Vol. 11, No. 9MOLECULAR AND CELLULAR BIOLOGY, Sept. 1991, p. 4771-47780270-7306/91/094771-08$02.00/0Copyright © 1991, American Society for Microbiology

Retinoic Acid-Induced Down-Regulation of the Interleukin-2Promoter via cis-Regulatory Sequences

Containing an Octamer MotifMARIA P. FELLI,' ALESSANDRA VACCA,' DANIELA MECO,1 ISABELLA SCREPANTI,12

ANTONIETTA R. FARINA,3 MARELLA MARODER,1 STEFANO MARTINOTTI,3ELISA PETRANGELI,4 LUIGI FRATI,1 AND ALBERTO GULINOl.3*

Department of Experimental Medicine, University of L'Aquila, L'Aquila, and Department of Experimental Medicine'and National Cancer Institute Genoa Biotechnology Section,2 University La Sapienza, and

Consiglio Nazionale delle Ricerche Biomedical Technology Institute,4 Rome, Italy

Received 22 March 1991/Accepted 24 June 1991

Retinoic acid (RA) is known to influence the proliferation and differentiation of a wide variety of transformedand developing cells. We found that RA and the specific RA receptor (RAR) ligand Ch55 inhibited the phorbolester and calcium ionophore-induced expression of the T-cell growth factor interleukin-2 (IL-2) gene.Expression of transiently transfected chloramphenicol acetyltransferase vectors containing the 5'-flankingregion of the IL-2 gene was also inhibited by RA. RA-induced down-regulation of the IL-2 enhancer is mediatedby RAR, since overexpression of transfected RARs increased RA sensitivity of the IL-2 promoter. Functionalanalysis of chloramphenicol acetyltransferase vectors containing either internal deletion mutants of the regionfrom -317 to +47 bp of the IL-2 enhancer or multimerized cis-regulatory elements showed that theRA-responsive element in the IL-2 promoter mapped to sequences containing an octamer motif. RAR alsoinhibited the transcriptional activity of the octamer motif of the immunoglobulin heavy chain enhancer. In spiteof the transcriptional inhibition of the IL-2 octamer motif, RA did not decrease the in vitro DNA-bindingcapability of octamer-1 protein. These results identify a regulatory pathway within the IL-2 promoter whichinvolves the octamer motif and RAR.

Retinoic acid (RA) plays crucial roles in the control ofnormal- and transformed-cell proliferation and differentia-tion and in the regulation of morphogenic potential duringembryonic development (6, 7, 42, 53). The molecular mech-anisms by which RA influences these processes are stillpoorly understood. However, following the interaction withspecific receptors belonging to the erbA oncogene-relatedsteroid-thyroid hormone receptor superfamily of nucleartranscription factors (reviewed in reference 11), RA has beenreported to influence cell genetic programs such as de-creased expression of proto-oncogenes and growth factors(i.e., c-myc, transforming growth factor a, k fibroblastgrowth factor, and b fibroblast growth factor) (9, 33). Thesechanges are often associated with RA-induced differentiationof malignant cells and correlate with a decrease in theirproliferative potential.RA has also been noted to affect the development of the

immune system, since thymic hypoplasia and subsequentimpairment of T-cell function have been observed in humansand in animal models after prenatal exposure to RA (14, 30,49). Among the multiple factors controlling the differentia-tion and proliferative processes of T cells, the T-cell growthfactor interleukin-2 (IL-2) plays a key role. Activation,differentiation, and growth of peripheral T lymphocytes aswell as of other lymphoid cells are controlled mainly by IL-2(23, 34, 35). Deregulated IL-2 production is involved inmaintaining the growth and the tumorigenicity of trans-formed T cells (17, 40, 59). IL-2 also plays an important rolein enhancing early thymocyte development, and it is ex-pressed by thymocytes early during embryonic ontogeny

* Corresponding author.

(10, 56). IL-2 gene expression is subjected to a cell-specificand developmentally regulated transcriptional control whichis accounted for by the cooperation of multiple molecularpathways resulting in the activation of the IL-2 gene pro-moter. We recently described an interference with such IL-2promoter-activating pathways by the glucocorticoid receptor(58), another member of the steroid-thyroid-RA receptorsuperfamily. To address the question of whether IL-2 generegulatory activity could be shared by members of thisreceptor family, we studied the effect of RA on IL-2 geneexpression and delineated the RA-responsive elements in theIL-2 promoter. In this work we report that RA decreases theIL-2 mRNA levels and transcriptional activity of the IL-2promoter in Jurkat T cells via a process that involves RAreceptor (RAR). Furthermore, we report that RA action isdirected through an IL-2 promoter region containing anoctamer motif. Thus, we identified a regulatory pathwaywithin the IL-2 promoter which involves two development-related elements, the octamer motif and RAR.

MATERIALS AND METHODS

Plasmids. Plasmid pIL2CAT contains the IL-2-flankingregion from -575 to +47 bp driving the expression of thechloramphenicol acetyltransferase (CAT) gene (51) and wasprovided by N. J. Holbrook. Plasmid p[-317/+47]IL2CATand the internal deletion mutants of the IL-2 enhancer-CATvectors represented in Fig. 4 (generous gifts of G. R.Crabtree) have been described elsewhere (18). Plasmidsp[-575]TK-CAT, pNFAT-TK-CAT, and pOct-TK-CATwere constructed by inserting into the BamHI site ofpBLCAT2 vector (31) (including the fragment at -105 bp ofthe thymidine kinase [TK] promoter, missing the octamer

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motif, and driving CAT gene expression) one copy of theIL-2 enhancer fragment at -575 to +47 bp in the antisenseorientation (p[-575]TK-CAT), three copies of the IL-2nuclear factor of activated T cells (NFAT) site (the fragmentat -294 to 261 bp of the IL-2 enhancer) (pNFAT-TK-CAT),and four copies of the proximal IL-2-octamer motif (thefragment at -96 to -66 bp) (pOct-TK-CAT), as syntheticoligonucleotides including a BamHI and a BglII restrictionsite at the 5' and 3' ends, respectively. Plasmid pBS-CAT-Octa (provided by J. R. Nevins) contains the octamer motifof the immunoglobulin heavy chain enhancer (IgH) drivingthe CAT gene through the enhancerless simian virus 40(SV40) early promoter (12). pAP-1-TK-CAT contains fivecopies of the collagenase AP-1 site in front of the TKpromoter (4) (a gift of M. Karin). Plasmid tk-TRE2p-CAT(provided by K. Umesono) contains the RA-responsiveelement driving the CAT gene through the TK promoter (57).Plasmid pSV2CAT (21) contains the SV40 early promoterand enhancer elements linked to the CAT gene. PlasmidshRARa, hRAR,3, and hRAR-y (kindly provided by P. Cham-bon) contain the cDNA coding for the human RARs underthe transcriptional control of the SV40 early promoter invector pSG5 (5, 29, 38). Plasmid pCH110 (Pharmacia, Upp-sala, Sweden) contains a functional lacZ gene, coding for,-galactosidase, under the transcriptional control of theSV40 early promoter.

Cell culture and DNA transfections. The human JurkatT-cell line (clone T77) was cultured in RPMI 1640 mediumsupplemented with 10% fetal calf serum and antibiotics(Flow Laboratories, Airshire, Scotland). Cells were treatedwith various concentrations of RA or Ch55 (kindly providedby K. Shudo) or 30 ng of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) per ml, 1 ,ug of A23187 (Sigma Chemicals, St.Louis, Mo.) per ml, or 1.5 ,ug of ionomycin (Calbiochem,San Diego, Calif.) per ml.

Transfections of lymphoid cells were carried out by theDEAE dextran method (39) as described previously (58).Cells were cotransfected with various plasmids together withpCH110 (as an internal control for transfection efficiency).Twenty-four hours after transfection, cells were treated withthe drugs indicated above, and after a further 24 h, cells wereharvested and protein extracts prepared for the CAT and3-galactosidase assays.RNA isolation and analysis. For RNA extraction, cells

were harvested and washed in ice-cold phosphate-bufferedsaline and RNA was isolated as described elsewhere (13).For Northern (RNA) blot analysis, 20 pg of RNA wasfractionated by formaldehyde gel electrophoresis and trans-ferred to Hybond-N (Amersham) filters. Filters were hybrid-ized with 32P-labeled probes in 50% formamide-5 x SSC (1 xSSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.05 Msodium phosphate (pH 7.2)-S5 x Denhardt's solution-0.2%sodium dodecyl sulfate (SDS)-10% dextran sulfate-0. 1 mg ofdenatured salmon sperm DNA per ml at 42°C for 24 h andthen washed in 2x SSC-0.5% SDS at room temperature and0.1 x SSC-0.5% SDS at 500C.CAT assay. The CAT assay was carried out as previously

described (21) by incubating 50 to 100 ,ug of cell lysateprotein with 0.1 ,uCi of ['4C]chloramphenicol (60 mCi/mmol;Amersham) in the presence of 9 mM acetyl coenzyme A(Sigma Chemicals) for different times at 370C. Acetylatedand unacetylated chloramphenicol were separated by thinlayer chromatography, and acetylation was quantified byautoradiography and liquid scintillation counting.

P-Galactosidase assay. A 20- to 30-,ug sample of the samecell lysate prepared for analysis of CAT activity was diluted

A) B)10

1L2

0.9kb- *i *

act in

RA (M) 0 0 O-8- 10

-< c

Z

E6---zJ *

n) 0 1 2 4TPACa - + + + * + + + hou rs

TPA+Ca+FIG. 1. Inhibition of IL-2 gene expression by RA. Jurkat cells

were treated with 30 ng of TPA per ml and 1 ,ug of A23187 per ml inthe absence (-) or in the presence (0) of the indicated amount ofRAfor 7 h (A) or 106 M RA for the times indicated (B). Isolated RNAwas analyzed by Northern blotting and hybridized with a random-primed 32P-labeled human IL-2 cDNA probe and, after stripping,,-actin probe.

in 100 mM NaPO4-10 mM KCl-1 mM Mg2SO4-50 mM3-mercaptoethanol, pH 7.0, and ,B-galactosidase activity wasdetermined spectrophotometrically at 420 nm by the hydro-lysis of o-nitrophenol-p-D-galactoside.

Gel mobility shift assays. Cells were lysed in 10 mMHEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid) (pH 7.9)-1.5 mM MgCl2-10 mM KCl-0.25 mM dithio-threitol-0.5 mM phenylmethylsulfonyl fluoride, and nucleiwere spun at 800 x g and extracted in 20 mM HEPES (pH7.9)-20% glycerol-0.42 M NaCl-0.2 mM EDTA-1.5 mMMgCl2-0.25 mM dithiothreitol-0.5 mM phenylmethylsulfo-nyl fluoride. Nuclear extract was cleared by centrifugation.Nuclear extracts (5 jig of protein) were incubated for 30 minat 20°C with 0.2 ng of the 32P-end-labeled fragment at -96 to-66 bp spanning the octamer motif of the IL-2 enhancer(5'-TTGAAAATATGTGTAATATGTAAAACATTT-3') in15 RI of a reaction buffer containing 2 jig of poly(dI-C), 20mM HEPES (pH 7.9), 20% glycerol, 0.2 mM EDTA, 0.25mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride,and 80 mM KCI, ± a 100-fold or 500-fold excess of specificor unspecific unlabeled oligonucleotide competitor, respec-tively. Complexes were separated on 4% polyacrylamidegels in 0.45 M Tris-borate-1 mM EDTA (pH 8.0) buffer.Specific and unspecific competitors were the same as theprobe and 5'-TCGAGACGAAAATCCACACTCATGAGTGTGCAACATATCG-3', respectively.

RESULTSRA inhibits IL-2 gene expression. The IL-2 gene is tran-

scriptionally activated by interaction of the antigen with theT-cell receptor, that generates several intracellular signalssuch as inositol 1,4,5-trisphosphate, calcium mobilization,and protein kinase C activation (2, 3). To study the interfer-ence of RA with intracellular events leading to IL-2 geneactivation, we bypassed T-cell receptor stimulation by treat-ing the human Jurkat T-cell line with the protein kinaseC-activating drug TPA and the calcium ionophore A23187 orionomycin. Figure 1 shows that RA treatment of Jurkat cellssignificantly decreases the expression of the IL-2 gene asearly as 2 h after TPA-A23187 cell treatment.RA inhibits transcription from the IL-2 promoter. IL-2

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1o0

*> 50-I-

0

A)RARa RAR.8 RARy

28S

4m

las-

IL2CAT

RA(M) 0 168lo- 10 6 10FIG. 2. Effect of RA on the transcriptional activity of the IL-2

enhancer. Four micrograms of pIL2CAT (IL-2 enhancer region at-575 to +47 bp) or pSV2CAT plus 1 ,ug of pCH110 (Pharmacia), asan internal control for transfection efficiency, was cotransfected intoJurkat cells. After 24 h, cells were stimulated with TPA and A23187or ionomycin without or with the indicated concentrations ofRA fora further 24 h. Cells were then harvested, and cell extracts assayedfor CAT or ,B-galactosidase as described in Materials and Methods.Results are expressed as the average (+ standard error from at leastfour experiments) percent CAT activity with respect to valuesobserved in RA-untreated, TPA-calcium ionophore-treated cells.CAT activities in RA-untreated cells were 34 + 5 and 2,530 ± 200pmollhlmg of protein (pIL2-CAT) and 400 + 50 and 2,687 ± 200pmol/h/mg of protein (pSV2CAT) without and with TPA-ionomycintreatment, respectively.

gene transcription is controlled mainly by cis-regulatorysequences located within 300 bp of the 5'-flanking region ofthe gene, that are responsible for its T-cell-specific antigenicstimulation (16). To define the IL-2 promoter sequencesresponsive to the modulatory activity of RA, we first usedthe pIL2CAT chimeric gene containing the region at -575 to+47 bp of the IL-2 gene driving the expression of the CATreporter gene (51). Following transient transfection ofpIL2CAT plus the SV40 promoter-o-galactosidase vector(pCH110) as an internal control for transfection efficiencyand for specificity, Jurkat cells were treated with TPA andcalcium ionophore in the presence or absence of RA. RAwas able to significantly inhibit the expression of pIL2CATinduced by TPA and calcium ionophore, but it did not affecteither pCH110 or pSV2CAT expression (Fig. 2).RA-induced down-regulation of the IL-2 promoter is medi-

ated by RAR. To study the role ofRAR in mediating the IL-2gene regulation by RA, we first investigated the presence ofRAR in Jurkat cells. Indeed, we detected the presence ofspecific RARa mRNA in Jurkat cells, while no RAR, norRAR-y mRNA was observed (Fig. 3A), in keeping withrecent observations of the presence of selected RAR tran-scripts in hematopoietic cells (15).We next studied the action of a bioactive benzoic acid

analog of RA, the compound Ch55, which does not bind tothe cellular RA-binding protein CRABP and has a higheraffinity for RAR than does RA (22). Ch55 was a morepowerful inhibitory agent of the TPA-ionomycin-inducedexpression of the IL-2 gene in Jurkat cells than was RA (Fig.3B).To further determine the role of RAR in regulating IL-2

gene transcription and to examine potential differences in thebiological activity of the three RARs cloned so far, wecotransfected Jurkat cells with expression vectors for humanRARa, RARP, and RARy and either p[-317/+47]IL2CATor tk-TRE2p-CAT containing two copies of the syntheticRA-responsive element TRE (thyroid/retinoic acid-respon-

C)

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tk-TRE2p CAT

B) RA Chss(HiM), 0.1TPACa - + + + +

.. ,*-0

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_+ -_+ _, -_+

PIL2 CAT

ag y - ad y

1zM 10yM

FIG. 3. RAR is involved in down-regulating IL-2 gene expres-sion. (A) Northern blot analysis of Jurkat cell RNA hybridized withcDNA probes of RARa, RAR,, and RARy (5, 29, 38). (B) Dot blotanalysis of 2, 1, and 0.5 jig of RNA isolated from Jurkat cells 7 hafter the treatments indicated and hybridized with a human IL-2cDNA probe. (C) pSG5 vectors expressing RARa, RARP, andRAR-y (2 jig each) were cotransfected with 4 p.g of tk-TRE2p-CATor 4 ,ug of pIL2CAT in Jurkat cells. After 24 h, cells were treatedwith TPA-ionomycin without or with 0.1 ,uM RA (tk-TRE2p-CAT)or the indicated concentrations of RA (pIL2CAT) for a further 24 h.Cell extracts were prepared 24 h later. The percent pIL2CATexpression relative to the CAT activity in TPA-ionomycin-treatedand RA-untreated cells is shown. TPA-ionophore-induced expres-sion of pIL2CAT in the absence of RA was 1,500 + 100, 1,750 +

150, 1,450 ± 140, and 1,570 ± 130 pmollh/mg of protein withoutvector and with RARa, RAR,B, and RARy expression vectors,respectively.

sive element) (57). Figure 3C shows that RA treatment ofJurkat cells increases the expression of tk-TRE2p-CAT,suggesting the presence of a functional RAR in these cells.Cotransfection of RARa, RARP, and RARy expressionvectors (5, 29, 38) significantly increased tk-TRE2p-CATactivity after RA administration. No differences in enhanc-ing activity were observed among the three RARs. Cotrans-fection of RARa and RARP induced a RA-dependent inhi-bition of p[-317/+47]IL2CAT activity to a higher extentwith respect to RAR-untransfected cells, while RAR-y wasless active. Taken together, the inhibitory activity of theRAR-specific ligand Ch55 and the increased cell sensitivityto RA after complementation with an exogenous recombi-nant RAR suggest that RAR plays a role in mediating theRA-induced down-regulation of the IL-2 promoter.

Delineation of RA-responsive cis elements. The data re-ported above show that RA interferes with intracellularsignals activating the antigen-responsive and T-cell-specificcis-regulatory elements lying in the 5'-flanking region of IL-2gene. This region is composed of multiple cooperating cis-regulatory elements which have been characterized on thebasis of their ability (i) to direct reporter gene expressionwhen multimerized in front of heterologous promoters, (ii) todecrease, if mutated, the IL-2 enhancer activity, or (iii) tobind several transacting factors, such as AP-1 (- 185 to -179bp and -157 to -140 bp), AP-3 and NFKB (-207 to -187bp), octamer (-256 to -240 bp and -86 to -65 bp), and aless identified protein called NFAT (-289 to -263 bp) (8, 18,24, 27, 47, 48, 50). To further delineate the RA-responsive

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Cl

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-zzz__ -RA +RA

ZiH 100 23

c(-317/-286) L2CAT ±.

A(-2731-263) I L2CAT NIIt(-2551-217) IL2CAT

A(-2081-174) IL2CAT

A(-1601-150) IL 2CAT WJ1A(-116/-88) I L 2CAT .

J-4 100 26

20 6

46 33

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/(-821-73) IL 2CAT I 1 1

A(-591-45)IL2CAT A h 30 10FIG. 4. Delineation of RA-responsive elements by analysis of wild-type IL-2 enhancer (fragment from -317 to +47 bp) or internal deletion

mutants (18) activating transcription of the CAT gene. The indicated plasmids (4 ,ug) plus 1 ,ug of pCH110 were cotransfected into Jurkat cells.Further cell treatment was as described in the legend to Fig. 2. Results are expressed as the average (+ standard error) percent CAT activityrelative to values observed in RA-untreated, TPA-calcium ionophore-treated cells. The open and closed bars and numbers on the right givethe TPA-Ca2+-induced activity of the deletion mutant relative to the 100% p[-317/+47]IL2CAT enhancer wild-type fragment in untreated(-RA) and RA-treated (+RA) cells, respectively. p[-317/+47]IL2CAT activities in RA-untreated cells were 40 ± 8 and 2,350 + 300pmol/h/mg of protein without and with TPA-ionomycin treatment, respectively.

elements in the IL-2 promoter, we examined the effect ofRAtreatment on several deletion mutants of the IL-2 generegulatory region driving the expression of the CAT gene. Inagreement with results previously reported (18, 19), muta-tions of the NFAT, distal octamer, and proximal AP-1 motifssignificantly reduced the activity of the enhancer, leaving,however, a residual activity due to unmutated elements (Fig.4). Deletion of the proximal octamer motif almost abolishedCAT expression (Fig. 4). Impairment of the inhibitory effectof RA on the enhancer transcriptional activity as comparedwith that of the wild-type enhancer was observed mostly inthe mutant carrying a deletion of the distal octamer motif(Fig. 4).

pBLCAT2 -5W)1L2- N FAT.TK CAT-TK-CAT

To further define the targets of RA action, we studied theability of the drug to inhibit the TPA-Ca2" ionophore-induced enhancer activity of multimers of the NFAT site,the IL-2 octamer motif, and the AP-1 site driving CAT genetranscription (Fig. 5). RA was able to inhibit the TPA-ionomycin-induced activation of CAT gene expression bymultimers of the octamer motif, while it did not affectsignificantly the enhancer activity of the other sites (Fig. 5).No activity ofRA on the transcriptional activity of any of theconstructs tested in the absence of TPA and ionomycin wasobserved. As for the intact IL-2 enhancer (Fig. 3C), cotrans-fection of RARa expression vector increased the RA-depen-dent inhibition of the transcriptional activity of the IL-2

AP1l01lTK-CAT OCT.TK CAT

c~50

U

RA(M) 0 105 -+ +- 018 id0101i6 O1661 106TPA-Ca - +- + -- + +

FIG. 5. RA inhibits the ability of multimers of the IL-2 octamer motif to activate the TK promoter. Four micrograms of pBLCAT2,p[-575]TK-CAT, pNFAT-TK-CAT, AP-1C.11-TK-CAT, or pOct-TK-CAT was transfected into Jurkat cells. After 24 h, cells were treated withTPA-ionomycin without and with RA, as described previously. p[-575]TK-CAT-transfected cells were either untreated (-) or received 10p.M RA (+). Results are expressed as the average (± standard error from at least three experiments) percent value relative to CAT activity(considered as 100%) observed in TPA-calcium ionophore-treated and RA-untreated cells. CAT activities without and with TPA-ionophoretreatment for each construct were, respectively, (pmol/h/mg of protein) 589 + 100 and 450 + 80 (pBLCAT2), 720 + 50 and 8,736 + 400(p[-575]TK-CAT), 420 ± 50 and 2,100 + 100 (pNFAT-TK-CAT), 142 + 30 and 7,410 + 100 (pAP-1-TK-CAT), and 237 + 25 and 1,816 ± 120(pOct-TK-CAT).

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IL2-Oct-CAT IgH-Oct-CAT

100

, 50

RARa - + -+ - + + + +,RA(pW O 1 10 0 ml I 10

L2-GGAIClEAAAGTTAbA4TwGATC:CTITAAAT

UH -GAATClAACTTTA WAiCATACCGlll

FIG.- 6. RA inhibits the transcriptional activity of octamer motifsof both IL-2 and IgH. Four niicrograms of pOct-TK-CAT (IL2-Oct-CAT, containing the IL-2 octamer motif) or pBSCAT-Octa (IgH-Oct-CAT, containing the octamer motif of the IgH) was transfectedinto Jurkat cells. Where indicated, RARax expression vector wasalso cotransfected. After 24 h, cells were treated with TPA-ionomy-cin without and with RA, as described previously. Results areexpressed as the average (-+ standard error) percent value relative toCAT activity (considered as 10Ot%) observed in TPA-calcium iono-phore-treated and RA-untreated cells. CAT activities without andwith TPA-ionophore treatment for each construct were, respec-tively, (pmol/hmg of protein) 300 -+- 35 and 2,010 -+- 150 (IL2-Oct-CAT), 240 -+- 30 and 1,350 -+- 97 (IL2-Oct-CAT plus RARax), 80 -+- 14and 450 +t 55 (IgH-Oct-CAT), and 70 -+- 15 and 395 +- 24 (IgH-Oct-CAT plus RARa). At the bottom of the figure, the sequences of theIL-2 and IgH regions containing the octamer motifs used in the CATvectors are indicated. Arrowheads indicate the ends of the syntheticoligonucleotides encompassing the IL-2 and IgH regulatory regions.One copy followed by the beginning of the second copy of the IL-2oligonucleotide is shown. The BamHI-BglII junctions are under-lined. The octamer-binding site is boxed.

octamer motif, suggesting that RAR is involved in mediatingthis effect (Fig. 6). All of these data, taken together, suggestthat RA action is directed mainly through the IL-2 enhancerregion containing the octamer motif. To study whether RAaction was directed through sequences coincident with theIL-2 octamer motif, we examined the effect of the RA-dependent activation of RARa upon the transcriptionalactivity of an octamer motif derived from a different en-hancer element. Figure 6 shows that RA significantly inhibitsthe TPA-A23187-activatable transcriptional activity of aCAT vector containing the octamer motif of IgH. This ciselement contains no significant homology with the IL-2octamer-CAT construct, except for the octamer binding site(Fig. 6). No significant activity of the IgH-Oct-CAT wasobserved in the absence of TPA and A23187 treatment.RA does not inhibit in vitro Oct-1 DNA-binding activity.

The octamer motif binds a number of specific nucleoproteinswhich are involved in its transcriptional activity (reviewed inreferences 28 and 43). Since we observed a RA effect on theIL-2 octamer motif, we next wanted to determine whetherRtA was able to affect the DNA-binding capability of IL-2-octamer binding protein(s). Figure 7 shows that nuclearextracts of Jurkat cells contain a factor binding the IL-2octamer motif. This factor has been recently identified as theOct-1 protein (27). In agreement with previous reports (18,50), Oct-1 protein DNA-binding activity is not induced byTPA-A23187 treatment but is present under basal condi-

0 TPA+Cd* TPA+Ca+RA $.01--' 1711 0

competitor S - - S NS - S NS f.-ft . _w h. ^-_.." -- _S-wco

t S* * .

FIG. 7. Gel retardation assay of nuclear extracts from untreatedJurkat cells (0) and Jurkat cells treated with TPA-ionomycin for 5 hwithout (TPA+Ca++) or with (TPA+Ca+++RA) 10 ,uM RA. Nu-clear extracts (5 jLg of protein) were incubated for 30 min at 20°Cwith 0.2 ng of the 32P-end-labeled fragment (-96 to -66 bp)spanning the octamer motif of the IL-2 enhancer (5'-TTGAAAATATGTGTAATATGTAAAACATTT7-3'). A 100-fold or 500-fold ex-cess of specific (S) or unspecific (NS) unlabeled oligonucleotidecompetitor was included in the binding reactions, as indicated inMaterials and Methods. Arrowhead indicates DNA-protein com-plex.

tions. This suggests that Oct-1 transcriptional activity uponthe IL-2 gene needs TPA-Ca2+-induced additional activatingsignals. Interestingly, RA treatment, which significantlydown-regulates octamer enhancer activity (Fig. 5 and 6),does not affect the levels of octamer DNA-binding activityunder the conditions tested in vitro (Fig. 7).

DISCUSSION

In this paper we addressed the question of whether RAregulates the expression of the IL-2 gene. We have shownthat RA is able to down-regulate IL-2 gene expression.Furthermore, we have shown that RA is able to interferewith signals generated by protein kinase C activation andintracellular calcium increase which transactivate cis-regu-latory sequences in the 5'-flanking region of the gene. Thisis, to our knowledge, the first report on the RA-inducedregulation of the IL-2 gene.The further question we addressed dealt with the molec-

ular mechanisms by which RA regulates IL-2 gene transcrip-tion. Several intracellular proteins (i.e., the cellular RA-binding protein CRABP and three major specific RARs, a, I,and y) have been reported to bind RA and to be likely

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involved in RA actions (5, 20, 25, 29, 38). The cloned RARscontain DNA-binding and ligand-binding domains structur-ally related to those of members of the steroid-thyroidhormone receptor superfamily of nuclear transcription fac-tors (5, 11, 20, 29, 38). We have recently described down-regulation of the IL-2 promoter by the glucocorticoid recep-tor, another member of the steroid-thyroid hormone-RAreceptor superfamily (58). Consequently, we wanted todetermine the role of specific RAR in RA-induced down-regulation of IL-2 gene transcription. On the basis of theRARa expression in Jurkat cells, the inhibitory activity of aRAR-specific ligand, and the increased sensitivity of the IL-2enhancer to RA by complementation of an exogenous over-expressed recombinant RAR, we suggest that RARa playsan important role in mediating the RA-induced down-regu-lation of the IL-2 gene.The presence of multiple RARs and their differential tissue

distribution, regulation, and developmentally determinedexpression (15, 61) suggest distinct receptor physiologicalroles. Our data also show that the transcriptional regulatoryactivities of the three RARs can be separated depending onthe target gene, since, while affecting to a similar extent theTRE element, RARot and RAR3 are more powerful inhibi-tory agents of IL-2 gene expression than is RARy. Aminoacid sequence comparison among the three RARs reveals ahigh degree of homology only for regions corresponding tothe DNA-binding and the RA-binding domains (5, 20, 29,38), suggesting that all three receptors share similar DNA-binding and ligand-dependent activation properties. Thisalso suggests that these domains are to some extent requiredto sustain the overall down-regulatory action on the IL-2promoter. In contrast, the N-terminal regions of RARs arecompletely divergent. The N-terminal region of steroid re-ceptors appears to play a role in specifying target genetranscription, probably through selective interactions withother cooperating nuclear factors (32, 55). Whether thedifference in the N-terminal regions of the three RARsaccounts for their quantitative differential activities upon theIL-2 enhancer through such a mechanism remains to beelucidated.

Negative gene regulation by steroid hormone receptornucleoproteins is suggested to be mediated either by recep-tor competition with positive transacting factors for bindingto overlapping cis elements or by interfering with suchpositive factors through protein-protein interaction (1, 26,46, 60). The IL-2 enhancer is composed of multiple cooper-ating cis elements (8, 18, 24, 27, 47, 48, 50). To delineate thecis element of the IL-2 enhancer whose transactivation wasinhibited by RA, we performed functional analyses of CATvectors containing either internal deletion mutants of theIL-2 regulatory region or multimerized cis elements. Wehave shown that the RA-responsive element in the IL-2promoter mapped to sequences containing an octamer motif.Originally found in the immunoglobulin enhancer and re-ported to be required for specific transcription in B lympho-cytes, the octamer motif has also a broader transcriptionalfunction since it is an essential element in the promoter ofubiquitously transcribed genes such as histone H2B and Usmall nuclear RNAs (reviewed in references 28 and 43). Anumber of octamer-binding proteins have been identified,which are expressed ubiquitously or in a cell-specific ordevelopmentally regulated manner (43, 54). Recently, aprotein indistinguishable from octamer-1 and recombinantoctamer-2 have been shown to bind to the octamer motifs ofthe IL-2 promoter and to contribute to the activation of itstranscriptional activity (27, 50). The inability of the consti-

tutively expressed Oct-1 protein to activate the IL-2 gene inthe absence of TPA-calcium-induced activating signals sug-gests that Oct-1 transcriptional activity in Jurkat cells needsthose additional signals. The need for additional factorsactivating Oct-1 function has also been recently supportedby the ability of the herpes simplex virus transactivatorVP16 to interact with Oct-1 and to confer on it transcrip-tional activity (52). The significant RA-induced down-regu-lation of the IL-2 octamer transcriptional activity we havereported here was not associated with altered levels ofoctamer-1 DNA-binding activity. This suggests that RAaffects those signals activating octamer protein function.Alternatively, RA might directly interfere with transactiva-tion of either the octamer motif or distinct although overlap-ping cis elements. Indeed, adjacent cis-regulatory sequencesmight have a role in determining RA action, since althoughthe SV40 enhancer contains both an octamer-binding siteoverlapping with independent SphI-SphII enhancer motifsand cooperating multiple surrounding cis elements (62), itstranscriptional activity is not inhibited by RA. Interestingly,cell-specific factors have been reported to determine thetranscriptional activities of either octamer or SphI-SphIImotifs of the SV40 enhancer (37). Whether the SV40 oc-tamer motif is active on its own in Jurkat cells or compen-sation by other elements can interfere with RA action on theoverall viral enhancer remains to be investigated. Furtherstudies are required to elucidate the mechanisms by whichRA down-regulates octamer motif-containing cis elements.However, these mechanisms appear to be distinct fromthose mediating the recently reported RA-induced decreaseof the enhancer transcriptional activity of the murine immu-noglobulin heavy chain octamer motif in the F9 embryonalcarcinoma cell line (12, 44). This effect has been suggested tobe a consequence of the decreased levels of octamer-bindingproteins (as well as of octamer mRNA levels) associatedwith the RA-induced differentiation of these cells (12, 44).Thus, we speculate that RA might affect the transcriptionalactivity of octamer motif-containing regions through multi-ple molecular mechanisms depending upon the combinato-rial context of cis elements or the cell-specific molecularpathways which lead to the activation of these regulatorysequences.

Dissection of the molecular pathways arising from theantigen-T-cell receptor interaction and transacting the IL-2promoter might be accounted for by selected physiologicalor pharmacological agents. Two of them are specificallyaffected by cyclosporin A and IL-1, which have been re-cently shown to selectively control IL-2 gene expression viathe NFAT and AP1 elements, respectively (19, 36, 41). Herewe have reported that IL-2 cis-regulatory elements contain-ing an octamer motif mediate the down-regulation of the IL-2gene induced by the physiological hormone RA. Althoughthe relevance to thymocyte development of our observationsbased on a T-cell lymphoma line needs to be elucidated, theidentification of a regulatory pathway controlling IL-2 geneand involving its octamer motif and RAR suggests that itmight be operating and developmentally regulated during thedifferentiation process of T lymphocytes. Indeed, the in-volvement of octamer proteins in development and celldifferentiation is suggested by their developmentally regu-lated expression in embryonic stem cells and during mouseembryogenesis and by their relationship with other membersof the POU (Pit-1, octamer, and Unc-86) family of transcrip-tion factors characterized by a homeobox-containing con-served DNA-binding domain (28, 43, 45). Given the roleplayed by IL-2 in thymocyte development (10, 56), these

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elements might combine to contribute to the molecularevents sustaining both the thymic hypoplasia observed inRA-induced embryopathy (14, 30, 48) and the T-cell differ-entiation process.

ACKNOWLEDGMENTS

We thank P. Chambon, G. R. Crabtree, N. J. Holbrook, M.Karin, J. R. Nevins, K. Shudo, and K. Umesono for kindlyproviding reagents; A. Hayday, N. J. Holbrook, D. L. Levens, andL. Staudt for critical reading of the manuscript; and F. Durante, F.Ortolani, and S. Ferraro for technical assistance.

This work was supported by grants from the National ResearchCouncil, Target Project on Biotechnology and Bioinstrumentationand Ingegneria Genetica, Italian Association for Cancer Research,and AIDS Program.

REFERENCES

1. Akerblom, I. E., E. S. Slater, M. Beato, J. D. Baxter, and P. L.Mellon. 1988. Negative regulation by glucocorticoids throughinterference with a cAMP responsive element. Science 241:350-353.

2. Albert, F., C. Hua, A. Trunah, M. Pierres, and A. M. Schmitt-Verhulst. 1985. Distinction between antigen receptor and IL-2receptor triggering events in the activation of alloreactive T cellclones with calcium ionophore and phorbol ester. J. Immunol.134:3649-3655.

3. Alcover, A., D. Ramarli, N. E. Richardson, H.-C. Chang, andE. T. Reinherz. 1987. Functional and molecular aspects ofhuman T lymphocyte activation via T3-Ti and Til pathways.Immunol. Rev. 95:5-37.

4. Angel, P., M. Imagawa, R. Chiu, B. Stein, R. J. Imbra, H. J.Rahmsdorf, C. Jonat, P. Herrlich, and M. Karin. 1987. Phorbolester inducible genes contain a common cis-element recognizedby TPA-modulated trans-acting factor. Cell 49:729-739.

5. Brand, N., M. Petkovich, A. Krust, P. Chambon, H. de The, A.Marchio, P. TioUlais, and A. Dejean. 1988. Identification of a

second human retinoic acid receptor. Nature (London) 332:850.6. Breitman, T. R., S. E. Selonick, and S. S. Collins. 1980.

Induction of differentiation of the human promyelocytic leuke-mia cell line (HL60) by retinoic acid. Proc. Natl. Acad. Sci.USA 77:2936-2940.

7. Brockers, J. P. 1989. Retinoids, homeobox genes and limbmorphogenesis. Neuron 2:1285-1294.

8. Brunvand, M. W., A. Schmidt, and U. Siebenlist. 1988. Nuclearfactors interacting with the mitogen-responsive regulatory re-

gion of the interleukin 2 gene. J. Biol. Chem. 263:18904-18910.9. Campisi, J., H. E. Gray, A. B. Pardee, M. Dean, and G. E.

Sonenshein. 1984. Cell-cycle control of c-myc but not c-ras

expression is lost following chemical transformation. Cell 36:241-247.

10. Carding, S. R., E. J. Jenkinson, R. Kingston, A. C. Hayday, K.Bottomly, and J. J. T. Owen. 1989. Developmental control oflymphokine gene expression in fetal thymocytes during T-cellontogeny. Proc. Natl. Acad. Sci. USA 86:3342-3345.

11. Carson-Jurica, M. A., W. T. Schrader, and B. W. O'Malley.1990. Steroid receptor superfamily: structure and functions.Endocr. Rev. 11:201-220.

12. Chellappan, S. P., and J. R. Nevins. 1990. DNA octamerelement can confer ElA trans-activation, and adenovirus infec-tion results in a stimulation of the DNA-binding activity ofOTF-1/NFIII factor. Proc. Natl. Acad. Sci. USA 87:5878-5882.

13. Chomczynski, P., and N. Sacchi. 1987. Single-step method ofRNA isolation by acid guanidinium thiocyanate-phenol-chloro-form extraction. Anal. Biochem. 162:156-159.

14. Cohen, M., A. Rubistein, J. K. Li, and G. Nathenson. 1987.Thymic hypoplasia associated with isotretinoin embryopathy.Am. J. Dis. Child. 141:263-266.

15. de The, H., A. Marchio, P. TioUais, and A. Dejean. 1989.Differential expression and ligand regulation of the retinoic acidreceptor a and P3 genes. EMBO J. 8:429-433.

16. Durand, D. B., M. R. Bush, J. G. Morgan, A. Weiss, and G. R.Crabtree. 1987. A 275-base pair fragment at the 5' end of the

interleukin 2 gene enhances expression from a heterologouspromoter in response to signals from the T cell antigen receptor.J. Exp. Med. 165:395-407.

17. Durand, D. B., M. Kamoun, C. A. Norris, N. J. Holbrook, J. S.Greengard, G. R. Crabtree, and J. A. Kant. 1986. Retroviralactivation of interleukin 2 gene in a gibbon ape T cell lymphomaline. J. Exp. Med. 164:1723-1734.

18. Durand, D. B., J. P. Shaw, M. R. Bush, R. E. Replogle, R.Belagaje, and G. R. Crabtree. 1988. Characterization of antigenreceptor response element within the interleukin-2 enhancer.Mol. Cell. Biol. 8:1715-1724.

19. Emmel, E. A., C. L. Verweij, D. B. Durand, K. M. Higgins, E.Lacy, and G. R. Crabtree. 1989. Cyclosporin A specificallyinhibits function of nuclear proteins involved in T cell activa-tion. Science 246:1617-1620.

20. Giguere, V., E. S. Ong, P. Segui, and R. M. Evans. 1987.Identification of a receptor for the morphogen retinoic acid.Nature (London) 330:624-629.

21. Gorman, C. M., L. F. Moffat, and B. H. Howard. 1982.Recombinant genomes which express chloramphenicol acetyl-transferase in mammalian cells. Mol. Cell. Biol. 2:1044-1051.

22. Hashimoto, Y., H. Kagechika, and K. Shudo. 1990. Expressionof retinoic acid receptors (RAR's). Biochem. Biophys. Res.Commun. 166:1300-1307.

23. Henny, C. S., K. Kuribayashi, D. E. Kern, and S. Gillis. 1981.Interleukin 2 augments natural killer cell activity. Nature (Lon-don) 291:335-338.

24. Hoyos, B., D. W. Ballard, E. Bohnlein, M. Siekevitz, and W. C.Greene. 1989. KappaB-specific DNA binding proteins: role inthe regulation of human interleukin 2 gene expression. Science244:457-460.

25. Jetten, A. M., and M. E. R. Jetten. 1979. Possible role of retinoicacid binding protein in retinoid stimulation of embryonal carci-noma cell differentiation. Nature (London) 278:180-182.

26. Jonat, C., H. J. Rahmsdorf, K. K. Park, A. C. B. Cato, S. Gebel,H. Ponta, and P. Herrlich. 1990. Antitumor promotion andantiinflammation: down-modulation of AP-1 (fos/jun) activity byglucocorticoid hormone. Cell 62:1189-1204.

27. Kamps, M. P., L. Corcoran, J. H. LeBowitz, and D. Baltimore.1990. The promoter of the human interleukin-2 gene containstwo octamer-binding sites and is partially activated by theexpression of Oct-2. Mol. Cell. Biol. 10:5464 5472.

28. Kemler, I., and W. Schaffner. 1990. Octamer transcriptionfactors and the cell type-specificity of immunoglobulin geneexpression. FASEB J. 4:1444 1449.

29. Krust, A., P. Kastner, M. Petkovich, A. Zelent, and P. Cham-bon. 1989. A third human retinoic acid receptor, hRAR--y. Proc.Natl. Acad. Sci. USA 86:5310-5314.

30. Lammer, E. J., T. D. Chen, R. M. Hoar, S. D. Agnish, P. J.Benke, J. T. Braun, P. M. Fernhoff, P. J. Grix, I. T. Lott, J. M.Richard, and S. C. Sun. 1985. Retinoic acid embryopathy. N.Engl. J. Med. 313:837-841.

31. Luckow, B., and G. Schutz. 1987. CAT constructions withmultiple unique restriction sites for the functional analysis ofeukaryotic promoters and regulatory elements. Nucleic AcidsRes. 15:5490.

32. Meyer, M. E., H. Gronemeyer, B. Turcotte, M. T. Bocquel, D.Tasset, and P. Chambon. 1989. Steroid hormone receptorscompete for factors that mediate their enhancer function. Cell57:433-442.

33. Miller, W. H. Jr., D. May, A. Li, J. F. Grippo, and E.Dmitrovsky. 1990. Retinoic acid induces down-regulation ofseveral growth factors and proto-oncogenes in a human embry-onal cancer cell line. Oncogene 5:511-517.

34. Mingari, M. C., F. Gerosa, G. Carra, R. S. Accolla, A. Moretta,R. H. Zubler, T. A. Waldmann, and L. Moretta. 1984. Humaninterleukin 2 promotes proliferation of activated B cells viasurface receptors similar to those of activated T cells. Nature(London) 312:641-643.

35. Morgan, D. A., F. W. Ruscetti, and R. C. Gallo. 1976. Selectivein vitro growth of T lymphocytes from normal human bonemarrow. Science 193:1007-1008.

36. Muegge, K., T. M. Williams, J. Kant, M. Karin, R. Chiu, A.

VOL . 1 l, 1991

on January 29, 2018 by guesthttp://m

cb.asm.org/

Dow

nloaded from

4778 FELLI ET AL.

Schmidt, U. Siebenlist, H. A. Young, and S. K. Durum. 1989.Interleukin-1 costimulatory activity on the interleukin-2 pro-moter via AP-1. Science 246:249-251.

37. Nomiyama, H., C. Fromental, J. H. Xiao, and P. Chambon.1987. Cell specific activity of the constituent elements of thesimian virus 40 enhancer. Proc. Natl. Acad. Sci. USA 84:7881-7885.

38. Petkovich, M., N. J. Brand, A. Krust, and P. Chambon. 1987. Ahuman retinoic acid receptor which belongs to the family ofnuclear receptors. Nature (London) 330:444 450.

39. Queen, C., and D. Baltimore. 1983. Immunoglobulin gene tran-scription is activated by downstream sequence elements. Cell33:741-748.

40. Rabin, H., R. F. Hopkins, F. W. Ruscetti, R. H. Neubauer, R. L.Brown, and T. G. Kawakami. 1981. Spontaneous release of afactor with properties of T cell growth factor from continuousline of primate tumor T cells. J. Immunol. 127:1852-1856.

41. Randak, C., T. Brabletz, M. Hergenrother, I. Sobotta, and E.Serfling. 1990. Cyclosporin A suppresses the expression of theinterleukin 2 gene by inhibiting the binding of lymphocyte-specific factors to the IL-2 enhancer. EMBO J. 9:2529-2536.

42. Roberts, A. B., and M. B. Sporn. 1984. Cellular biology andbiochemistry of the retinoids, p. 209-286. In M. B. Sporn, A. B.Roberts, and D. S. Goodman (ed.), The retinoids, vol. 2.Academic Press, Inc., New York.

43. Schaffner, W. 1989. How do different transcription factorsbinding the same DNA sequence sort out their jobs? TrendsGenet. 5:37-39.

44. Scholer, H. R., R. Balling, A. K. Hatzopoulos, N. Suzuki, and P.Gruss. 1989. Octamer binding proteins confer transcriptionalactivity in early mouse embryogenesis. EMBO J. 8:2551-2557.

45. Scholer, H. R., A. K. Hatzopoulos, R. Balling, N. Suzuki, and P.Gruss. 1989. A family of octamer-specific proteins presentduring mouse embryogenesis: evidence for germline-specificexpression of an Oct factor. EMBO J. 8:2543-2550.

46. Schule, R., P. Rangarajan, S. Kliewer, L. S. Ransone, J. Bolado,N. Yang, I. M. Verma, and R. M. Evans. 1990. Functionalantagonism between oncoprotein c-jun and the glucocorticoidreceptor. Cell 62:1217-1226.

47. Serfling, E., R. Barthelmas, I. Pfeuffer, B. Schank, S. Zarius, R.Swoboda, F. Mercurio, and M. Karin. 1989. Ubiquitous andlymphocyte-specific factors are involved in the induction of themouse interleukin 2 gene in T lymphocytes. EMBO J. 8:465-473.

48. Shaw, J. P., P. J. Utz, D. B. Durand, J. J. Toole, E. A. Emmel,and G. R. Crabtree. 1988. Identification of a putative regulatorof early T cell activation genes. Science 241:202-205.

49. Shenefelt, R. E. 1972. Morphogenesis of malformations in ham-sters caused by retinoic acid: relation to dose and stage attreatment. Teratology 72:103-118.

50. Shibuya, H., and T. Taniguchi. 1989. Identification of multiplecis-elements and trans-acting factors involved in the induced

expression of human IL-2 gene. Nucleic Acids Res. 17:9173-9184.

51. Siebenlist, U., D. B. Durand, P. Bressler, N. J. Holbrook, C. A.Norris, M. Kamoun, J. A. Kant, and G. R. Crabtree. 1986.Promoter region of interleukin-2 gene undergoes chromatinstructure changes and confers inducibility on chloramphenicolacetyltransferase gene during activation of T cells. Mol. Cell.Biol. 6:3042-3049.

52. Stern, S., M. Tanaka, and W. Herr. 1989. The Oct-1 homoe-odomain directs formation of a multiprotein-DNA complex withthe HSV transactivator VP16. Nature (London) 341:624-630.

53. Strickland, S., and M. Mahdavi. 1978. The induction of differ-entiation in teratocarcinoma stem cells by retinoic acid. Cell15:393-403.

54. Suzuki, N., H. Rohdewohld, T. Neuman, P. Gruss, and H. R.Scholer. 1990. Oct-6: a POU transcription factor expressed inembryonal stem cells and in the developing brain. EMBO J.9:3723-3732.

55. Tora, L., H. Gronemeyer, B. Turcotte, M. P. Gaub, and P.Chambon. 1988. The N-terminal region of the chicken proges-terone receptor specifies target gene activation. Nature (Lon-don) 333:185-188.

56. Toribio, M. L., J. C. Gutierrez-Ramoz, L. Pezzi, M. A. R.Marcos, and A. C. Martinez. 1989. Interleukin 2-dependentautocrine proliferation in T-cell development. Nature (London)342:82-85.

57. Umesono, K., V. Giguere, C. K. Christofer, M. G. Rosenfeld,and R. M. Evans. 1988. Retinoic acid and thyroid hormoneinduce gene expression through a common responsive element.Nature (London) 336:262-265.

58. Vacca, A., S. Martinotti, I. Screpanti, M. Maroder, M. P. Felli,A. R. Farina, A. Gismondi, A. Santoni, L. Frati, and A. Gulino.1990. Transcriptional regulation of the interleukin 2 gene byglucocorticoid hormones. Role of steroid receptor and antigen-responsive 5' flanking sequences. J. Biol. Chem. 265:8075-8080.

59. Yamada, G., Y. Kitamura, H. Sanoda, H. Harada, S. Taki, R. C.Mulligan, H. Osawa, T. Diamantstein, S. Yokoyama, and T.Taniguchi. 1987. Retroviral expression of the human interleukin2 gene in a murine T cell line results in cell growth autonomy andtumorigenicity. EMBO J. 6:2705-2709.

60. Yang-Yen, H. F., J. C. Chambard, Y. L. Sun, T. Smeal, T. J.Schmidt, J. Drouin, and M. Karin. 1990. Transcriptional inter-ference between c-jun and the glucocorticoid receptor: mutualinhibition of DNA binding due to direct protein-protein interac-tion. Cell 62:1205-1215.

61. Zelent, A., A. Krust, M. Petkovich, P. Kastner, and P. Cham-bon. 1989. Cloning of murine a and 1B retinoic acid receptors anda novel receptor predominantly expressed in skin. Nature(London) 339:714-717.

62. Zenke, M., T. Grundstrom, H. Matthes, M. Wintzerith, C.Schatz, A. Wildeman, and P. Chambon. 1986. Multiple sequencemotifs are involved in SV40 enhancer function. EMBO J.5:387-397.

MOL. CELL. BIOL.

on January 29, 2018 by guesthttp://m

cb.asm.org/

Dow

nloaded from