Cell, Vol. 63, 1267-1276,

December

21, 1990, Copyright

0 1990 by Cell Press

Activation of the Ovalbumin Gene by the Estrogen Receptor Involves the Fos- Jun Complex Marie-Pierre Gaub, Maria Bellard, lsabelle Scheuer, Pierre Chambon, and Paolo Sassone-Corsi Laboratoire de Genetique Moleculaire des Eucaryotes CNRS Unite de Biologie Moleculaire et de Genie Genetique I’INSERM lnstitut de Chimie Biologique Faculte de Medecine 11, rue Humann 67085 Strasbourg Cedex France

du de

Summary The proximal region of the ovalbumin gene promoter contains a half-palindromic estrogen-responsive element (ERE) that mediates cell-specific frans-activation by the estrogen receptor (ER). We show that the ovalbumin ERE binds a ubiquitous nucleoprotein complex containing oncoproteins c-Pas and c&n. Mutations altering the estrogen inducibility of the promoter prevent the complex formation, which is, however, found in the presence and absence of ER and estradiol. Mutagenesis indicates that the sequence S-TGGGTCA-3’, containing the half-palindromic ERE, is responsible for induction by phorbol esters of the ovalbumin promoter and is a target for c-fos and c-jun frans-activation. Transfection experiments reveal that c-fos, c-jun, and ER coactivate the ovalbumin promoter. Direct ER interaction with the target sequence is not required, since an ER deleted for its DNA binding domain is functional in the coactivation with c-fos and c-jun. Our data indicate a convergence of hormonal induction and activation of signal transduction pathways at the transcriptional level. Introduction Control of initiation of transcription is the first step in gene expression and plays a major role in the physiology of cells and organisms. In this respect, the chicken ovalbumin gene has been an intensively studied model system because of both the importance of its protein product in growth physiology (Oka and Schimke, 1969; Kohler et al., 1969) and the complex hormonal regulation of its expression (for references see Palmiter et al., 1978; LeMeur et al., 1981). Indeed, the expression of the ovalbumin gene is controlled at the transcriptional level by all four classes of steroid hormones: estrogens, progestins, glucocorticoids, and androgens (for references see Palmiter et al., 1978; LeMeur et al., 1981; Chambon et al., 1984). Ovalbumin levels are also regulated by the activation of specific signal transduction pathways with different second messengers, such as increased levels of intracellular CAMP

involving stimulation of adenylate cyclase (Evans et al., 1981; Evans and McKnight, 1984). The ovalbumin regulatory region appears to include several distal sequences located up to 8 kb upstream from the transcription initiation site. This large region includes several sites of DNAase I hypersensitivity whose appearance is hormonally regulated (Kaye et al., 1986; Bellard et al., 1986). Detailed mutational analysis of the first 500 bp of this regulatory region revealed that it contains both positive and negative control elements. The negatively acting sequences are located in a distal segment between positions -425 and -132 (Gaub et al., 1987; Sanders and McKnight, 1988; Pastorcic et al., 1989). On the other hand, the proximal promoter region appears to contain positive elements, which also confer cell-specific expression (Dierich et al., 1987; Tora et al., 1988). Two separate sequences 5’-GGTCA-3’ have been identified that could dictate at least part of the hormonal regulation of the gene, since they correspond to half-palindromic elements of the consensus recognition site for estrogen receptor (ER) binding (estrogen-responsive element [ERE]; see Table 1). Whereas the proximal of these two sequences, located between positions -47 and -43, is a functional ERE, the distal one (positions -77/-73) is not (Tora et al., 1988). The distal element appears to be part of the site recognized by the COUP transcription factor, another member of the steroid receptor superfamily (Sagami et al., 1986; Wang et al., 1989). It is noteworthy that, as indicated in Table 1, a halfpalindromic ERE could also constitute part of a phorbol ester (TPA)-responsive element (TRE or TPA-RE). In particular, the -471-43 GGTCA element of the ovalbumin promoter is preceded by a TG dinucleotide resulting in the sequence TGGGTCA, which differs only in one nucleotide from a canonical AP-1 recognition site (TGAGTCA) (Table 1; Figure 1A). The transcription factor AP-1 corresponds to a number of polypeptides ranging between 30 and 55 kd, which are the products of the jun and fos gene families (Bohmann et al., 1987; Curran and Franza, 1988; Ryder et al., 1988, 1989; Rauscher et al., 1988b; Sassone-Corsi et al., 1988b; Hirai et al., 1989). As a heterodimer, Fos and Jun proteins bind to the AP-1 site with high affinity and regulate transcription of genes containing this DNA element (Curran and Franza, 1988). The Fos-Jun association involves the leucine zipper domain of both proteins (LandSchulz et al., 1988; Kouzarides and Ziff, 1988; SassoneCorsi et al., 1988c; Halazonetis et al., 1988; Nakabeppu et al., 1988; Turner and Tjian, 1989; Schuermann et al., 1989; Gentz et al., 1989) which appears to be required for dimerization and precise orientation of the DNA binding basic domains (O’Shea et al., 1989; Neuberg et al., 1989; Busch and Sassone-Corsi, 1990). Because of the sequence similarity of the ovalbumin ERE and a TRE, we examined the ovalbumin promoter regulation by phorbol esters, c-fos, and c-jun. We report here studies that demonstrate that the TGGGTCA element

Cdl 1266

Table

1. Sequence

Responsive

Element

Consensus

ERE

Ovalbumin

Comparison

between

(RE)

VDS-RE

ERE

(somatostatin) (osteocalcin)

Palindromic

structures

Sites Responsive Sequence

TPA-RE CAMP-RE

Target

are highlighted.

to Second

(5-3’)

Kumar

TGGGTCA

Tora et al., 1966 Lee et al., 1967

TGACGTCA --

Yamamoto

GxCeGGGTGA --

Morrison

The canonical

ERE sequence

-30

-20

WT.58

bCCTGTGG~~C~TTCA~~c~*~A~**cccc~~~~c~c*~c~*~~~~c~~~~-

MU1 9

--TGA

MUT MUT MUT YUT MUT

B

-40

is derived

-~~~~4~~

1 2 3 4

(C) Analysis extract. The 2-4) Mut4

et al., 1968 et al., 1989

from Beat0 et al. (1989).

(Dierich et al., 1987; Tora et al., 1988) that corresponds to the sequence GGTCA located between positions -47 and -43(Figure 1A). An oligodeoxynucleotidefrom -1 to -58 of the ovalbumin promoter was used in gel retardation studies with nuclear extracts from different cell types and tissues. The binding obtained using nuclear extracts from chicken embryo fibroblasts (CEFs) and oviduct tissue is shown in Figures 1B and 1C (lane I), respectively. The upper nucleoprotein complex, which appears as a doublet using CEF extract, was specifically competed by adding excess wild-type -l/-58 unlabeled oligodeoxynucleotide (Figure lB, lanes 2-4). Competition experiments with various mutated -11-58 oligodeoxynucleotides indicated that the GGTCA sequence is implicated in specific complex formation, as shown by using excess unlabeled MUT2 oligonucleotide with both CEF and oviduct extracts (Figure 18, lanes 11-13; Figure lC, lanes 2-5). Interestingly, MUTl, in which the first T preceeding the GGTCA motif is mutated, also failed as efficient competitor (Figure lB, lanes 8-10). This indicates that the sequence TGGGTCA, more than just the half-palindromic GGTCA, may be responsible for specific formation of the nucleoprotein complex. Oligonucleo-

-10

+&

Figure 1. Localization Sequences Binding Nuclear Proteins

of Ovalbumin Promoter Fibroblast and Oviduct

(A) Sequence of the wild type (WT-58) and mutated sequences containing the ovalbumin gene 5’-flanking region. The TATA box and AGC GGTCA half-palindrome are represented as GAT boxes. The dotted part of the box includes A-CG nucleotides that when added to the GGTCA sequence, give a heptapalindrome closely C MUT 2 MUT 4 MUT 6 MUT 9 MUT 1 MUT 2 MUT 3 MUT 4 MUT 6 related to a TRE (see text). (B) Competition binding assays with various -~~~ promoter mutants. Labeled wild-type (W-T-58) oligodeoxynucleotide (0.2 ng; lanes l-22) was incubated with CEF nuclear extract (5 ug of proteins) in the absence (-; lane 1) or in the presence of 2 ng, 20 ng, or 100 ng of unlabeled oligodeoxynucleotide as a competitor (corresponding to 10, 100, or 500 times excess; see Experimental Procedures). Competitor oligonucleotides were wild type (WT-58, lanes 2-4) or mutated Mut9 (lanes 5-7) Mutl (lanes 8-10). Mu12 (lanes 11-13) Mu13 (lanes 14-16) 12 3 4 5 6 7 8 910 5 6 7 s 9 10 11 12 13 14 15 16 17 l&l 19 20 21 22 Mu14 (lanes 17-19) or Mut6 (lanes 20-22). The arrow indicates the specific complexes (as a doublet). of nucleoprotein complexes formed between labeled wild-type (WT-58) oligonucleotide and proteins (5 kg) from chick oviduct nuclear incubation was in the absence (-; lane 1) or in the presence of 10, 100, or 500 times excess of unlabeled oligonucleotide Mu12 (lanes (lanes 5-7). or Mu16 (lanes e-10). An arrow denotes the specific slow-mobility complex.

1 2 3 4 6

WT-58

et al., 1966

TGAGTCA --

Binding of Ubiquitous Nuclear Proteins to the Half-Palindromic Ovalbumln ERE We have previously reported that the proximal region of the ovalbumin promoter contains a “cell-specific” ERE

-50

and to Steroids

References

Results

-56

(TPA and CAMP)

GGTCANNNTGACC --

(-49/-43) mediates the frans-activation of the ovalbumin promoter by c-fos, c-iun, and the ER. Mutations of this element abolish induction by TPA and forskolin, and impair trans-activation by ER, c-fos, and/or c-jun. Moreover, specific binding in vitro of nuclear factors to the TGGGTCA element is not affected by the presence of the ER in cultured cells. We show that the nucleoprotein complex formed in vitro with this element contains c-Fos and c-Jun proteins and can be assembled with nuclear extracts from a variety of cells. Strikingly, an ER deleted for its DNA binding domain is still able to coactivate with c-fos and c-jun. Our results indicate a new molecular pathway of estrogen-induced expression, which involves other transcription factors and does not require direct ER DNA binding.

A

Messengers

CTA

CT--T

Fos, Jun, and Estrogen 1269

Receptor

Oviduct IIIr----lII^

5 labeled

oligo

C

B

A Kidney

z In

Hela

CEF

%

:

-HE0 /II_

5,

>N(D> @J(D:N u2D=N(D : ’ •~L~k-L+~-L~~

$wIw 355B

F33l-33l-33+33 3zl3zr3rz3Iz

Figure

+HEO

-HE0

‘HE0

III :

2

WTI-l/-56)

competitor

_m_-

I 1 2

3 4

5 6 7

6 9101112

1234

,

2345676910

tides containing other mutations in the -1 to -58 region were efficient competitors in our assay, indicating that sequences 3’-flanking the TGGGTCA element are not directly involved in the specific complex formation. Binding of Ubiquitous Protein(s) to the TGGGTCA Sequence Is ER Independent We already pointed out the similarity between the TGGGTCA sequence (positions -49/-43) with a TRE (TGAGTCA) (see Introduction and Table 1). We next examined whether the ovalbumin element might bind proteins from other cell types. Specific complex formation was observed with extracts from CEF and HeLa cells, chicken oviduct, and kidney tissues (Figure 2A). The formation of the complex was impaired with all the extracts tested when the GGTCA sequence was mutated (Figure 2A, MUT2 oligodeoxynucleotide in lanes 2, 5, 8, and 11). These results indicate that a ubiquitous protein(s) binds to the ovalbumin ERE and suggest that ER cell-specific trans-activation may involve additional nuclear factors. It is well documented that ER present in nuclear extracts binds readily to a palindromic ERE (Kumar and Chambon, 1988). However, a half-palindromic ERE binds the ER only very weakly when compared with binding to a palindromic ERE (Kumar and Chambon, 1988; S. Mader and L. Tora, unpublished data). Hence we tested whether the ovalbumin half-palindromic ERE would bind the receptor upon estradiol induction in ER (HEO)-transfected CEFs. There was no modification in the complex formed with the GGTCA element upon ER addition (Figure 28, compare lanes 1 and 3). This result was confirmed under a variety of experimental conditions in the incubation reaction (data not shown). However, as expected, an ER-ERE complex was readily formed when a perfect palindromic ERE was used (Figure 28, compare lanes 2 and 4). Thus the ovalbumin TGGGTCA element does not behave as a canonical ERE element but, instead, forms a complex with a ubiquitous protein(s) that is apparently not affected by the presence of the ER. Competition experiments using the homologous unlabeled fragment and extracts from estra-

2. Tissue

Specificity

of Complexes

(A) Labeled wild-type WT(-V-56) or mutated oligodeoxynucleotides Mut2 and Mu16 (see Fiaure 1) were incubated with nuclear extracts f&m chicken oviduct (lanes l-3), chicken kidney (lanes 4-6), CEF (lanes 7-9), or HeLa cells (lanes 1012). The specific complex is shown by an arrow. (B) Labeled oligodeoxynucleotide WT(-V-56) or ERE (see text and Kumar et al., 1987) was incubated with nuclear extract from CEFs (lanes 1-2) or with extracts prepared from CEFs transfected with HE0 (lanes 3-4). Note that ERE shows a higher mobility complex than with the WT(-V-56) (asterisk). (C) Formation of specific slow-mobility complexes between the wild-type WT(-V-58) oligodeoxynucleotide and nuclear proteins from CEFs either not transfected (lanes 1-5) or transfected with HE0 (lanes 6-lo), in the presence of increasing amounts of WT(-V-58) oligonucleotide as a competitor.

diol-treated cells, transfected or not with HEO, also indicated that the binding affinity of the complex to the TGGGTCA element was unchanged by the presence of the ER (Figure 2C). These results are in apparent contrast with Tora et al. (1988), who showed direct recognition of the ovalbumin ERE by the receptor DNA binding domain. Although we cannot exclude the possibility that the ER binds to the half-ERE in some circumstances, we note that the binding experiments of Tora et al. (1988) involved a nonphysiological large excess of bacterially produced ER DNA binding domain. The data presented here, on the other hand, clearly demonstrate the binding of a ubiquitous, ER-independent nucleoprotein complex. The Ovalbumin Half-Palidromic ERE Binds c-Fos and c&n Oncoproteins Since the ovalbumin -49/-43 region differs from a TRE only in one nucleotide, we examined whether binding to a canonical AP-1 binding site (TGAGTCA) would be competed by the ovalbumin TGGGTCA element. Using CEF nuclear extracts, we found that TRE-specific binding was efficiently competed by the -331-58 ovalbumin promoter region at concentrations of unlabeled DNA that would also compete binding to the homologous -33/-58 region (Figure 3, compare lanes l-4 and 5-8). Binding to a CAMPresponsive element was also efficiently competed by the -331-58 promoter region (not shown). However, binding of the ER to a palindromic ERE was not competed by excess unlabeled -33/-58 ovalbumin oligonucleotide (Figure 3, lanes 13-16) when using extracts from HEOtransfected cells. Conversely, binding to the ovalbumin -331-58 probe was tested using the unlabeled palindromic ERE as a competitor. Again, no competition was observed (Figure 3, lanes g-12), confirming that the nucleoprotein complex formed with the ovalbumin -33/-58 region has characteristics distinct from those of a classical ER-ERE complex. It has been shown that addition of anti-Fos and antiJun antibodies to nuclear extracts from several cell types blocks the formation of a TRE nucleoprotein complex (Dis-

Cdl 1270

si

labeled oligo

;;

g I

-n competitor

t

WT(-33/-58) -A-d

12345678

9 10 11 12 13 14 15 16

Figure 3. The Specific Complexes Formed between CEF Nuclear Proteins and Oligonucleotides Carrying Specific Responsive Elements Labeled oligodeoxynucleotides were WT(-33/-58) (lanes I-4). containing the ovalbumin gene 5’-flanking region (see Figure lA), and metallothionein II TRE (lanes 5-8) (Angel et al., 1987). Oligoprobes were incubated with CEF nuclear extract in the absence (-) or in the presence of increasing amounts of unlabeled WT(-33/-58) oligodeoxynucleotide as a competitor. Complex formation between labeled wild-type oligodeoxynucleotide WT(-33/-58) and nuclear proteins from CEFs transfected with estradiol receptor expression vector HE0 in the absence (-; lane 1) or presence of increasing amounts of ERE, an oligodeoxynucleotide containing an estradiol-responsive element as a competitor (lanes 9-12). In lanes 13-16 are shown complexes formed on labeled ERE oligonucleotide in the absence (-; lane 13) or in the presence of WT(-331-58) unlabeled oligonucleotide as a competitor (lanes 14-16).

tel et al., 1987; Rauscher et al., 1988a; Sassone-Corsi et al., 1988a). Thus, we tested nuclear extracts from CEFs, HeLa cells, and oviduct tissue with these antibodies. In all cases, we found that treatment with both anti-Fos and antiJun antibodies inhibited complex formation with the ovalbumin TGGGTCA sequence. An example of these studies using CEF extracts is shown in Figure 4 (lanes 2-4). Control experiments using preimmune sera, excess antigenic peptide, or an unrelated antibody (an antibody against Myb protein; Boyle et al., 1985) indicated the specificity of our assay (lanes 5-7). These results, combined with the competition experiments, clearly show that the ovalbumin TGGGTCA element binds c-Fos and c-Jun oncoproteins and that the ER does not bind stably to this sequence. The TGGGTCA Element Is Responsive to Second Messengers The above results (Figures l-4) indicate that the ovalbumin ERE -49/-43 sequence bears several characteristics of a TPA-responsive element, forming a nucleoprotein complex containing both Fos and Jun oncoproteins. We thus tested whether the same ovalbumin element would be responsive to phorbol esters and forskolin. Treatment

1234567 Figure 4. Both Fos and Jun Proteins on the Gvalbumin TGGGTCA Site

Participate

in Complex

Formation

Nuclear extract from CEF cells contains specific binding activity (lane 1) that is inhibited by preincubation with anti-Fos (M2) and antiJun (PEP-l) antibodies (lanes 2-4). Preincubation with M2 antibody plus a 5-fold excess of the M peptide (Curran et al., 1985) leads to no binding inhibition (lane 5). Preimmune PEP-l antiserum (Bohmann et al., 1987) is inefficient in inhibiting binding (lane 6). A nonspecific antibody (against the oncoprotein Myb) is not able to inhibit binding (lane 7). Binding reactions were performed as described in Experimental Procedures using the WT(-V-58) ovalbumin oligoprobe.

of cells in culture with a tumor-promoting phorbol ester, such as TPA, activates the protein kinase C pathway, whereas activation of adenylate cyclase by forskolin leads to the induction of the CAMP-dependent protein kinase (Nishizuka, 1988). Reporter plasmids (Figure 1A) carrying wild-type or mutated ovalbumin promoter regions linked to the bacterial chloramphenicol acetyltransferase (CAT) gene (Tora et al., 1988) were transfected into CEF cells. The inducibility by estrogens (tested by cotransfection of the ER expression vector HE0 in the presence of estradiol [E2]), TPA, and forskolin was examined. As shown in Figure 5, the ovalbumin promoter is inducible by ER-E2, TPA, and forskolin. The sequences required for the induction by estradiol and TPA are centered in the region -49/-43 (MUTl and MUT2). Induction by forskolin is abolished using MUT2, but not MUTl, reporter genes. This difference with respect to estradiol and TPA induction might be due to different sequence requirements for CAMP-dependent stimulation (Yamamoto et al., 1988). Thus the same element of the ovalbumin promoter is a target for hormonal stimulation and two different signal transduction pathways.

Fc& Jun,

and Estrogen

Receptor

Figure 5. Effect of Estradiol ity of Ovalbumin Promoter

Receptor, TPA, and Forskolin Recombinants

on the metallothionein TRE (Sassone-Corsi et al., 1988b; Chiu et al., 1988). More interestingly, a clear increase was detected when HE0 was cotransfected with either c-fos or c-jun. Furthermore, additive activation was obtained when c-fos, c-jun, and HE0 were coexpressed (see table in Figure 6). No detectable stimulation of trans-activation was obtained with HE0 expressed in the absence of estradiol or when the MUT2 reporter gene was used (data not shown). These observations confirm the notion that the element centered around the TGGGTCA sequence has the properties of both a TRE/AP-1 site and an ERE. In addition, when inducers of signal transduction pathways (TPA and forskolin) were used in conjunction with cotransfected fransactivators, a marked increase in specific activation was observed (Figure 6). In particular, the effect of TPA on c-jun and HE0 is remarkable. TPA augmented c-jun- and HEO-independent rrans-activation by a factor of 2.5, whereas when c-jun and HE0 were cotransfected, TPA enhanced their effect by about lo-fold (see table in Figure 6). To ensure that the sequences centered around the TGGGTCA are responsible for the coactivation, and to exclude the possible involvement of other ovalbumin promoter sequences present in WT-58, we generated an additional construct, ovAdACAT. In this recombinant, the ovalbumin -58/-33 element is linked to the adenovirus type 2 major late promoter (MLP) TATA element (positions -34/-l) (see Figure 7A). Cotransfection of ovAdCAT in CEF cells with c-fos, c-jun, and HE0 resulted in activated transcription levels analogous to WT-58 (see Figure 7A, lower panel; see also Figure 78, lower panel). These data confirmed that the sequences required for the coactivation are limited to the region centered on the AP-1 site.

on the Activ-

CEF cells were cotransfected with a chimeric plasmid (pCAT-03) containing the wild-type (WT-58) or mutated ovalbumin gene 5’-flanking sequences (see Figure 1A) together with HEO. To test the effect of TPA or forskolin. these products were added directly to the medium in concentrations of 2 x lo-’ M and 2 x 10-s M, respectively. Cultures were maintained for 46 hr and then assayed for CAT activity with 6-galactosidase calibration as described in Experimental Procedures.

Coactivation by the ER, c-fos, and c-jun To determine whether the ovalbumin TGGGTCA -49/-43 element is a target for frans-activation by c-fos and c-iun, we performed cotransfection experiments in CEFs (Figure 6). The table in Figure 6 shows a summary of the data, which were obtained as averages of several experiments, similar to those displayed in the bottom part of the figure. Activation was observed with all three trans-regulators: C-/OS, c-jun, and estrogen receptor (HEO). Induction by c-fos was higher than with c-jun, in contrast with previously reported data using a canonical TRE sequence (Sassone-Corsi et al., 1988b). This could be due to a relatively low endogenous Fos level in CEF cells or to intrinsic properties of the ovalbumin sequence. Cooperativity between c-fos and c-jun was seen, as expected from results

‘ORSKOLIN

++++

6.5

15.7

53.6

7.9 44.2

50.6 148.0 5.4

19.1

104.4

33.5

172.4

55.8

182.2

++++

Figure 6. pans-Activation by c-fos, c-jun, and the Estrogen Receptor (HEO) Is Cooperative

1 TPA+FORSKOLIN

2.4

2.2

HE0 FOS JUN WA FORSKOUN

ER Effect on a Canonical AP-1 Site The experiments described in the previous sections (see also Figures 1,4, and 6) indicate that the ovalbumin TGGGTCA element has all the characteristics of an AP-1 site. Recently, however, Risse et al., (1989) introduced an A-G mutation in the third position of the collagenase TRE (TGAGTCA converted to TGGGTCA) and found that FosJun binding was strongly impaired. The reason for this ap-

++

++

++++

++++++++

++++++++ ++++++.I+++++++++

+ +

++++++++++++++ ++ rr*

++

++

++

~* e

I

b

+

aI

-*rrro

++

Using the wild-type (WT-58) construct (see Figure 5 and Experimental Procedures) as a reporter plasmid, trsns-activation by c-fos, c-jun, and HE0 was tested in transfected CEF cells. The bans-activation values reported in the top panel are the averages of several experiments having less than 15% deviation from the mean value. A representative experiment is presented in the bottom panel. Correct RNA initiation was tested by Sl nuclease mapping (C. Rochette-Egly, unpublished data). In cotransfections a 2.51 ratio of reporter plasmid to expression vector (c-fos or c-jun) was used. When more than one Vans-activator was used, the ratio was 25:l:l and 2.5:0.5 for HEO. The same total DNA amount was transfected in each experiment.

Cdl 1272

A -1 I 1 CAT

-33 OY A&AT

ov -50

Adenovirus

/ \ TGGGTCA

MLP

-34

rrm

Q

-em

rttittti JUN FOS HE0 TPA

12345676 + + +

++++ + + +++++

9

10

+

+ +

+

B -55

-50 WT.56 MutlO MutlP

-1 I

-30

I

ov

TGTGGGTGGGTCACAATT I I I ~GTGGGTG~GTCACAAT~ III II I III AAAGCATGAGTCAGACAC

OV

[

CAT

r

r

-II----

JUN

Figure

7. Effect

FOS

HE0

JUN HE0

FOS HE0

of ER on AP-1 Sequences

(A) The -%I/-33 region of the ovalbumin promoter is responsive to fos, jun, and HE0 when inserted in a heterologous environment. On the top is indicated the structure of ovAdCAT, a plasmid that contains the -5S/-33 ovalbumin region linked to the adenovirus type 2 major late promoter -34/-l region and to the CAT gene. The lower panel shows a representative CAT assay after transfection in JEG-3 cells of ovAdCAT in conjunction with fos, jun, and ER expression vector. Quantification of several experiments with ovAdCAT is indicated in the lower panel of (8). (B) Mutants MutlO and Mu112 are equivalent to m-56 except that the ovalbumin ERE region is mutated as indicated. The lower panel indicates the results after several transfections in CEF cells.

parent discrepancy could be a crucial role played by the sequence flanking the AP-1 sites. Alternatively, differences in the binding properties of the AP-1 molecules present in the nuclear extracts used here (Figures 1-3) and of in vitro generated Fos and Jun proteins used by Risse et al. (1989) are conceivable. We next asked whether a canonical AP-1 site might re-

spond to the ER in a manner similar to the ovalbumin element. To test a canonical TRE in a totally homologous ovalbumin environment, we generated two additional mutants (MutlO and Mut12 in Figure 78). MutlO is equivalent to WT-58, but the TGGGTCA sequence is converted by a single G-A mutation into a perfect palindrome, TGAGTCA, equivalent to the one present in the collagenase promoter. Mut12 is a more drastic mutation since, as MutlO, it contains the AP-1 site converted into TGAGTCA, and in addition the sequences flanking the palindromic core have also been substituted with those of the collagenase promoter. Mut12 could be considered, in fact, as a fusion promoter in which the collagenase TRE is inserted upstream of the ovalbumin TATA element. Both MutlO and Mut12 were tested by cotransfection experiments in CEF cells with fos, jun, and HEO. When compared with the ovalbumin unaltered construct WT-58, we noticed that MutlO responds in a similar manner (lower panel of Figure 78) to trans-activation by fos, jun, HE0 and by their combination. Thus, a canonical AP-1 site can be the target of ER activation and costimulation with fos and jun. However, analysis of Mut12 in the same assay indicated that, whereas ER responsiveness was conserved, coactivation with fos was drastically reduced. These results suggest a possible role of flanking sequences in the coactivation phenomenon and clearly indicate that a canonical AP-1 site can be the target of ER activation. Coactivation by fos, jun, and HE0 appears to require a short sequence (see also the results with the ovAdCAT construct; Figure 7A) and the intact AP-1 site. Coactivation Does Not Require DNA Binding of the ER We are interested in the mechanism by which c-fos, c-jun, and ER coactivate transcription from the ovalbumin promoter. Two possibilities can be considered, taking into account that c&s and c-Jun directly bind to this target sequence (Figure 4): the ER cooperates via weak binding to a neighbor DNA element; or coactivation involves some ER-Fos-Jun interactions without binding of the ER to the DNA. To test these hypotheses we used HEll, an estrogen receptor mutant that lacks the DNA binding function but is wild type for the ligand binding and transcriptional activation functions (see Kumar et al., 1988, 1987; Tora et al., 1989). Remarkably, when cotransfected with c-fos, HE11 coactivated as efficiently as HE0 (Figure 8). The coactivation was much weaker with c-jut?, but the synergistic effect of TPA in conjunction with c-jun and HE0 (see table in Figure 6) was clearly conserved when HE11 was cotransfected with c-jun (Figure 8). Note, however, that as expected, HE11 did not activate transcription from the WT58 reporter gene when used alone, in contrast to HEO. These data show that the specific DNA recognition by ER is not required for coactivation. Discussion Regulation of ovalbumin gene transcription in oviduct tubular gland cells, whose growth and differentiation are under estradiol control, is complex and subject to induc-

i$Jun,

and Estrogen

Receptor

70.

60-

HE0 HE11 FOS JUN TPA

Figure 8. HEll, ates with c-fos

I +

+++ +++ +

an ER Mutated

+

+

+ +++ +++++

in the DNA Binding

Domain,

Cooper-

Cotransfections in CEF cells were performed using the CAT reporter (W-r-58). HE11 (Kumar et al., 1997) is as efficient as HE0 in cooperating with c-fos in trans-activation. HE11 is inactive by itself or in conjunction with c-jun.

tion by hormones and second messengers (Kohler et al., 1969; Palmiter et al., 1976; Chambon et al., 1964; Evans et al., 1964; Gaub et al., 1967; and references therein). Therefore, the ovalbumin promoter is a paradigm for the study of the regulatory interplays involved in the activation of specific signal transduction pathways. We are interested in elucidating the molecular processes that are involved in the conversion of short-term effects of extracellular signals into long-term cellular responses, such as cell growth and differentiation. In this network, nuclear oncoproteins such as Fos and Jun could form links between an incoming signal and gene regulation, by converting an extracellular signal into a change in the genetic program (Verma and Sassone-Corsi, 1987; Vogt and Bos, 1989). Fos and Jun proteins are constituents of the transcription factor AP-1 (Rauscher et al., 1988b; Chiu et al., 1988; Sassone-Corsi et al., 1988b), which interacts with a specific promoter palindromic element that is a target for activation by phorbol esters. All the members of the Fos and Jun families of proteins are likely to form heterodimers, thus dramatically increasing the possibilities of combinatorial gene regulation. In this context it is noteworthy that Fos-Jun also binds to and activates CAMP-responsive elements (Hoeffler et al., 1989; Sassone-Corsi et al., 1990) suggesting the presence of cross-talk mechanisms at the transcriptional level. Transcriptional activation by members of the steroidjthyroid receptor family also involves palindromic target se-

quences (see Table 1; Green and Chambon, 1988; Evans, 1988). Some recent reports, however, have suggested that transcriptional stimulation triggered by steroid receptors could be mediated by half-palindromic recognition sequences (Tora et al., 1988; Berg, 1989). Such stimulation may involve cooperativity with adjacent promoter elements (Martinez and Wahli, 1989; Ponglikitmongkol et al., 1990). We report here that the ovalbumin half-palindromic ERE is a unique target for coactivation by the ER and the oncoproteins c-Fos and c-Jun. We have shown that the ovalbumin ERE forms a complex in vitro whose migration is apparently not affected by the presence of the ER (Figures l-3). This complex contains both Fos and Jun oncoproteins (Figure 4). Therefore, the -49/-43 ovalbumin sequence could be considered as a multiple estrogen- and second messenger-responsive element, although the ER appears not to act directly on the sequence. The ovalbumin induction by the estrogen receptor may thus involve other transcription factors, such as AP-1. At least in this case, transcriptional activation by a steroid receptor could be indirect, since it is mediated by other frans-acting factors distinct from ER. In this context, it is noteworthy that steroid receptors are also repressors of transcription for certain genes (Ackerblom et al., 1988; Sakai et al., 1988; Meyer et al., 1989); at the least in the case of the proliferin gene, the elements required for phorbol ester stimulation and glucocorticoid repression localize to the same regulatory region (Mordacq and Linzer, 1989). Interestingly, the repression is dependent on the glucocorticoid receptor, which binds to a region that includes an AP-1 site (Mordacq and Linzer, 1989). In this respect, the case of the osteocalcin promoter may also be relevant, since both the vitamin D3-responsive element and a canonical AP-1 site are located within a short 20 bp doubly palindromic structure (Morrison et al., 1989) (see Table 1). Recent data by Schtile et al. (1990) in fact, indicated a possible alternate recognition of the palindromic site by AP-1 and vitamin D3 receptor, resulting in transcriptional repression. In the case presented here, on the other hand, the ER activates transcription and does not appear to bind to the AP-1 site. Our data indicate that the regulatory functions exerted by the estrogen receptor are likely to be both direct and indirect. Direct activation is well documented and requires tight binding of the receptor to an ERE as a homodimer (Kumar and Chambon, 1988). Indirect regulation appears to require other factors themselves bound to the responsive element. Indeed, using an ER mutant that lacks the DNA binding function (HEll; Figurea), we found that it still coactivates with c-fos with an efficiency similar to that of the wild-type ER (HEO). In contrast, c-jun does not appear to coactivate efficiently with HE11 (Figure 8) unless TPA is added to the transfection. The reason for this difference is unclear at this stage and will be explored in future studies. How coactivation by the ER, c-fos, and c-jun is achieved without ER binding to the responsive element remains to be investigated. Several hypotheses can be formulated to explain the observations reported in this work. For example, ER, Fos, and Jun may directly interact to generate

Cdl 1274

transcriptional activation. Alternatively, the ER may activate another gene whose product might modulate FosJun function. Or alternatively, ER may titrate a putative Fos-Jun inhibitor, thus generating an increased AP-1 activity. Further studies are required to distinguish among these possibilities, and experiments are in progress to analyze the molecular mechanisms involved in this phenomenon. We believe that the detailed study of the present system will improve our understanding of the links existing between hormonal regulation mediated by nuclear receptors, signal transduction mediated by membrane receptors, and modulation of gene expression. Experimental

Procedures

Expression Vectors and Reporter Plasmlds HEO, wild-type (WT-58) and mutated (MUTl to MUTG) ovalbumin gene promoter fragments and the corresponding CAT reporter genes (ovAdCAT included) were as described (Tora et al., 1988). MUT9 was constructed accordingly. c-fos and c-jun expression vectors have been already described (Sassone-Corsi et al., 1988b). Cell Transfections and CAT Assays CEF primary cultures were prepared from 9-11-day-old chicken embryos according to Solomon (1976). CEFs and HeLa cells were maintained in Dulbecco’s modified Eagle’s medium without phenol red sup plemented with 5% dextran-coated charcoal-treated fetal calf serum (Dierich et al.. 1987). Cells were transfected at 40%-80% confluency using the calcium phosphate technique. For CAT assay experiments after overnight incubation with the precipitate, cells were washed and incubated in medium containing 0.5% fetal calf serum. When required, 10-s M estradiol, 2 x lo-’ M TPA, or 2 x 10m5 M forskolin was added after the cells were washed. CAT assays were carried out 24 hr later as described by Webster et al. (1988). The enzymatic reaction lasted 2 hr. with a second addition of 0.4 mM acetyl-CoA after 1 hr of incubation. In the figures the notation HE0 represents experiments where estradiol was added together with the expression vector HEO. Preparation of Nuclear Extracts and DNA Blndlng Assays CEF cells transfected or not with HE0 expression vector were incubated for 1 hr with 10-s M estradiol just before two washes with cold saline buffer (PBS), harvested, and lysed with 15 strokes with a Dounce B in buffer A (20 mM Tris-HCI [pH 81, 20 mM KCI, 1 mM MgCIz, 1 mM DTT, 0.3 mM PMSF with protease inhibitors 15 uglml each of leupeptin, aprotinin, pepstatin, chymostatin, and antipain]). After centrifugation at 1500 x g the crude nuclei pellet was washed twice and then resuspended in the high salt buffer B (same as buffer A but 0.8 M KCI and 25% glycerol). After 20-30 strokes with a Dounce B, extracts were incubated at 4°C for 30 min. After centrifugation at 18,000 x g (30 min). the supernatant was dialyzed at 4OC against buffer C (20 mM Tris-HCI [pH 81, 30 mM KCI, 25% glycerol, 1.5 mM MgClz, 0.5 mM DTT, and 0.3 mM PMSF), aliquoted, and frozen in Nz. Proteins were quantified by the method of Bradford (1976). Oviducts and kidneys from 8-day-old chickens stimulated by estradiol were removed, minced in cold PBS, and washed extensively to remove erythrocytes and treated as described above. Mobility shift assays were performed as in Garner and Revzin (1981) using the double-stranded oligodeoxynucleotides WT-58, and MUTl to MUT9 described in Figure IA, which have been used for constructing the CAT reporter plasmids. Whole-cell extract, usually 5 pg, was incubated in a 20 PI reaction mixture containing 20 mM Tris-HCI (pH 7.5) 100 mM KCI, 1 mM MgCIz, 0.1 mM EDNA, 0.5 mM DTT, 10% glycerol, 10-s M estradiol, 4 ug of poly(dl-dC), and 0.2 ng (=20,000 cpm) of double-stranded, end-labeled, synthetic oligodeoxynucleotide corresponding to the wild-type -1 to -58 ovalbumin promoter sequence or mutated sequences as described in Figure 1A. Poly(dl-dC) and whole-cell extracts were first incubated at O°C for 30 min before adding the end-labeled oligodeoxynucleotide. For competition experiments, lo-500 times more of a double-stranded oligodeoxynucleotide was added to the reaction mixture just before the addition of the end-labeled oligodeoxynucleotide. Free DNA and

DNA-protein complexes were resolved on a 5% polyacrylamide gel in 0.5x TBE (45 mM Tris-borate, 45 mM boric acid, 2 mM EDTA). Antlbodies to Fos and Jun Affinity-purified antibodies to the M2 peptide of mouse Fos have been described (Curran et al., 1985). Anti-Jun PEP-l peptide antiserum was a kind gift of T. J. Bos and P Vogt (Bohmann et al., 1967). Inhibition of DNA-protein complex binding with antibodies was performed by adding the antisera to extracts of the purified proteins and incubating for periods from 2 hr to overnight at 4OC. No disruption of the nucleoprotein complex binding was observed when a B-fold excess of M2 peptide was added together with the antibody to the extract or to the purified proteins. Acknowledgments Address correspondence to Dr. R Sassone-Corsi. We thank F. Bellard, L. Tora, C. Rochette-Egly, A. Dierich, T. J. Bos, and P Vogt for gifts of recombinants and material; A. Staub, F. Ruffenach, M. Digelmann, and F. Schlotter for technical help at various stages; and the secretarial and illustration staff for assistance in preparing the manuscript. This work was supported by grants from CNRS, INSERM. and the Association pour la Recherche sur le Cancer. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “edvertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

February

5, 1990; revised

September

3, 1990.

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Added

In Proof

Evidence for a Pos-Jun inhibitor has been recently obtained: Auwerx, J., and Sassone-Corsi, R (1991). IP-1: a dominant inhibitor of Pos-Jun whose activity is modulated by phosphorylation. Cell, in press.