators(0.5 ug)

reporter(12 ug)

fold activation

20 40 60 80 100 120 1»

1 "'

2

3

4 PG

5

6 _

7 --

6

9pGY-

10 PGY

11

12

r̂ iri i,plxB-MIC ^

p2xB-MIC XYl T

p3xB-MIC ft,

P4XB-MIC feo

pSxB-MIC *>

1 PMC |

plxB-MIC

-FOS PM-MC

-JUN p3xB-MIC

p4xB-MIC

P5XB-WC

>QEa••

Figure 2. Transactivation of Chimeric Promoters by Fos Plus Jun.

The effect of the concatemerized AP1/GCN4 binding sites in thechimeric promoters alone on CAT activities is shown in lanes 1 to6. The degree of transactivation of these promoters by Fos plusJun is shown in lanes 7 to 12. In this last case, the pGY-FOS:pGY-JUN ratio is 9:1. One microgram of pBI121 was added to eachplasmid mixture. The three symbols correspond to three inde-pendent experiments. The CAT assays were normalized accord-ing to the GUS activity. In both blocks, the quantification of theinduction is expressed as the ratio of chloramphenicol acetyltrans-ferase activities of each sample versus the pMIC sample (boxed).The CAT activity of the pMIC+pGY-FOS/pGY-JUN sample issimilar to those of the nonstimulated series. The columns repre-sent the average-fold activation values. The autoradiogram cor-responds to the CAT assay of one experiment. The abbreviationsCM and AcCM stand for nonacetylated chloramphenicol and1-acetyl plus 3-acetyl chloramphenicol, respectively.

Fos and Jun Stimulate an HMW Glutenin Promoter

We have previously isolated the Glu-1 Dy12 allele codingfor an HMW glutenin of the wheat cultivar Camp Remy.The 1364-bp sequence flanking the open reading frame atits 5' side was subcloned and sequenced. This promoteris highly homologous to shorter HMW glutenin ones al-ready published (P. Hilson, C. Lejour, D. de Froidmont, P.du Jardin, and J.-M. Jacquemin, manuscript in prepara-tion). As shown in Figure 1 B, the glutenin reporter plasmidspPGC1, pPGCS, and pPGC7 contain promoter sequencesending at position -5 and starting at positions -1364,-646, and —433, respectively, relative to the glutenininitiation codon. Two AP1/GCN4 binding sites (A/GTGAGTCAT) are located at positions -571/-563 and-592/-5S4 of this promoter (Figure 1 B).

The transfections with the three glutenin reporterplasmids alone gave rise to low transcriptional activitiesdecreasing with the glutenin promoter size, as illustratedin Figure 4. Taking the values of the shorter one (-433/-5) as a unit value, the sequences -646/-S (in pPGCS)and —1364/—5 (in pPGC1) correspond to relative activitiesof 2.5 and 7.5, respectively (lanes 1 to 3).

In the presence of Fos and Jun, the activity of the —433/-5 fragment was not changed, whereas the -646/-S and-1364/-5 promoters gave rise to relative activities of 7.4(2.8-fold activation) and 19.0 (2.6-fold activation), respec-tively. These results indicate that the Fos and Jun factorswere able to stimulate HMW glutenin promoter regionsharboring AP1 /GCN4 binding sites located more than 500bp from the putative transcription start site.

to the B consensus in vivo and to their subsequent inter-actions with the plant transcription machinery, we con-structed two plasmids expressing mutant proteins. pGY-FDC contains the murine c-fos proto-oncogene lacking itsC-terminal acidic transcription activation domain and pGY-CDL codes for a c-y'un-derived protein in which the leucinezipper region has been deleted. It was previously shownthat the CDL protein fails to bind DMA in vitro and thatboth FDC and CDL fail by themselves to transactivatereporter plasmids containing AP1 binding sites in mam-malian cells (Hirai et al., 1990).

In cotransfected plant protoplasts, the fos or jun trun-cated genes in the 35S expression cassette (pGY-FDCand pGY-CDL) behaved like the pGY1 negative control(Figure 3); each one of them alone (data not shown) orboth together (lanes 6 and 7) did not transactivate thereporter plasmid. Furthermore, the truncated Fos failed tocooperate with Jun (lanes 2 and 3), and the C-terminal-deleted Jun did not significantly increase transactivationby Fos (lanes 4 and 5).

activators(0.5 ug>

reporter(12 ug)

1 pGY-FOS / pGY-JUN

2 pGYl / pGY-JUN

3 pGY-FDC / pGY-JUN

d pGY-FOS / pGYl p5xB

5 pGY-FOS / pGY-CDL

6 pGY-FDC / pGY-CDL

7 HGY]

•1•

-MIC |

•fl

^ >

CM AcCM

Figure 3. Transactivation Properties of Truncated Fos and JunProteins.

In all samples, the reporter plasmid is the most inducible p5xB-MIC, and FOS-type:JUN-type plasmid ratios are 9:1. The threesymbols correspond to three independent experiments. The CATassays were normalized according to the total protein concentra-tion. The quantification of the induction is expressed as the ratioof chloramphenicol acetyltransferase activities of each sampleversus the pGY1 sample (boxed). The columns represent theaverage-fold activation values. The autoradiogram correspondsto the CAT assay of one experiment. The abbreviations CM andAcCM are defined in the legend to Figure 2.

Fos and Jun Transactivate Genes in Plant Cells 655

Plasmids

1

2

3

4

5

6

controlor

activators(0.5 ug)

-[-pGYl

-i-

-r-

pGY-FOSpGY-JUN

_J _

reporter '(12 ug)

FPSCT|PPGC5

pPGCl

|pPGC7|

pPGCS

pPGCl

fold activation

CM AcCM

Figure 4. Transactivation of the HMW Glutenin Promoter by FosPlus Jun.The promoter activity of each 5'-flanking region alone is shown inlanes 1 to 3. The transactivation of these by Fos plus Jun (plasmidratio 9:1) is shown in lanes 4 to 6. The three symbols correspondto three independent experiments. The CAT assays were nor-malized according to the GUIS activity. In both blocks, the quan-tification of the induction is expressed as the ratio of chloram-phenicol acetyltransferase activities of each sample versus thepPGC7 sample (boxed). The CAT activities for pPGC7 in theabsence or in the presence of Fos plus Jun are similar. Thecolumns represent the average-fold activation values. The auto-radiogram corresponds to the CAT assay of one experiment. Theabbreviations CM and AcCM are defined in the legend to Figure 2.

DISCUSSION

The present work demonstrates that the Fos and Junmammalian oncoproteins, forming a heterodimer that con-stitutes the major component of the AP1 transcriptionfactor, can stimulate plant promoters containing AP1 rec-ognition sites when expressed in Nicotiana plumbaginifoliacells. These transactivations were achieved with nativeheterologous transcription factors, whereas a previousreport showed that GAL4 derivatives induce chimeric pro-moters in N. tabacum protoplasts when deprived of a largeinternal portion of the protein (Ma et al., 1988).

In vitro analyses revealed that Jun homodimers have alow affinity for AP1 binding sites compared with the Fos-Jun heterodimers. On the contrary, Fos by itself is unableto form homodimers and, hence, to bind DNA (Nakabeppuet al., 1988; Neuberg et al., 1989; Hirai et al., 1990). In ourplant system, cotransfection with the Fos or Jun codingplasmid led to significant transactivation of a chimericpromoter containing five AP1/GCN4 binding sites. Thissuggests that at least the Fos protein can associate withendogenous plant transcription factor(s), as is the case inmammalian transient expression experiments (Setoyamaet al., 1986). The activity of Jun protein may be related toits capacity to form homodimers that bind to DNA and mayactivate transcription, although we cannot exclude that itforms heterodimers with endogenous plant protein(s) anal-ogous to Fos.

A Fos derivative deprived of its C-terminal acidic acti-vation domain (FDC), or a Jun derivative lacking the leucinezipper region necessary for dimerization (CDL), are inactivein tobacco as in mammalian cells (Schuermann et al., 1989;Hirai et al., 1990). Together with the functionality of thenonmutated factors, those data strongly suggest that thesame elements of Fos and Jun proteins are involved indimerization, in DNA binding, and in transcription activationin mammalian and plant cells.

We showed that the Fos and Jun mammalian factorsare also able to activate a genuine plant promoter. The-646/-S and the -1364/-5 HMW glutenin promoter re-gions, containing two AP1/GCN4 binding sites located atpositions -592/-S84 and -571/-563, were transacti-vated by Fos and Jun, whereas the -433/-S region wasnot. As in mammalian genes (Dynan, 1989), plant pro-moters could be composed of multiple short modulesmediating together cellular responses to developmental orphysiological stimuli. The presence of AP1/GCN4 bindingsites in consensus regions of related prolamin promoterstogether with our demonstration that they can mediatetranscription activations in plant cells suggest that theseelements could be involved in the regulation of plant geneexpression.

This hypothesis is supported by the recent finding thatplant genes code for predicted proteins containing a struc-tural domain similar to the DNA binding domain of theleucine zipper class of transcription activators. Two ofthem are involved in tissue specificity: Opaque 2 probablyregulating the zein expression in maize (Hartings et al.,1989) and TGA1 a binding to a DNA sequence conferringroot expression in tobacco (Benfey et al., 1989; Katagiriet al., 1989). Two others, the wheat HBP-1 (Tabata et al.,1989) and the tobacco TGA1b (Katagiri et al., 1989), bindto regulatory elements of the histone gene promoters.

METHODS

Reporter and Activator Plasmids

All reporter plasmids were Bluescript(+)KS derivatives. In thechimeric promoters, a 52-bp fragment identical to a region of theCaMV 35S promoter extending from position —46 to +6 relativeto the transcription start site (Odell et al., 1985) and generatedwith two synthetic oligonucleotides was placed in the BluescriptSmal site. A BamHI 2.0-kb fragment containing the caf gene fusedto the nos polyadenylation signals (caf-nos ter, Masson andFedoroff, 1989) was then added in 3' to give pMIC. The region ofan HMW glutenin promoter extending from position -575 to -559was synthesized as a double-stranded oligonucleotide called Bconsensus, flanked by the 5'-protruding ends of the Hindlll site(see Figure 1A). Concatemerized copies of this oligonucleotidewere introduced upstream of the CaMV 35S minimal promoter inthe pMIC Hindlll site to give p1xB-MIC to p5xB-MIC, accordingto the copy number of oligonucleotides inserted. The orientation

656 The Plant Cell

of the copies in each plasmid was determined by sequencing. All copies were intact except for the central oligonucleotide in p5xB- MIC in which the two last Ts, in the transcription sense, were deleted.

In the HMW glutenin series, the 5’43‘ deletants were obtained from Exolll-ExoVII digestions. The 3’ end of the promoter regions is located at position -5 and originated from the Klenow enzyme treatment of a digested BstXl restriction site overlapping the first ATG in the genomic clone. A BamHl linker was introduced in the BstXI-blunted site and used for the introduction of the above- mentioned car-nos ter cassette to give the PGC plasmids.

In the activator plasmids, open reading frames were subcloned in the 35s expression cassette of pGYl , which corresponds to the previously described pDH51 (Pietrzak et al., 1986) in which the Ncol site was replaced by an Xhol linker. A 0.96-kb BamHl fragment containing a v-fos gene, hybrid of FBJ-MSV and FBR- MSV fos genes, was extracted from pE3002 (Schuermann et al., 1989) and introduced in the pGYl BamHl cloning site (pGY-FOS); an Rsrll-Scal 1.12-kb fragment containing the mouse c-jun coding region (Ryseck et al., 1988) was blunted by treatment with the Klenow fragment of Escherichia coli DNA polymerase I and intro- duced in the pGYl Smal cloning site (pGY-JUN); two Hindlll- BamHl fragments of 0.87 kb and 1.24 kb, coding for the mouse Fos protein lacking its acidic domain and for the mouse Jun protein lacking its leucine zipper, were both extracted from a mammalian expression vector (Hirai et al., 1990), blunt end repaired with the Klenow fragment, and introduced between the pGYl Smal and BamHl sites (pGY-FDC and pGY-CDL, respectively).

Transient Expression Assays

lsolation of Nicotiana plumbaginifolia leaf-derived protoplasts and Ca(NO&-PEG-mediated DNA transfer were performed as de- scribed elsewhere (Negrutiu et al., 1987). After a 24-hr culture period, pelleted protoplasts were lysed in glucuronidase (GUS) extraction buffer. Each sample was ground, and after centrifuga- tion the supernatant was used for chloramphenicol acetyltransfer- ase (CAT) (Gorman et al., 1982), GUS (Jefferson et al., 1987), or protein assays (Bradford, 1976). GUS activities were measured by fluorogenic assays. CAT activities were determined by 14C scintillation counting .

The lysate amounts used in the CAT reactions concerning the activation by Fos and/or Jun were normalized by two parameters: (1) The GUS activity originating from the 35s promoter-GUS coding sequence plasmid (pB1121) served as interna1 control for experiments in which the ratio of Fos and Jun expression plasmids was constant. It gave a global estimation for the transfection efficiency and the residual mortality inherent in the PEG technique. (2) CAT assays were normalized according to protein concentra- tions for experiments in which the activator plasmid ratio varied. Hence, systematic deviations that could result from Fos or Jun interaction with the full 35s promoter present in the plasmid pB1121 were avoided.

ACKNOWLEDGMENTS

We thank Drs. Patrick Masson, Eric Verdin, Françoise Salomon, Richard Kettmann, and Arsène Burny for discussions and critical

reading of the manuscript. We are grateful to Dr. loan Negrutiu and his team for the introduction to transient expression tech- niques and to Drs. Marcus Schuermann, Rolf Müller, and Maciej Pietrzak for providing plasmids. This work was supported by the Belgian National Incentive Program on Fundamental Research in Life Sciences (610 23), and by fellowships from the Fonds National de Ia Recherche Scientifique of Belgium (to P.H.) and from the lnstitut pour I’Encouragement de Ia Recherche Scientifique dans I’lndustrie et I’Agriculture (to D.deF.).

Received February 28, 1990; revised April30, 1990.

REFERENCES

Bartels, D., and Thompson, R.D. (1986). Synthesis of mRNAs coding for abundant endosperm proteins during wheat grain development. Plant Sci. 46, 117-125.

Benfey, O.D., Ren, L., and Chua, N.-H. (1989). The CaMV 35s enhancer contains at least two domains which can confer different developmental and tissue-specific expression patterns.

Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.

Colot, V., Bartels, D., Thompson, R.D., and Flavell, R.B. (1989). Molecular characterization of an active wheat LMW glutenin gene and its relation to other wheat and barley prolamin genes. MOI. Gen. Genet. 216, 81-90.

Curran, T., and Franza, B.R., Jr. (1988). Fos and Jun: The AP-1 connection. Cell 56, 395-397.

Dynan, W.S. (1 989). Modularity in promoters and enhancers. Cell 58, 1-4.

Fisher, J.A., Giniger, E., Maniatis, T., and Ptashne, M. (1988). GAL4 activates transcription in Drosophila. Nature 332,

Forde, B.G., Heyworth, A., Pywell, J., and Kreis, M. (1985). Nucleotide sequence of a B I hordein gene and the identification of possible upstream regulatory elements in endosperm storage protein genes from barley, wheat and maize. Nucl. Acids Res.

Gorman, C., Moffat, L., and Howard, B. (1982). Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. MOI. Cell. Biol. 2, 1044-1054.

Greene, F.C. (1983). Expression of storage protein genes in developing wheat (Triticum aestivum L.) seeds: Correlation of FINA accumulation and protein synthesis. Plant Physiol. 71,

Halford, N.G., Forde, J., Anderson, O.D., Greene, F.C., and Shewry, P.R. (1 987). The nucleotide and deduced amino acid sequences of an HMW glutenin subunit gene from chromosome 1 B of bread wheat (Triticum aestivum L.) and comparison with those of genes from chromosomes I A and 1D. Theor. Appl. Genet. 75, 1 17-1 26.

EMBO J. 8,2195-2202.

853-856.

13,7327-7339.

40-46.

Fos and Jun Transactivate Genes in Plant Cells 657

Harshman, K.D., Moye-Rowley, W.S., and Parker, C.S. (1988). Transcriptional activation by the SV40 AP-1 recognition element in yeast is mediated by a factor similar to AP-1 that is distinct from GCN4. Cell 53, 321-330.

Hartings, H., Maddaloni, M., Lazzaroni, N., Di Fonzo, N., Motto, M., Salamini, F., and Thompson, R. (1989). The 0 2 gene which regulates zein deposition in maize endosperm encodes a protein with structural homologies to transcriptional activa- tors. EMBO J. 8,2795-2801.

Hirai, S.4, and Yaniv, M. (1989). Jun DNA-binding is modulated by mutations between the leucines or by direct interaction of Fos with the TGACTCA sequence. New Biol. 1, 181-191.

Hirai, S.4, Bourachot, B., and Yaniv, M. (1990). Both Jun and Fos contribute to transcription activation by the heterodimer. Oncogene 5,39-46.

Hurst, H.C., and Jones, N.C. (1 987). ldentification of factors that interact with the E1 A-inducible adenovirus E3 promoter. Genes Dev. 1,1132-1 146.

Jefferson, R.A., Kavanagh, T.A., and Bevan, M.W. (1987). GUS fusions: (3-Glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 3901-3907.

Johnson, P.F., and McKnight, S.L. (1 989). Eukaryotic transcrip- tional regulatory proteins. Annu. Rev. Biochem. 58, 799-839.

Jones, N.C., Rigby, P.W.J., and Ziff, E.B. (1988). Trans-acting protein factors and the regulation of eukaryotic transcription: Lessons from studies on DNA tumor virus. Genes Dev. 2,

Kakidani, H., and Ptashne, M. (1988). Ga14 activates gene expression in mammalian cells. Cell 52, 161 -1 67.

Katagiri, F., Lam, E., and Chua, N.-H. (1 989). Two tobacco DNA- binding proteins with homology to the nuclear factor CREB. Nature 340, 727-730.

Kouzarides, T., and Ziff, E. (1989). Leucine zippers of fos, jun and GCN4 dictate dimerization specificity and thereby control DNA binding. Nature 340, 568-571.

Kreis, M., Williamson, M.S., Forde, J., Schmutz, D., Clark, J., Buxton, B., Pywell, J., Marris, C., Henderson, J., Harris, N., Shewry, P.R., Forde, B.G., and Miflin, B.J. (1986). Differential gene expression in the developing barley endosperm. Philos. Trans. R. SOC. Lond. 8314,355-365.

Lambert, P.F., Dostatni, N., McBride, A.A., Yaniv, M., Howley, P.M., and Arcangioli, B. (1989). Functional analysis of the Papilloma virus E2 trans-activator in Saccharomyces cerevisiae. Genes Dev. 3,38-48.

Landschultz, W.H., Johnson, P.F., and McKnight, S.L. (1 988). The leucine zipper: A hypothetical structure common to a new class of DNA binding proteins. Science 240, 1759-1 764.

Lech, K., Anderson, K., and Brent, R. (1988). DNA-bound Fos proteins activate transcription in yeast. Cell 52, 179-1 84.

Lucibello, F.C., Neuberg, M., Hunter, J.B., Jenuwein, T., Schuermann, M., Wallich, R., Stein, B., Schonthal, A., Herrl- ich, P., and Miiller, R. (1988). Transactivation of gene expres- sion by fos protein: lnvolvement of a binding site for the tran- scription factor AP-1. Oncogene 3, 43-51.

Ma, J., Przibilla, E., Hu, J., Bogorad, L., and Ptashne, M. (1988). Yeast activators stimulate plant gene expression. Nature 334,

Masson, P., and Fedoroff, N.V. (1989). Mobility of the maize

267-281.

631 -633.

suppressor-mutator element in transgenic tobacco cells. Proc. Natl. Acad. Sci. USA 86,2219-2223.

Metzger, D., White, J.H., and Chambon, P. (1988). The human oestrogen receptor functions in yeast. Nature 334, 31 -36.

Mitchell, P.J., and Tjian, R. (1 989). Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science 245,371 -378.

Montminy, M.R., and Bilezikjian, L.M. (1987). Binding of a nuclear protein to the cyclic-AMP response element of the somatostatin gene. Nature 328, 175-178.

Nakabeppu, Y., Ryder, K., and Nathans, D. (1988). DNA binding activities of three murine Jun proteins: Stimulation by Fos. Cell

Negrutiu, I., Shillito, R., Potrykus, I., Biasini, G., and Sala, F. (1987). Hybrid genes in the analysis of transformation condi- tions. I. Setting up a simple method for direct gene transfer in plant protoplasts. Plant MOI. Biol. 8, 363-373.

Neuberg, M., Schuermann, M., Hunter, J.B., and Miiller, R. (1989). Two functionally different regions in Fos are required for the sequence-specific DNA interaction of the Fos/Jun protein complex. Nature 338, 589-590.

Odell, J.T., Nagy, F., and Chua, N.-H. (1985). ldentification of DNA sequences required for activity of the cauliflower mosaic virus 35s promoter. Nature 313, 810-81 2.

Pietrzak, M., Shillito, R., Hohn, T., and Potrykus, 1. (1986). Expression in plants of two bacterial antibiotic resistance genes after protoplast transformation with a new plant expression vector. Nucl. Acids Res. 14, 5857-5868.

Rahman, S., Kreis, M., Forde, M.G., Shewry, P.R., and Miflin, B.J. (1 984). Hordein-gene expression during development of the barley (Hordeum vulgare) endosperm. Biochem. J. 223,

Ryseck, R.-P., Hirai, S.I., Yaniv, M., and Bravo, R. (1988). Transcriptional activation of c-jun during the GO/Gl transition in mouse fibroblasts. Nature 334, 535-537.

Schuermann, M., Neuberg, M., Hunter, J.B., Jenuwein, T., Ry- seck, R.-P., Bravo, R., and Miiller, R. (1989). The leucine repeat motif in Fos protein mediates complex formation with Jun/AP-1 and is required for transformation. Cell 56, 507-516.

Sellers, J.W., and Struhl, K. (1 989). Changing Fos oncoprotein to a Jun-independent DNA-binding protein with GCN4 dimeri- zation specificity by swapping “leucine zippers.” Nature 341,

Setoyama, C., Frunzio, R., Lian, G., Mudryj, M., and de Crom- brugghe, B. (1 986). Transcriptional activation encoded by the v-fos gene. Proc. Natl. Acad. Sci. USA 83, 321 3-321 7.

Struhl, K. (1 987). The DNA-binding domains of the jun oncoprotein and the yeast GCN4 transcriptional activator protein are func- tionally homologous. Cell 50, 841-846.

Struhl, K. (1 988). The JUN oncoprotein, a vertebrate transcription factor, activates transcription in yeast. Nature 332, 649-650.

Struhl, K. (1 989). Molecular mechanisms of transcriptional regu- lation in yeast. Annu. Rev. Biochem. 58, 1051-1 077.

Tabata, T., Takase, H., Takayama, S., Mikami, K., Nakatsuka, A., Kawata, T., Nakayama, T., and Iwabuchi, M. (1989). A protein that binds to a cis-acting element of wheat histone genes has a leucine zipper motif. Science 245, 965-967.

Vogt, P.K., and Bos, T.J. (1990). Jun oncogene and transcription

55,907-91 5.

31 5-322.

74-76.

658 The Plant Cell

factor. Adv. Cancer Res., in press. 331 6-331 9. Vogt, P.K., Bos, T.J., and Doolittle, R.F. (1987). Homology

between the DNA-binding domain of the GCN4 regulatory pro- tein of yeast and the carboxy-terminal region of a protein coded

Webster, N., Jin, J.R., Green, S., Hollis, M., and Chambon, P. (1988). The yeast US& is a transcriptional enhancer in human HeLa cells in the presence of the GAL4 trans-activator. Cell52,

for by the oncogene jun. Proc. Natl. Acad. Sci. USA 84, 161-167.

DOI 10.1105/tpc.2.7.651 1990;2;651-658Plant Cell

P Hilson, D de Froidmont, C Lejour, S Hirai, J M Jacquemin and M Yanivsites in plant cells.

Fos and Jun oncogenes transactivate chimeric or native promoters containing AP1/GCN4 binding

 This information is current as of September 23, 2020

 

Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

eTOCs http://www.plantcell.org/cgi/alerts/ctmain

Sign up for eTOCs at:

CiteTrack Alerts http://www.plantcell.org/cgi/alerts/ctmain

Sign up for CiteTrack Alerts at:

Subscription Information http://www.aspb.org/publications/subscriptions.cfm

is available at:Plant Physiology and The Plant CellSubscription Information for

ADVANCING THE SCIENCE OF PLANT BIOLOGY © American Society of Plant Biologists