t3 receptor suppression of spl-dependent transcription from the

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, h e .

Vol. 268, No. 21, Issue of July 25, pp. 16065-16073,1993 Printed in U.S.A.

T3 Receptor Suppression of Spl-dependent Transcription from the Epidermal Growth Factor Receptor Promoter via Overlapping DNA-binding Sites*

(Received for publication, February 24, 1993)

Jun Xu$, Karol L. Thompson§, Lee B. ShephardQ, Laurie G. Hudsonll, and Gordon N. Gill§11 From the Departments of $Chemistry and §Medicine, University of California, San Diego, La Jolla, California 92093 and the llDepartment of Pharmacology, Northwestern University School of Medicine, Chicago, Zllinois 6061 1

Expression of the human epidermal growth factor receptor (EGFR) gene is inhibited by ligand-activated thyroid hormone receptor (T3R). Binding sites for Spl and for the T3R-retinoid X receptor (RXR) complex overlap in a functional core of the EGFR promoter. Spl inhibited binding of the T3R complex to this 36- base pair (bp) EGFR element in vitro but did not affect binding of the T3R complex to a positive thyroid hormone response element (TRE). In Drosophila SL2 cells, which lack Spl and T3R, function of the EGFR promoter was strongly dependent on Spl. Spl-dependent promoter function was inhibited by ligand-activated T3R but not by mutant T3R defective in DNA or T3 binding. RXR increased the extent of inhibition. S p l enhanced activity of the 36-bp element placed 5’ to a minimal TATA promoter and this enhancement was also repressed by T3R. Mutations in the 36-bp element were unable to separate Spl and T3R functions. How- ever, addition of a second half-site 5‘ to the existing site in an inverted repeat configuration created a positive TRE. In the absence of ligand, T3R inhibited S p l stimulation from this altered element; addition of T3 reversed the inhibition. When a dimeric TRE is separated from Spl-binding sites strong synergism was observed. The nature and location of the TRE thus strongly influence biological responses. A TRE site in the EGFR promoter that overlaps an Spl-binding site inhibits S p l function but is unable to direct positive function.

The promoter of the human epidermal growth factor receptor (EGFR)’ is GC-rich and lacks characteristic CAAT and TATA boxes (1). Multiple nuclear protein-binding sites have been identified 5‘ to the ATG translation start site (1,2), and a number of factors are reported to regulate transcription of the EGFR promoter in vitro (3-5). In vivo positive effectors include EGF, cyclic AMP, and 12-0-tetradecanoyl-phorbol 13-acetate (6-8); negative effectors include ligand-activated

* These studies were supported by National Institutes of Health Grant DK13149 and by a Johnson and Johnson Focused Giving Award. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1) To whom correspondence should be addressed Dept. of Medicine, University of California at San Diego, La Jolla, CA 92093-0650.

The abbreviations used are: EGFR, epidermal growth factor receptor; T3R, thyroid hormone receptor; RAR, retinoic acid receptors; bp, base pair(s); RXR, retinoid X receptor; TRE, thyroid hormone response element; EMSA, electrophoretic mobility shift assay; WT, wild-type; MHC, myosin heavy chain.

thyroid hormone and retinoic acid receptors (TSR and RAR) (9). A proximal 36-bp segment (-112 to -77 bp relative to the ATG translation start site) has been characterized as a minimal promoter element and has also been demonstrated to function as an enhancer (10). This 36-bp element binds T3R and RAR with high affinity (9). Direct high affinity binding of T3R to this DNA requires a nuclear accessory protein which was isolated from HeLa cell nuclei (11) and subsequently identified immunologically as retinoid X receptor a (RXRa). RXR proteins form functional heterodimers with T3R, RAR, and the vitamin D receptor with each member of the heterodimer binding to half of the direct or inverted repeat DNA sequences that constitute positive hormonal response elements (12-15). However, the T3R.RXR heterodimer contacts the EGFR promoter DNA primarily on a single 7-bp site that is homologous to half of the positive dimeric T3 response element (TRE) present in the rat growth hormone promoter (11, 16, 17).

T3R can decrease basal transcription from a positive TRE in the absence of hormone and produce a superactivation when T3 is added (18,19), but the mechanism by which ligand- dependent repression by TsR occurs is largely uncharacter- ized. The nature of the TRE and its context appear to be important determinants of the biological response to T3. T3R binds immediately adjacent to the TATA box in negative TREs in the rat growth hormone and human TSHa genes (20, 21). Interference with the basal transcription machinery has been proposed but not directly tested. A direct repeat of the core T3R binding motif without spacing which occurs in the TSHp promoter confers transactivation in the absence of T3 and repression in the presence of T3 (22). Naturally occur- ring splice variants of the a form of TSR inhibit T3R activity through protein-protein interactions, either forming inactive heterodimers (23) or competing for limiting transactivating factors (24).

In the present study we have investigated the mechanism through which TBR inhibits EGFR promoter function. Meth- ylation protection analysis indicated that the binding sites for the T3R. RXR complex and the transcription factor Spl overlap; T3R. RXR and Spl competed for binding to the proximal promoter fragment of EGFR in uitro. Upon cotransfection of Spl and T3R expression plasmids into Drosophila SL2 cells which lack Spl and T3R (25, 26), ligand-activated T3R inhibited Spl-dependent EGFR promoter-driven luciferase expression. Cotransfection of an RXRa expression plasmid increased the extent of inhibition. Because Sp l is required for basal transcription from GC promoters that lack a TATA box (27, 281, interference with the activity of Spl provides an effective mechanism for repression of basal transcription. Competition for DNA binding between Spl and T3R is pro-

16065

16066 Td, RXR, and Spl Regulation of EGF Receptor Promoter

posed to be the mechanism through which the observed repression occurs.

MATERIALS AND METHODS

Construction of Reporter and Expression Plasmids-Both strands of the wild-type and mutant EGF receptor promoter 36-bp elements corresponding to -112 to -77 bp were synthesized as 22-mer oligonucleotides using an Applied Biosystems 380B DNA synthesizer. The oligonucleotides were annealed, filled in by the Klenow fragment of DNA polymerase I, and ligated into the blunt-ended Hind111 site of the luciferase expression vector pSVOALA5' (29) to generate the promoter activity reporter plasmids. Extended 5' EGFR promoter fragments were placed in pSVOALA5' as previously described (8). In order to mutate the central GC box in the -153- to -19-bp fragment of the EGFR promoter, two oligonucleotides were synthesized. One corresponded to -115 to -86 bp of the promoter with base pair alterations of GGG to TTT at position -97 to -95. The other corresponded to 2271 to 2250 bp of the SV40-derived sequences of the reporter plasmid p(N-S)-LUC (8) with the unique ApaI site changed to a KpnI site. Mutagenesis was performed as described by Deng and Nickoloff (30). Mutant ApaI undigestible plasmid was isolated and transformed into competent cells. Mutants that contained the TTT substitution in the GC box were then identified by sequencing.

A minimal TATA box promoter reporter plasmid, mTK-LUC, was prepared by digesting the Herpes simplex virus thymidine kinase promoter with BamHI and BglII to excise the -37- to +52-bp fragment. The fragment was purified by low-melting agarose gel electro- phoresis and ligated into the polylinker region of the luciferase expression vector pXP2 (31), which was digested with the same restriction enzymes. The plasmid TAQ-LUC (mPrl-LUC), which contains the -36 to +33 fragment of the promoter of the rat prolactin gene, was obtained from Dr. Christopher Glass, University of Cali- fornia, San Diego (32). mTK-LUC and mPrl-LUC each contain a TATA box, but no other known regulatory elements (32, 33). Blunt- ended 36-bp wild-type and mutant EGFR promoter elements were inserted into the blunt-ended BamHI site of mTK-LUC and into the blunt-ended XhoI site of mPrl-LUC to generate the heterologous promoter-enhancer reporter plasmids. Other reporter plasmids have been described (12, 31). All constructions were verified by dideoxy- nucleotide sequencing (34).

pPacSpl, pADHpga1, and pPacU+NdeI plasmids were a generous gift of Drs. Erica Pascal and Robert Tjian, University of California at Berkeley (25). The coding region of T3RP was excised with XhoI and XbaI from pT3RP-14 (35) and subcloned into the XhoI- and BamHI-digested pPacU+NdeI expression vector plasmid to yield pPacT3R. Plasmid pPacT3RAC was constructed using XhoI and PuuII to prepare a T3R that terminates at the codon for amino acid 335 (T3RAC) and deletes 121 carboxyl-terminal amino acids. The cDNA coding for the mutant T3RP which lacks amino acids 100-171 was amplified by polymerase chain reactions from parental plasmid pTPA100-171 (35). The polymerase chain reaction product was then inserted into NdeI- and BanHI-digested pPacU+NdeI to generate pPacT3RADBD. A similar strategy was used to construct pPacT3RS and pPacRXR from parental plasmid pSV2hTRPS and pRShRXRa, respectively (26,36). The mutant T3RS has Thr deleted at codon 332, which destroys ligand binding activity (36). Sequencing confirmed that the cDNA sequences were inserted in the correct open reading frame and the desired mutations were retained. The expression plasmid pCMVPgal was previously described (8).

Methylation Protection Assays-Oligonucleotides corresponding to -112 to -77 bp in the EGFR promoter with 10-bp overhangs were 5"labeled with [Y-~'P]ATP and T4 polynucleotide kinase, annealed to the unlabeled opposite strand, and filled in with dNTPs and Klenow enzyme. DNA was methylated with dimethyl sulfate and repeatedly precipitated. Spl was purified from HeLa cell nuclei by chromatography on wheat germ agglutinin followed by DNA affinity chromatography (37, 38). Human T3R,8 was expressed in bacteria under control of the T, promoter in the PET-3A vector (39) and purified by heparin-agarose chromatography (11). The hRXRa cDNA was cloned into the bacterial expression vector pRSETA (Invitrogen, San Diego, CA), which creates a fusion protein with a polyhistidine sequence at the NHz terminus. pRShRXRa (26) was digested with EcoRI to release the RXRa cDNA sequence. The isolated insert was digested with Smd and ligated into the blunt-ended BamHI site of pRSETA, such that translation of RXRa initiated at amino acid 45. RXRa was expressed in Escherichia coli strain BL21 (DE3) and

purified over heparin-agarose as described for the T3RP (11). Meth- ylation protection assays were performed as described ( l l ) , and products were run on an 8% sequencing gel.

Auidin-Biotin Complex DNA Binding Assays-Zn vitro DNA binding assays (80 pl) contained T3R (90 fmol of T3-binding site equiva- lents calculated from Scatchard analysis (40) of bacterially synthesized T3RP purified by heparin-agarose chromatography), [1Z51]T3 (300 fmol), RXR purified from HeLa cell nuclei through the Mono Q chromatography step ( l l ) , 10-168 ngof pure Spl (Promega, Madison, WI), 0.2 pg/ml of poly(d1-dC) and 350 fmol of biotinylated DNA- binding sites. Before use, Spl was dialyzed into buffer containing 25 mM HEPES/KOH, pH 7.5, 20% glycerol, 0.1% Nonidet P-40, 50 mM KCl, 10 mM ZnS04, 0.5 mM MgC12, and 1 mM dithiothreitol. The HeLa nuclear Mono Q fraction contained RXRa as determined by immunoblotting with an antipeptide antibody specific for the a form of human RXR ((41) generously supplied by Jacqueline Dyck, The Salk Institute) and by the ability of this antibody to supershift the T3Rp. MonoQ fraction heterodimer in electrophoretic mobility gel shift assays (data not shown). Components were incubated 10 min at 23 "C prior to addition of biotinylated DNA; reactions were continued for 45 min and protein-DNA complexes precipitated with strepavidin- agarose (16). Kinetic constants were calculated using the Ligand Program written by G. A. McPherson, Biosoft, Cambridge, United Kingdom.

Transfection and Assay of Enzyme Actiuities-HeLa cells were maintained in Dulbecco's modified Eagles's/F-12 (1:l) medium supplemented with 5% calf serum. Transfections were performed as described by Chen and Okayama (42) except that the calcium phosphate-DNA was mixed with pooled cells after incubation at room temperature for 30 min. Ten pg of reporter plasmid and 10 pg of pCMVPgal were used per 10 ml of culture and plated into three 60- mm diameter dishes. Cells were harvested after 48 h.

Schneider line 2 (SL2) cells were grown in Schneider's Drosophila medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum. Cells were seeded overnight before transfection at a density of 2 X lo6 cells/60-mm dish containing 5 ml of medium. Alternatively, 5 X IO5 cells were seeded with 1 ml of medium/well of a 12-well dish. Transfections were carried out as described (43). Plasmids were suspended in 0.25 M CaC12, and an equal volume of 2 X HEBS buffer (42 mM HEPES, pH 7.1, 274 mM NaC1, 9.4 mM KC1, 2.8 mM Na2HP04, and 0.2% dextrose) was added dropwise. The mixture was incubated at room temperature for 30 min before adding it to cells. Transfected cells remained undisturbed until harvest 48 h later.

Luciferase activity was measured as described by de Wet et al. (29). Aliquots of cell extract were added to an assay reaction containing 100 mM potassium phosphate, pH 7.8,5 mM ATP, and 15 mM MgSO4 in a volume of 0.35 ml. Reaction were initiated by injection of 0.1 ml of 1 mM luciferin, and light readings were integrated over 10 s with a Monolight 2110 luminometer. &Galactosidase was measured as described by Norton and Coffin (44).

Electrophoretic Mobility Shift Assays (EMSA)-T,R.RXR binding

their ability to compete for binding to the WT 36-bp element using specificity to mutant EGFR promoter sequences was analyzed by

EMSA. T3R and RXR were incubated in 25 mM potassium phosphate, pH 7.7, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 4% glycerol, and 250 pg/ml bovine serum albumin for 10 min at 23 "C. Poly(d1- dC) (50 pg/ml), unlabeled competitor DNA, and labeled WT probe were added, and incubation was continued for 20 min. Samples were loaded onto 6% acrylamide gels and run for 2.5 h at 4 "c in 0.5 X TBE (25 mM Tris-borate, 0.5 mM EDTA). Gels were exposed to X-

ray film for 2 days at -70 "C.

RESULTS

Overlapping Ta- and Spl-binding Sites in the Proximal EGFR Promoter-A 36-bp sequence in the EGFR promoter, which contains two in uiuo transcription start sites (l), also functions as an enhancer when placed in front of TATA- containing promoters (10). Four Spl-binding sites have been identified with DNase I footprinting in the EGFR promoter with the most proximal site located in this 36-bp region between -110 and -84 bp (2). T o accurately define the site of Spl binding within the 36-bp element, methylation protection assays were carried out. Spl contacted 9 G residues on both strands within an 11-bp site extending from -100 to -90 (Fig. 1). This region of Spl interaction is essential for

Ta, RXR, and Spl Regulation of EGF Receptor Promoter 16067

A SP1

.94 -. .loo +

m c -100 - + -102

-77

B ""-" ~

5' GCGTCCGCC TCCC GCCTCCCCGCCAACGCCA 3' -112

CGCAGGC +GGCGGTTGCGGT +!I- 0 0 0 0 0 - -: 0

FIG. 1. Comparison of Spl- and TsR-binding si^ on the 36-bp EGFR promoter element. A, methylation protection of the EGFR promoter element by Spl and by T3R.RXR complexes. An- nealed oligonucleotides corresponding to sequences from -112 to -77 bp in the EGFR promoter were 5"labeled on the upper or lower strand, methylated with dimethyl sulfate, and used in EMSA with Spl or T3R plus RXR. L-TI (100 nM) was added to the indicated reactions prior to electrophoretic separation of bound from free DNA. The arrows indicate the methylated guanines that interfere with protein binding. B, overlap of binding sites for Spl and T,R-RXR on the EGFR promoter element. G residues protected by Spl (0) and by T3R.RXR (e) are indicated. The regions of protein-DNA interaction are boxed, and the area of overlap is shaded.

promoter and enhancer activities in vivo. Fig. 2 shows the effects of placing six 3-bp substitutions in the 36-bp element, all designed to decrease the GC content. Mutations M3 and M4, which change residues between -97 and -92 in the central GC box, severely impair both promoter and enhancer functions of the 36-bp element. The M3 and M4 substitutions decrease promoter function by 75 and 78%, respectively, and decrease enhancer function by 88 and 89%, respectively. Dis- ruption of other CCGCC sequences at -108 to -104 and -88 to -84 had only small effects on activity of the element. As previously reported (lo), the M1 mutation reproducibly increased promoter activity but decreased enhancer activity by -20%.

By comparison, methylation protection of the 36-bp EGFR promoter element by the T3R RXR complex indicated con- tact with G residues at -102, -100, -98, -97, and -96 bp (Fig. 1). Identical results were obtained using T3R.TRAP heterodimers (11) and T3R homodimers (data not shown).

A. EGF Receptor PromoterEnhancer -112 G C G T C C G F F ~ C . 3 F T C ~ F ~ ~ ~ ~ T F C . F ~ ~ ~ C ~ C G C C A -77 " TTTTTT "" TTTTTT TATTTT M1 M2 M3 M4 M5 M6 Mutations

B. Promoter Mapping

I

100

50

n WT M1 M2 M3 M4 M5 M6

C. Enhancer Mapping

WT M1 M2 M3 M4 M5 2xM6 2xWT

FIG. 2. Effects of mutations in the 36-bp EGFR element on promoter and enhancer functions. A, sequence of the 36-bp EGFR promoter element with the indicated 3-bp changes. Each mutation decreases GC-content of the fragment. B, promoter activity. A single copy of the indicated 36-bp elements was placed in front of the luciferase reporter gene, and activity was determined following transfection into HeLa cells. Activity of the wild-type 36-bp element which was set as 100% equaled 4.4 X IO3 light units of lu~iferase/OD~~~ unit of &galactosidase. Similar results were obtained using CV1 cells. Results are obtained from three experiments with triplicate points in each. C, enhancer activity. The indicated 36-bp elements were placed in front of the mTK promoter. Activity of constructions containing a single copy of wild-type 36-bp element equaled 5.9 X lo' light units of lu~i fe rase /OD~~~ unit of @-galactosidase while activity of two copies of the 36-bp element (2xWT) equaled 1.4 X lo5 light units of luciferase/ODllr unit of P-galactosidase. Wild-type activities were set as 100%. The activities of mutant enhancers are shown relative to the activities of the wild-type constructions with the same number of copies of enhancer elements. Results from one experiment with standard errors are representative of four experiments of similar design.

There is thus, significant overlap of the Spl- and T3R complex-binding sites on the 36-bp EGFR DNA element in the region that is essential for promoter and enhancer function. Addition of T3, which increased mobility of the heterodimer complex slightly in EMSA (data not shown), did not change the G residues contacted (Fig. 1).

Competition of Spl and TJ3 - RXR for Binding to the EGFR Promoter Element-The finding that DNA-binding sites for Spl and the T3R. RXR complex extensively overlapped suggested that the two proteins would compete for binding to the 36-bp EGFR promoter fragment. This hypothesis was directly tested in uitro using avidin .biotin complex DNA-binding assays. Binding of T3R to the 36-bp element was stimulated 20-fold by addition of a partially purified fraction from HeLa nuclei that contains RXRa (11). Increasing amounts of pure Spl progressively decreased the amount of '251-labeled Tf.

16068 T& RXR, and Spl Regulation of EGF Receptor Promoter

T3R.RXR complex that bound to the 36-bp element (Fig. 3). Based on a KO of 0.9 nM for T3R.RXR complex binding to the 36-bp EGFR promoter element (9) and a molecular mass for Spl of 100 kDa (45), a Kr of 3.2 nM for Spl was calculated from two separate experiments using varying amounts of "'1. T3. T3R.RXR. The affinities of the two proteins for the EGFR 36-bp element are similar. The Kr is less than the KO for Spl binding to a canonical Spl site contained in a 155-bp restriction fragment but is comparable to the KD for binding to the same consensus site in a 30-mer oligonucleotide (46). Binding of either "'I.T3.T3R or "'I.T3.T3R-RXR to the palindromic T3 response element was not inhibited by Spl, indicating that the inhibition was specific to the EGFR promoter element. Similar results were obtained using 35S-labeled T3R synthesized in uitro (data not shown). These results indicate that Spl and the T3R. RXR complex compete for binding to overlapping DNA sites in the EGFR promoter.

Regulation of EGFR Promoter Function by S p l and T$ in Vivo-To examine the role of Spl in transcription directed by the EGFR promoter, extended (-1100 to -19 and -485 to -19 bp) and proximal (-153 to -19 bp) fragments of the human EGFR gene 5' region were inserted into the luciferase reporter vector and transfected into Drosophila Schneider SL2 cells which are deficient in Spl (25). An Spl expression plasmid under control of the actin 5C promoter was cotransfected. Expression directed by each of the EGFR 5' sequences was strongly stimulated by Spl (Fig. 4A). The activity of the extended promoters that contain multiple Spl-binding sites was stimulated 97-132-fold by Spl. Although the proximal promoter fragment contains only a single Spl site (2), its activity was stimulated 18-fold. Changing GGG to TTT at residues -97 to -95 in the -153 to -19-bp proximal promoter within the Spl footprint reduced Spl-dependent activity by -75% but did not abolish it. These results provide evidence that the expression of EGFR is strongly dependent on Spl and are in agreement with the proposed role for Spl to sequester the transcription apparatus on TATA-less promoters (27, 28).

The observations that Spl- and T3R-binding sites are overlapping in the proximal portion of the EGFR promoter and Spl and T3R. RXR competed for binding to the 36-bp element in uitro suggested that the in vivo inhibitory effects of T3R (9) are due to interference with Spl function. This hypothesis was tested by cotransfection of Spl and T3R expression plasmid constructions which both use the actin 5C promoter. Fig. 4B shows ligand-dependent inhibition of Spl-stimulated transcription of the proximal EGFR promoter luciferase construction by T3R. T3 alone had no significant effect, and T3R

1

-9.0 -8 5 -8 0 -7.5

J-ogIsP~l FIG. 3. Inhibition of binding of the TsR.RXR complex to

the EGFR promoter by Spl. lZ5I-Labeled T3, TaR, and RXR were incubated at 23 "C for 10 rnin with the indicated amounts of purified Spl. Biotinylated DNA was added and protein-DNA complexes isolated on strepavidin beads. Background obtained in the absence of biotinylated DNA was subtracted from all data points. 100% binding to the EGFR 36-bp DNA and to the TREpal DNA equaled 5.4 and 8.6 fmol of '261-T3, respectively.

in the absence of ligand did not decrease Spl-stimulated reporter gene expression. Addition of T3 in the presence of T3R resulted in a significant decrease in Spl-stimulated transcription. Near maximal inhibitory effects were observed in the presence of T3 with a ratio of pPacT3R.pPacSpl of 1. Increasing amounts of pPacT3R to a 4-fold excess over pPacSpl resulted in little further inhibition suggesting that a factor other than T3R was limiting. Cotransfection of RXR in the same expression vector, which did not affect expression alone, resulted in further T3-dependent repression. Addition of 9-cis RA, a ligand for RXR (47), had a small effect alone and augmented T3-dependent repression in the presence of both TZR and RXR. In the presence of a 4-fold excess of T3R and RXR, T3 plus 9 4 s RA resulted in -85% inhibition of Spl-stimulated EGFR promoter function (Fig. 4B, right). The in vivo requirement for RXR parallels in uitro DNA binding studies which demonstrate enhanced binding of TSR. RXR heterodimers to the EGFR promoter element (11).

To investigate the requirement for DNA binding in the inhibitory effects of T3R, a mutant T3R, T3RADBD, which lacks the DNA binding domain (9), was placed in the pPac vector and cotransfected with Spl. As shown in Fig. 4C, T3RADBD had no effect on Spl-stimulated transcription from the EGFR promoter. These results indicate a requirement for DNA binding and argue against a squelching mechanism (48). The requirement for T3 was investigated using T3RAC, a COOH-terminal truncation mutant that is defective in T3 binding and dimerization (35) and T3RS, a mutant lacking Thr332 that is defective in T3 binding (36). Neither T3RAC nor T3RS inhibited Spl-stimulated EGFR promoter activity confirming ligand-dependent inhibition. T3R alone, without or with T3, or with RXR did not affect transcription from the EGFR promoter indicating that T3R cannot substi- tute for Spl (Fig. 4B). These results indicate that ligand- activated T3R inhibits EGFR promoter expression by interference with the action of Spl.

Effects of Spl and T3R on the Enhancer Function of the 36- bp EGFR 5' Element-When assayed in HeLa cells the EGFR 36-bp proximal promoter element functions as a strong enhancer. When placed 5' to a Herpes simplex virus thymidine kinase promoter that contains only a TATA box (mTK), a single copy of the 36-bp element in the forward orientation enhanced mTK activity 50-fold, and two copies enhanced promoter activity 110-fold (data not shown). The 36-bp element similarly enhanced activity of a minimal TATA-containing promoter derived from the rat prolactin gene; in both contexts the 36-bp element was active in both forward and reverse orientations. Primer extension verified that transcription initiated from the thymidine kinase promoter confirming that the 36-bp element functioned as an enhancer and not as a second promoter (data not shown). To determine whether the enhancer function of the 36-bp EGFR element was regu- lated by Spl, activity was measured in SL2 cells. Fig. 5A shows that enhancer function of the 36-bp EGFR element was strongly stimulated by Spl. Mutations M3 and M4 that alter the central CCCGCC sequence abolished the ability of this element to function as an Spl-stimulated enhancer while mutations placed outside the Spl footprint did not affect Spl- stimulated activity (MI is shown). Cotransfection of the T3R expression plasmid inhibited Spl-stimulated enhancer activity (Fig. 5B). Inhibition was -50% at a 21 ratio of T3R to Spl expression plasmids. Addition of T3 gave an additional 10% inhibitory effect, but dependence on ligand was less than in the promoter context. Cotransfection of the RXR expression plasmid with Spl resulted in a small inhibitory effect in the presence of 9-cis RA. Cotransfection of T3R and RXR

T3R, RXR, and Sp l Regulation of EGF Receptor Promoter 16069

SPl - + - + - + - + EGFRP 5' element -1 100 to -19 -485 to -19 -153 to -19 -153 to -19. rnui

25

L-T3 I

" + - + - + + + 94s RA Spl (ng DNA) 0 10 10 T3R(ng DNA) 0 20 20 0 10102040 RXR(ng DNA) 0 20 20 0 0

" _ " " "

" + + - + - + - + - +

10 10 20 40 20 40

J

and treatment with both T3 and 9-cis RA resulted in stronger inhibition with an -75% decrease in Spl-stimulated activity. The inhibitory effect of T3R required both the DNA- and ligand-binding domains of the receptor. As shown in Fig. 5B T3RADBD and T3RAC did not suppress Spl-stimulated activity. T3RS, which can form dimers (36), was inhibitory (data not shown), supporting the lack of ligand dependence in the enhancer context. In the absence of Spl, T3R and RXR had no significant effect, confirming that T3R and T3R. RXR do not stimulate EGFR promoter or enhancer activity.

To investigate whether the opposing effects of Spl and T3R in vivo reflect competition for binding to the overlapping sequences in the EGFR promoter that occurs i n uitro, several additional mutations were made in the 36-bp element in a r attempt to separate effects of T3R and Spl. Mutant 36-bp elements were placed in front of the mTK promoter and the effects of Spl and T3R assayed in cotransfection experiments using SL2 cells. Deletions into the 5' end of the Spl footprint which overlaps the binding of the T3R.RXR complex, abolished Spl-stimulated activity (Fig. 6). Several base substitutions in the 5' end of the T3R.RXR-binding site, which did not interfere with Spl-stimulated activity, failed to abolish inhibitory effects of T3R. Three copies of an agtc to gggt substitution, which exhibited strong (32-fold) stimulation by Spl , were also strongly inhibited (by 70%) by T3R. One mutation (ACCG) significantly impaired both Spl and T3R responses.

All mutations which retained the ability to be inhibited by T3R also bound T3R as assayed by EMSA (Fig. 6C). T,R. RXR heterodimers bound to the 32P-labeled 36-bp element were competed by unlabeled wild-type or mutant probes. Effective competition by 10-ng probe was seen with all probes tested except the M3 mutant which also disrupts activation by Spl. It was thus not possible to functionally separate Sp l and T3R effects by DNA mutations due to extensive overlap of their binding sites.

Effects of Creating a Dimeric TRE in the EGFR Promoter- To further analyze the interactions between Spl and T3R in uiuo, the 36-bp element was mutated to create a dimeric TRE. Because the identified half-site in the 36-bp element is homologous to half of the rat growth hormone imperfect palindrome TRE (16, 17), the 36-bp element was changed to contain the dimeric inverted repeat rGH sequence with the second half located 5' to the T3R. RXR footprint (Fig. 7, bottom). This mutation does not affect the core Spl-binding site. The mutant element was fused to mTK-luciferase, and activity was assayed in SL2 cells. Whereas T3R without or with T3 had no effect on the WT 36-bp element, the mutant element functioned as a T3-responsive enhancer (Fig. 7). This effect was specific for W T T3R and was not observed with the inactive mutant T3RAC. Sp l still stimulated activity of the mutant element. Cotransfection of increasing amounts of the T3R expression plasmid resulted in progressive and complete inhibition of Spl-stimulated activity. Addition of T3 reversed the inhibitory effects of T3R. These results indicate that T3R activates transcription through the inverted repeat element. In contrast T3R cannot function as a ligand-dependent acti-

1 pg of the -153- to -19-bp EGFR promoter-luciferase reporter plasmid, 1 pg of pADH@gal, and 10 ng of pPacSpl without or with 20 ng of the indicated mutant T3R. ALIBD = T3RA100-171 defective in DNA binding; S = T3RS with deletion of Thr332; AC=T,RAC trun- cated at residue 335 in the COOH terminus. Triplicate wells of transfected cells were used for each data point; where indicated, cells were treated with 100 nM L-T3. Activity of the reporter plasmid with pPacSpl and without L-T3 was set to 100, which equaled 7.5 X lo8 light units of luciferase activity/OD414 unit of 0-galactosidase activity min".

16070 Ta, RXR, and Spl Regulation of EGF Receptor Promoter

I A. I

Spl - - + - + - + - + - + 36 bp - element 2xWT 2xM1 3xM3 4xM4

L-T3 " + - + 9.~18 RA

SPl + T3R 0 + + 0 RXR

T

- +

+ +

"

+ + 0 +

- + - + + +

+

- + - +

+ + + ADBD AC +

FIG. 5. Effects of Spl and TsR on EGFR enhancer function in vivo. A, selective activation by Spl of wild-type and mutant 36- bp EGFR gene 5' elements. The indicated number of wild-type or mutant 36-bp elements were inserted into the mTK-LUC reporter plasmid in the forward orientation. Quadruplicate 60-mm dishes of SL2 cells were transfected for each data point. For each dish 2.5 pg of the indicated reporter plasmid, 2.5 pg of pADHj3gal,5 pg of pUCl8 DNA without or with 10 ng of pPacSpl were used. Activity of mTK- LUC without Spl was set to 1, which equaled 1.3 X IO' light units of luciferase a c t i ~ i t y / O D ~ ~ ~ unit of 0-galactosidase.min-'. Spl-dependent increases using 2xWT and 2xM1 were significant at p < 0.001. B, T3R inhibition of Spl-stimulated luciferase expression from 2xWT. mTK-LUC. In 12-well dishes, each well of SL2 cells was transfected with 0.33 pg of reporter plasmid, 1 pg of pADHBgal, and 0.67 pg of pUC18, without or with 3.3 ng of pPacSpl expression plasmid and without or with 6.7 ng of pPacT3R, pPacT3RADBD, or pPacT3RAC. Where indicated, 6.7 ng of pPacRXR was added. Triplicate wells of cells were treated without or with 100 nM L-T3 and/or 100 nM 9-cis RA. Activity of the reporter plasmid alone was set to 1, which equaled 3.3 X 10' light units of luciferase a c t i ~ i t y / O D ~ ~ ~ unit of @-galactosidase activity. min".

vator on the site present in the WT 36-bp element. In the absence of ligand, binding of T3R to both the mutant and native sites resulted in inhibition of Spl-stimulated activity.

Respmses to T3 and Spl in Other Promoter Contexts-A positive TRE from the myosin heavy chain (MHC) gene was placed 5' to an extended thymidine kinase promoter that contains two Spl-binding sites (12,49). Fig. 8 shows that in the presence of T3R, T3 stimulated activity of the MHC-TK promoter 6-fold in SL2 cells. Spl stimulated activity 3-fold in agreement with previous reports (25). Expression of both T3R and Spl increased activity 27-fold in the presence of T3.

A. -112 GCGTCCGCCCMGTCCCCQCCTCQCCQCCAACGCCA -77 ......

B. Mutation +/- SQl +/- T3R

9 . 2 2 . 8 0 . 9 0 .9 1 . 0 1 . 0 5 . 1

19 .1 5.4

32.4 0.7

0 . 6 1 . 4 nt nt nt nt 0.7 0 .7 0 .4 0 .3 1 . 3

FIG. 6. Activity of Spl and TsR on mutant 30-bp EGFR gene 5' elements. A, wild-type 36-bp element sequence and base- pair substitution and deletion mutations. Dots indicate base pairs involved in deletions. Lower case letters indicate substitutions. Ouer- l ine indicates Spl-binding site; underline indicates T3R complex- binding site. B. activity of the mutant 36-bp EGFR 5' element. The indicated number of copies of the 36-bp mutations were inserted into mTK-LUC. Four 60-mm dishes of SL2 cells were transfected with 10 pg of the indicated reporter plasmid, 10 pg of pADHBga1, 20 pg of pUC18, without (-) or with (+) 40 ng of pPacSpl, and without (-) or with (+) 80 ng of pPacT3R. Analysis of 3XM2 used 80 ng of pPacSpl and 160 ng of pPacT3R. Half of the dishes were treated with 100 nM L-T3. The average of duplicates is shown. +Spl , ratio of activity with Spl compared to activity without Spl. 22'8, ratio of activity with Spl and T3.T3R compared to activity with Spl alone. nt, not tested. C, competition of T3R. RXR binding to 32P-labeled 36- bp element by wild-type ( WT) or mutant-unlabeled probes by EMSA. Either 2 or 10 ng of unlabeled competitor was added per reaction. Only the portion of the gel with the bound heterodimer is shown.

Mutant T3R defective in DNA or ligand binding were inactive either alone or in the presence of Spl. These results indicate that when T3R- and Spl-binding sites are non-overlapping, Spl and ligand-activated T3R synergistically enhance promoter activity.

In contrast ligand-activated T3R inhibited Spl-stimulated activity of the SV40 promoter (Fig. 9). In SL2 cells expression of an SV40 promoter-luciferase construction was strongly dependent on Spl which increased activity over 200-fold upon cotransfection with pPacSpl (data not shown). Ligand-activated T3R decreased this strong Spl-enhanced activity by -30%, and RXR increased the extent of inhibition to -65%. This effect was specific for active T3R with mutant T3R showing no effect. Examination of the six GC boxes present in the SV40 promoter sequence (50) revealed that at least one of these, box 6, GGGCGGGACT, contains a potential overlapping TRE half-site.

DISCUSSION

Transcriptional repression can occur through several mech- anisms, including competition for DNA-binding sites, heter- odimerization of active proteins with inactive forms, and competition for limiting transactivating proteins (reviewed in

TSR, RXR, and S p l Regulation of EGF Receptor Promoter 16071

U 5. 5 .- .- U

2 Q > m Q

.- L - a

S-, I 9 , I

0

7 4

7 0 r

3 s 5 2 4 u 3

.- .- > 4 L

2

1

0 1

0

L-T3 - + - + - + - + - + - + - + - + - + - i

Spl(ngDNA) 0 0 0 0 0 10 10 10 10 10

T3R(ngDNA) 0 5 10 20 0 0 5 10 20 0 T3FlAC(ng DNA) 0 0 0 0 20 0 0 0 0 20

-112 GCGTCCGCCCGAGTCCCCGCCTCGCC... ... TgaGgtCacGTCCCCGCCTCGCC ... FIG. 7. Effects of Spl and TsR on the EGFR promoter con-

taining a rat growth hormone TRE mutation. Oligonucleotides were designed to create a dimeric TRE without disturbing the Spl- binding site in the 36-bp element. The mutated sequence is shown below the EGFR wild-type sequence with base substitutions designed to resemble the rat growth hormone TRE shown in lower case. The ouerbar marks the Spl footprint. Two copies of this construction were inserted into mTK-LUC to generate a reporter plasmid. Each 60-mm dish of SL2 cells was transfected with 2.5 pg of this reporter plasmid, 2.5 pg of pADH@gal, 5 Fg of pUC18, and varying amounts of the pPacSpl, pPacTsR, and pPacT3RAC. Triplicate dishes of cells were treated without or with 100 nM L-T3. Activity of the reporter plasmid alone was set to 1, which equaled 1.6 X 10’ light units of luciferase ac t i~ i ty /OD~~~ units of @-galactosidase activity. min”.

L-T3 . + . + - + . + . + . + . +

SPl o o + + + + + T3R 0 WT 0 WT ADBD S AC

FIG. 8. Synergistic effects of Spl and TsR on the MHC-TK- LUC. Each well of SL2 cells was transfected with 1 pg of the reporter plasmid MHC-TK-LUC, 1 pg of pADHpgal, 0 or 5 ng pPacSpl, and 0 or 10 ng of pPacT3R, pPacT,RADBD, pPacT,RS, or pPacT3RAC as indicated. Triplicate wells of cells were treated without or with 100 nM L-T3. Activity of the reporter plasmid alone was set to 1, which equaled 1.5 X lo7 light units of luciferase ac t i~ i ty /OD~~~ units of p- galactosidase activity. rnin”.

Refs. 51, 52). Complex interactions involving more than one of these principles may also occur a t “composite” DNA response elements (53). Analysis of the inhibitory effect of ligand-activated T3R on the EGFR promoter indicates that repression results from interference with Spl. Because S p l is required for basal transcription from GC promoters that lack a TATA box (27, 28), interference with the activity of Sp l provides an effective mechanism for repression of basal transcription. The primary mechanism appears to involve competition for DNA-binding sites.

The binding site for the T3R.RXR complex in the EGFR promoter overlaps the proximal Spl-binding site. Both sites

OSIs RA L-T3 - + - + - + . + - + - +

+ SPl T3R RXR

+ + + + + + - WT W ADBD S AC

FIG. 9. Effects of Spl and TsR on the SV40 promoter. Each well of SL2 cells was transfected with 0.17 pg of the lucerifase reporter plasmid pSLuc2 (31) which contains the SV40 promoter, 1 pg of pADHpga1, 0.83 pg of pUC18,3.3 ng of pPacSpl, without or with 6.7 ng of pPacT3R, pPacT3RADBD, pPacTaRS, or pPacT3RAC, and without or with 6.7 ng of pPacRXR. Triplicate wells of cells were treated without or with 100 nM L-T3 and/or 100 nM 9-cis RA where indicated, Activity was defined as light units of luciferase activity/ OD414 units of @-galactosidase activity. min”.

are contained in a 36-bp sequence that can function autono- mously as an Spl-dependent promoter or enhancer. Spl competes with T3R. RXR for high affinity binding to this DNA element. Formation of a non-DNA-binding complex between T3R. RXR and Spl appears unlikely because Sp l failed to inhibit T3R homodimer or T3R. RXR heterodimer binding to a palindromic TRE. Drosophila SL2 cells which lack Spl and T3R (24, 25) were used to assess interactions in uiuo. T3R inhibition of Spl-dependent transcription from the EGFR promoter element confirmed T3R interference with Sp l action. RXR increased the extent of inhibition in vivo in agreement with the effect of RXR to enhance T3R binding to the EGFR promoter in vitro. Additionally, mutant T3Rs which lack a DNA-binding domain were inert.

Because the T3R- and Spl-binding sites overlap extensively it was not possible to selectively abolish T3R activity. Thirteen mutations were introduced into the 36-bp EGFR promoter region. These mutations either abrogated Spl-stimulated reporter gene expression or failed to affect either Sp l stimulation or T3R repression. In other promoters binding to adjacent but non-overlapping DNA-binding sites can inhibit the activity of Spl. In all genital papilloma viruses, an Spl consensus motif is spaced by a single nucleotide from the E2-binding site distal to the TATA box (54). E2 protein competes effi- ciently with S p l for binding in vitro and intact E2 bindings sites are necessary for negative transcriptional regulation by E2 in vivo (54). Binding of factors to adjacent but non- overlapping sites similarly inhibits the activity of Spl on the human growth hormone and low density lipoprotein receptor genes (55, 56). The present study indicates that ligand-activated T3R can also interfere with Sp l function in other promoter contexts. The SV40 promoter that contains six GC boxes is strongly stimulated by Spl. Ligand-activated T3R inhibited S p l stimulation of this promoter which contains at least one potential T3R-binding half-site that overlaps with Spl-binding sites. Because Sp l does not appear to act synergistically on the SV40 promoter (57, 58), interference with one Spl-binding site is expected to give incomplete repression. Repression by T3R on this promoter was ligand-dependent, specific for an intact T3R, and enhanced by RXR.

Interestingly, high affinity binding of TsR to the EGFR promoter which is strongly dependent on RXR in uitro, maps

16072 T,R, RXR, and Spl Regulation of EGF Receptor Promoter

to a single half-site (11). Although RXRs are Zn2+-finger motif DNA-binding proteins which occupy half-sites of dimeric positive hormone response elements as heterodimers with T3R, RAR, and vitamin D receptor (12-15), methylation interference footprinting revealed interaction of the hetero- dimeric T3R. RXR with G residues in only a half-site in the EGFR promoter. This does not exclude binding to adjacent bases, and the strong stimulation of T3R binding by RXR suggests that there is a binding site for RXR not detected by methylation interference. RXR half-sites may be GC-rich, detected poorly by methylation interference, and located up- stream of T3R half-sites (62). When the 36-bp EGFR promoter element containing this site was analyzed in an enhancer context in front of a TATA box, ligand-activated T3R failed to stimulate transcription. Mutation of the 36-bp element to create a dimeric palindromic TRE resulted in a 6- fold increase in reporter gene transcription in response to ligand-activated T3R. These results suggest that, when bound to the EGFR promoter site, T3R is in a conformation that is inactive. When bound to the palindromic site T3R is able to function as a positive transactivator. Because partial effects of T3R occurred in SL2 cells without exogenous RXR, the nature of the stimulatory and inhibitory complexes formed in uiuo remains unknown. T3R may form homodimers which have decreased binding ability relative to heterodimers or the Drosophila homolog of RXR, ultraspiracle (59), may form heterodimers with T3R in SL2 cells. Either the amount of ultraspiracle is limiting or interactions with T,R are weak. hRXR enhanced repression of both promoter and enhancer functions of the EGFR promoter element by T3R in SL2 cells.

Both spacing and orientation between the core DNA-binding motifs of dimeric sites determine receptor selectivity and transcriptional responses (22, 60). T3R gave a positive transactivation response on a palindromic mutant rat growth hormone TRE but this was lost when a 3-bp spacer was inserted (17). Without spacing, a direct repeat corresponding to a negative TRE in the mouse TSHP gene conferred transactivation by TsR in the absence of T3 but repression in the presence of T3 (22). On the EGFR promoter-enhancer T3R alone was inactive in both the presence and absence of T3 indicating the binding site does not direct transactivation analogous to the palindrome with 3 bp spacing (17). The inhibitory effect was seen only as inhibition of Spl-stimulated transcription.

On the mutant palindromic TRE in the EGFR promoter where the 3' half overlaps the Spl-binding site, unliganded T3R strongly inhibited Spl stimulation of EGFR promoter- enhancer function. Addition of T3 restored activation by TBR that was not additive with Spl. When positive elements are overlapping, either T3R or Spl can enhance transcription but the two do not act together. In contrast, when dimeric TRE and Spl sites are separated as in the MHC-extended TK- LUC construction, strong positive synergism between Spl- and ligand-activated T3R was observed. The arrangement of T3R- and Spl-binding sites will thus determine their activities on transcription from TATA promoters. In the GC-rich EGFR promoter, which lacks a TATA box, inhibition of Spl function is equivalent to interference with basal transcription which depends on Spl for assembly of the transcription complex.

Both cell surface growth factor receptors and nuclear hormone receptors play important roles in growth and develop- ment. Interplay between these two classes of receptors provides for a complex level of control. There is evidence that suggests in uiuo regulation of EGFR expression by TBR. In rats EGFR mRNA levels are low during fetal life and during

the first week of postnatal life and then begin to rise. Treat- ment with L-T4 during the first 6 days of postnatal life decreased EGFR mRNA levels at day 7 -5-fold (61). These findings are compatible with the mechanism described in this study. In tissues that contain multiple cell types, responses will depend on the concentration of receptors, of transcription factors, of hormones, and on the organization of binding sites for regulatory factors in particular genes.

Acknowledgments-We thank Drs. Ron Evans, Christopher Glass, Erica Pasquale, Robert Tjian, and Bruce Weintraub for generous gifts of plasmids and Richard Heyman for 9-cis RA.

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