Communication Vol. 268 No. 8 Issue of March 15 p 5353-53561993 THE JOURNAL OF BIOLOGICAL CHEMISTRY

0 1993 by The American Society for Biochemistry and Mbfechar Biology, Inc. Printed in U. S. A.

Glucocorticoid Receptor-CAMP Response Element-Binding Protein Interaction and the Response of the Phosphoenolpyruvate Carboxykinase Gene to Glucocorticoids*

(Received for publication, November 16, 1992) Enyu ImaiSQ, Jeffrey N. MinerT, John A. MitchellSII, Keith R. YamamotoT, and Daryl K. GrannerS** From the $Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee 37232-0615 and the TDepartment of Biochemistry and Biophysics, University of California, San Francisco, California 94143-0448

The phosphoenolpyruvate carboxykinase (PEPCK) gene encodes the rate-limiting enzyme in gluconeogen- esis. Glucocorticoids enhance PEPCK gene expression through a multicomponent regulatory complex. We show that a full response to glucocorticoids requires two DNA segments: 1) a glucocorticoid response unit (GRU), centered at about position -400, which con- tains two accessory factor elements (AF1 and AF2) and two glucocorticoid receptor binding sites (GR1 and GR2), and 2) a basal promoter/cyclic AMP response element (E/CRE) at about position -90, which binds the transcription factor CREB. A protein-protein in- teraction was observed in vitro between GR and CREB that might account for the role of the E/CRE in the glucocorticoid response of the PEPCK gene.

Glucocorticoid hormones and cyclic AMP increase the rate of transcription of the PEPCK’ gene (1, 2), and each does so through a complex DNA element (3-5). For example, hor- monal regulation at the GRU requires not only the glucocor- ticoid receptor bound at two contiguous sites, GR1 and GR2, but also nonreceptor factors bound at two adjacent sites, AF1 and AF2 ( 5 ) . Thus, the GRU is a “composite” GRE, a locus

* This work was supported in part by Health and Human Services Grants DK35107, DK20593 (to the Vanderbilt Diabetes Research and Training Center), and CA20535. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

J Recipient of training support from an American Diabetes Asso- ciation Mentor-based grant.

(1 Recipient of postdoctoral traineeships from the American Cancer Society and the Leukemia Society.

** To whom correspondence should be addressed Dept. of Molec- ular Physiology and Biophysics, Vanderbilt University Medical Cen- ter, Nashville, TN 37232-0615. Tel.: 615-322-7004; Fax: 615-322-7326.

The abbreviations used are: PEPCK, phosphoenolpyruvate car- boxykinase; GR, glucocorticoid receptor; GRU, glucocorticoid re- sponse unit; GRE, glucocorticoid response element; E/CRE, basal promoter/cyclic AMP response element; CREB, cAMP response ele- ment binding protein; HRE, hormone response element; bZIP, basic zipper.

at which the activity of specifically bound GR depends on other nearby DNA-binding factors (6, 7). In contrast, GR- mediated enhancement at “simple” GREs is conferred solely by the bound receptor. Indeed, AF1 and AF2 also serve other functions. AF1 includes a retinoic acid response element (8, 9) and an HNF-4 element’ that may be involved in tissue- selective expression of hepatic genes (10); AF2 functions as an insulin response sequence and as a phorbol ester response sequence (11,12). The interposition of the insulin and phorbol ester response sequences within the GRU may account for the inhibition of the glucocorticoid response by insulin and phorbol esters at the PEPCK promoter (2, 11-13).

The response of the PEPCK gene to cyclic AMP is similarly complex. Control is mediated primarily through an E/CRE at -90 that is both a part of the basal promoter and a cyclic AMP response element (3, 4). The E/CRE binds the tran- scription factor CREB, which is phosphorylated by the cyclic AMP-dependent protein kinase in response to elevated cyclic AMP (14, 15); phosphorylation appears to stimulate the ac- tivity of CREB without altering its affinity for the E/CRE (16). In addition, the P3(I) element at -240 is required in certain cell contexts for a maximal response of the PEPCK promoter to cyclic AMP (17). Interestingly, P3(I) binds the transcription factor C/EBP, but not CREB, and mutation of both P3(I) and E/CRE abolishes responsiveness of the PEPCK promoter to cyclic AMP (17). Thus, a full response of the PEPCK gene to cAMP requires both the E/CRE and P3(I), with associated binding proteins. In this paper we now show that glucocorticoid responsiveness of the PEPCK gene involves a functional interaction between the GRU and the E/CRE, perhaps through a physical association of the gluco- corticoid receptor with CREB.

EXPERIMENTAL PROCEDURES

Cell Culture and Transfection-H4IIE rat hepatoma cells were grown as described previously (18). Eighteen hours before experimen- tation, the cells were placed in serum-free Dulbecco’s modified Eagle’s medium. Transfection was accomplished by the calcium phosphate co-precipitation method, as described previously (8, 13).

Plasmids-The glucocorticoid receptor expression plasmid pSVGRl was co-transfected with various reporter plasmids where indicated. The plasmid pGRUTK was created by ligating the HindIII- XbaI fragment of pPG44 (Ref. 19; -600 to -200 of the PEPCK gene) into the polylinker site of the TKCAT vector. pGRETK was created by ligating a single copy of GRE 1.3 from the MMTV promoter

GATCGA-5’) into the polylinker site of the TKCAT vector. The series of internal deletion plasmids employed in the experiment illustrated in Fig. 3 were described by Quinn et al. (4).

Radiolabeled CREB, c-Jun, and A c-Jun-[35S]Methionine-labeled CREB, c-Jun, and Ac-Jun were produced by in vitro transcription (T7 polymerase for CREB, and SP6 polymerase for c-Jun and Ac- Jun; Promega) and in vitro translation in reticulocyte lysates (Pro- mega). The CREB and c-Jun expression vectors were provided by M. Montminy and R. Turner, respectively. Ac-Jun was prepared by digestion of the c-Jun expression vector with PstI, which cleaves the DNA at a position corresponding to amino acid 222. This results in a C-terminal truncation that eliminates the bZIP (DNA binding) region of the protein and serves as a negative control (20).

Immunoprecipitation-The [35S]methionine-labeled CREB, c-Jun, or Ac-Jun proteins were incubated for 30 min at 30 “C with cytosolic extracts isolated from HeLa cells infected with either wild type vaccinia virus, or with a GR-expressing recombinant virus (provided

* R. Hall, F. Sladek, and D. K. Granner, unpublished observation.

(5”AGCTTGTTACAAACTGTTCT-3’, 3”ACAATGTTTGACAA-

5353

5354 Functional GRU and CRE Interaction by H. Stunnenberg; Ref. 21). After incubation, 10 volumes of HEGNO5O (10 mM Hepes, pH 8.0, 1 mM EDTA, 10% glycerol, 50 mM NaCI) + 0.1% Triton X-100 was added. The mixture was then precleared by the addition of preswollen, prewashed protein A-Seph- arose (100 mg/ml) to 0.03% of the final volume; the Sepharose was removed by centrifugation after 30 min a t 4 "C. GR-specific mono- clonal antibody (BUGR2, provided by R. Harrison; Ref. 22) and another aliquot of protein A-Sepharose were then added, and the slurry was incubated a t 4 "C for 1 h with mixing. Sepharose was centrifuged, quickly washed four times in 20 volumes of HEGN050 + 0.1% Triton X-100, transferred to fresh tubes, and washed once in HEGN050. Precipitated proteins were eluted with sample buffer and analyzed by SDS-polyacrylamide gel electrophoresis, fluorography, and autoradiography.

RESULTS AND DISCUSSION

Glucocorticoid hormones and cyclic AMP each induce PEPCK mRNA in H4IIE rat hepatoma cells (Fig. 1). These effects are additive, and at early times are due to an increased rate of transcription of the PEPCK gene (1,2). As mentioned above, the effect of each inducer is mediated through a com- plex DNA element. A functional interaction between these two complex elements, the PEPCK GRU and E/CRE, was implied by experiments in which we compared the activity of the GRU on two different promoters. In particular, we tested the intact PEPCK promoter (from -600 to +69, in pPL9) and a fusion of the PEPCK -600 to -200 region to the thymidine kinase promoter (pGRUTK; Fig. 2). The latter

1 2 3 . 4 PEPCK -I

7 -

e - Fold Induction 1.0 5.0 4.3 9.3

FIG. l. Dexamethasone and CAMP induce accumulation of PEPCK mRNA. H4IIE rat hepatoma cells were incubated in serum- free Dulbecco's modified Eagle's medium containing no additive (lane I), 0.1 mM 8-(4-~hlorophenylthio)cAMP (lane 2 ) , 0.5 p~ dexameth- asone (lane 3) , or both (Lune 4 ) for 6 h. Total RNA was isolated and quantitated using a primer extension assay. Data from this repre- sentative experiment are expressed as the -fold increase of PEPCK mRNA in treated cells compared to cells that received no treatment (control). Differences in the amount of total RNA added to the primer extension assay are accounted for by expressing the ratio of PEPCK mRNA to that of calmodulin mRNA, which is not affected by either of these inducers. The ratios of PEPCK + calmodulin mRNA were 0.4, 2.0, 1.7, and 3.7 for lanes 1-4, respectively.

PPL9 9 0 2 1 3

PGRUTK 19fO.J * l8OiO9

pTK.CAT 1% C I ,

1 .Of 0.l

FIG. 2. The GRU functions poorly through the thymidine kinase promoter. Ten pg of the plasmids illustrated and 5 pg of the glucocorticoid receptor expression plasmid pSVGR1 were cotrans- fected into H4IIE cells by the calcium-phosphate method. After transfection the cells were placed in serum-free medium in the pres- ence or absence of 0.5 p~ dexamethasone for 18 h. The -fold induction of chloramphenicol acetyltransferase activity is expressed as mean S.E. for a minimum of five independent transfections. The construc- tion of the plasmids pGRUTK and pGRETK is described under "Experimental Procedures." The glucocorticoid response unit is lo- cated from base pairs -455 to -349 and includes two glucocorticoid receptor binding sites e), AF1 ( ), and AF2 ( 4). The basal pro- moter region of PEPCK gene includes a CAAT box ( :), E/CRE ( ), and a TATA box ( ).

construct contains the entire GRU but lacks the PEPCK basal promoter, which consists of a CAAT box, the E/CRE, and a TATA box (Ref. 4 and Figs. 2 and 3). Basal and dexamethasone-induced activity was measured in H4IIE cells cotransfected with these constructs and a GR expression vector. In contrast to pPL9, which was induced 9-fold in response to dexamethasone, pGRUTK was induced less than 2-fold (compare to pTKCAT; Fig. 2). The thymidine kinase promoter is not limiting, since a construct in which a simple GRE was ligated to pTKCAT yielded an 18-fold induction (pGRETK; Fig. 2). These results implied that some element in the PEPCK promoter between -200 and +69 is required for maximal activity of the GRU.

We found that internal deletions of either the CAAT box and the E/CRE together (pID6; Fig. 3) or of the E/CRE alone (pID3; Fig. 3) produced a marked reduction in the glucocor- ticoid response; each was stimulated approximately 3-fold by dexamethasone, whereas the intact promoter (pPL9) was stimulated 9-fold (Fig. 3). In contrast, glucocorticoid induc- tion was unaffected by deletion of the CAAT box alone (pID11; Fig. 3) or of sequences between the E/CRE and the TATA box (-75 to -40; pID7; Fig. 3). The latter region has been proposed as a glucocorticoid response element (3); how- ever, we failed to detect either independent GRE activity (5, 19) or a contribution to GRU activity by this segment (Fig. 3).

These results showed that maximal glucocorticoid induc- tion of the PEPCK gene requires an intact GRU and E/CRE and suggested the possibility that the proteins that bind to these elements might interact. The factors that associate with AF1 and AF2 have not yet been identified, but it is clear that the receptor binds to two sites in the GRU (GR1 and GR2) and that CREB binds to the E/CRE (4,5,17). To test whether the receptor and CREB interact, [35S]methionine-labeled CREB was mixed with extracts of HeLa cells infected with a GR-expressing recombinant vaccinia virus, or with wild type vaccinia virus. After incubation, the receptor and associated

c.QmMu -600 -5W -400 -3W ,200 . l o 0 * Blul &a-

PR9 - : R n f " Ld. 1 W f O 8 9 6 t l 3 l 9.0r1.3

plD6 4 52.6 140121 2.8tO.2

plD3 E e~ 57110 l78 t38 3 .6 r08

plDl1 - 59118 48Ofl52 9 .3~2.8

plD7 132a7 1083t112 8.4213

GI1 CAAT T.\rA CRE

FIG. 3. The PEPCK proximal promoter region is involved in glucocorticoid regulation through the GRU. The various internal deletion mutations of PEPCK proximal promoter region analyzed are illustrated a t left. The components of the glucocorticoid response unit (GRU) and the basal promoter of the PEPCK gene are illustrated as in Fig. 2. The boundaries of the deletions in the various constructs are as follows: pID6, -129/-86; pID3, -95/-86; pID11, -log/-101; pID7, -75/-40. Ten pg of the individual constructs were cotransfected into H41IE cells with 5 pg of glucocorticoid receptor expression vector (pSVRG1) and 2.5 pg of an internal control (PRSVL-AD~'), which was used to monitor transfection efficiency. The CaPO., co-precipitation method was used for transfection. After transfection the H4IIE cells were placed in serum-free medium in the presence or absence of 0.5 p~ dexamethasone for 24 h. Basal level expression of CAT activity of the various constructs, corrected for transfection efficiency, was compared to that of the wild type vector, which was assigned a value of 100% in each experiment. The basal promoter elements were mapped in experiments using CV-1 cells (41, whereas all the experiments reported in this paper were done using H4IIE rat hepatoma cells. This may account for the difference in basal activity noted in the two experiments. The ratio of CAT activity in dexamethasone induced versus control cells is expressed as the mean & S.E. of four experiments for the four members of the ID series and seven experiments for pPL9.

Functional GRU and CRE Interaction 5355

proteins were immunoprecipitated with a monoclonal anti- body directed against GR (22). The immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis and au- toradiography. As shown in Fig. 4, labeled CREB was precip- itated in the presence (lune 9) but not in the absence of GR (lane 10). As expected from previous work (23), co-precipita- tion of a GR.c-Jun complex was also observed (lanes 7 and 8) , whereas labeled AcJun (a C-terminal truncation of cJun that lacks the DNA-binding region) was unable to form a complex with GR (lanes 11 and 12). Although quantitation of such assays is difficult, it appears that the GR- CREB complex may form more efficiently than does the GR. Jun complex. In contrast to earlier studies (23), these reactions did not involve chemical cross-linking agents.

Because both the receptor and CREB are DNA-binding proteins, it was conceivable that their co-immunoprecipita- tion resulted from association of the proteins with contami- nating DNA in the extracts rather than with each other. Lai and Herr (24) demonstrated that such protein-DNA interac- tions could be eliminated by the addition of ethidium bromide, a DNA-intercalating agent. Unfortunately, we found that ethidium bromide inhibited the interaction of our monoclonal antibody with the receptor, making the ethidium bromide test ineffective for our purposes. However, several additional lines of argument suggest strongly that the receptor and CREB interact directly. First, depletion of contaminating DNA with micrococcal nuclease or DNase I had no effect on the effi- ciency of coprecipitation. Second, the amounts of coprecipi- tated protein were insensitive to the addition of high levels of nonspecific DNA. Third, the reactions were carried out with low concentrations of CREB and receptor in the presence of a vast excess of other proteins; the artifact pointed out by Lai and Herr (24) was observed only a t very high concentrations of the “interacting” proteins.

Minimal DNA sequences that constitute functional HREs have been defined for several hormones (25). These generally are less than 20 base pairs in length and are defined by their ability to bind a factor that regulates transcription. This factor is the intracellular receptor in the case of the steroid/

INPUT IMMUNOPPT GR + - + - + - + - + - + - cJun + + - - - - + + “ ” CREB - - + + - - - - + + - - AcJun - - - - + + ” “ + +

-68

-43

. 29

1 2 3 4 5 6 7 8 9 101112 FIG. 4. GR and CREB interact in vitro. Radiolabeled CREB,

cJun, and AcJun were incubated in extracts with or without unlabeled GR, as described under “Experimental Procedures.” An aliquot was removed and analyzed by SDS-polyacrylamide gel electrophoresis (INPUT, lanes 1-6). The remainder of each reaction was subject to immunoprecipitation with an anti-GR monoclonal antibody (22), as described under “Experimental Procedures.” Proteins were eluted from the protein A-Sepharose with sample buffer and analyzed by SDS-polyacrylamide gel electrophoresis, fluorography, and autoradi- ography. The results of this procedure are shown in lanes 7-12 under ZMMUNOPPT (immunoprecipitation). The relative migration of CREB is slightly slower that that of Jun, as expected from their molecular mass (43 and 36 kDa, respectively). The migration of standard proteins in this system (bovine serum albumin, 68 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 29 kDa) is shown at right. Ac-Jun, a truncated form of c-Jun, migrates much more rapidly than either of the other proteins.

thyroid family of hormones, and a nonreceptor, intracellular protein, such as CREB, in the case of hormones that bind to receptors located in the cell surface. Although such simple response elements can autonomously enhance transcription upon association of their cognate factors, they lack the ver- satility of more complex regulatory regions whose behavior is defined by the interactions of two or more distinct protein factors (6, 7). Many types of physiologic regulation require counter-balancing positive and negative control mechanisms, and certain situations may necessitate that either the positive or negative activity be dominant. In addition, the concerted actions of several different hormones on a gene may result in additive or synergistic effects.

Composite HREs, which contain sequences recognized both by receptors and by non-receptor factors, appear capable of just this sort of regulation (6,7). For example, communication between the transcription factor AP1 and various members of the steroid/thyroid hormone receptor family has been described in the regulation of the proliferin and collagenase genes by glucocorticoids (23, 26-28), the stromelysin gene by retinoic acid (29), the ovalbumin gene by estrogens (30), and the osteocalcin gene by vitamin D and retinoic acid (31-33). Direct interaction of AP1 with particular receptors has been demonstrated in some of these cases and implied in others. One composite element, plfG, a 25-base pair sequence from the proliferin gene promoter, is recognized both by GR and by AP1, a transcription factor comprised of c-Jun homodimers or c-Jun/c-Fos heterodimers. The ratio of c-Fos and c-dun activities determines whether glucocorticoids have no effect, a positive effect, or a negative effect on the proliferin gene. Thus, in the absence of AP1, dexamethasone has no effect on plfG; however, the hormone stimulates transcription when c-Jun is in excess relative to c-Fos and represses transcription when c-Fos is in excess of c-Jun. A physical association between GR and Jun, through this assembly of contiguous elements, appears to be involved in composite regulation of the proliferin gene (23).

The GRU in the PEPCK gene is itself a composite element in which factors bound a t two accessory factor sites, AF1 and AF2, are essential for activity of GR bound to two immediately adjacent sites (5). We now show that a full response to glucocorticoids requires both the GRU and E/CRE in a func- tional interaction that is accomplished over a distance of about 300 base pairs. That is, the activity of the composite GRE itself can be further modulated by physical association with additional factors bound a t remote sites. I t will be interesting in future work to determine whether these inter- actions, as well as others that have been inferred or detected a t loci that contain DNA binding sequences for only one of the putative interacting factors (see Refs. 6,7, 23, and 26-33) confer their effects via a single molecular mechanism or via multiple distinct pathways.

Our finding of an interaction between the glucocorticoid receptor and CREB may be considered unsurprising, given the known structural relationship of CREB and Jun (15), and it is possible that other members of the superfamily of bZIP proteins form similar interactions. Nevertheless, it is also clear that GR does not simply associate with all bZIP proteins (23).3 The detailed mechanism of the interaction between these members of two different families of transcription fac- tors remains to be investigated.

Cyclic AMP and glucocorticoids influence many of the same physiologic processes. In some cases one or the other of these effectors plays a permissive role, and numerous additive and synergistic responses have been described (34). A functional

M. Vivanco and K. R. Yamamoto, unpublished observation.

5356 Functional GRU and CRE Interaction

relationship between the glucocorticoid and CAMP response elements, through interaction of the proteins that bind to them, may be one way that these physiologic effects are achieved.

Acknowledgments-We thank the colleagues cited in text for pro- viding materials used in this study, T. Weil and R. O'Brien for a critical review of the manuscript, R. Printz for help in making the figures, and D. Caplenor for preparation of the manuscript.

REFERENCES 1. Lamers, W. H., Hanson, R. W., and Meisner, H. M. (1982) Proc. Natl.

2. Sasaki, K., Cripe, T. P., Koch, S. R., Andreone, T. L., Petersen, D. D., Acad. Sci. U. S. A . 79,5137-5141

3. Short. J. M.. Wvnshaw-Boris. A,. Short. H. P.. and Hanson. R. W. (1986) Beale, E. G., and Granner, D. K. (1984) J. Biol. Chem. 259,15424-15251

J. Biol. Chemr 261,9721-9726

D. K. (1988) Mol. Cell. Biol. 8. 3467-3475

. .

4. Quinn, P. G., Wong, T. W., Magnuson,, M. A,, Shabb, J. B., and Granner,

5. Imai, E.,'Stromstedt,P.-E., QGnn,, P . G., Carlstedt-Duke, J., Gustafsson, J.-A., and Granner, D. K. (1990) Mol. Cell. Biol. 10,4712-4719

6. Miner, J. N., and Yamamoto, K. R. (1991) Trends Biochern. Sci. 16,423- 426

7. Lucas, P. C., and Granner, D. K. (1992) Annu. Reu. Biochem., 6 1 , 1131- 1173

8. Lucas, P. C., O'Brien, R. M., Mitchell, J. A., Davis, C. M., Imai, E., Forman, B. M., Samuels, H. H., and Granner, D. K. (1991) Proc. Natl. Acad. Sci. U. S. A. 88. 2184-2188

9. Lucas, P. C., Forman, B. M., Samuels, H. H., and Granner, D. K. (1991)

10. Sladek, F. M., Zhong, W., Lai, E., and Darnell, J. E., Jr. (1990) Genes &

11. O'Brien, R. M., Lucas, P. C., Forest, C. D., Magnuson, M. A,, and Granner,

12. O'Brien, R. M., Bonovich, M. T., Forest, C. D., and Granner, D. K.. (1991)

Mol. Cell. Biol. 11 , 5164-5170

Deu. 4,2353-2365

D. K. (1990) Science 249,533-537

Proc. Natl. Acad. Sci. U. S. A. 88,6580-6584

13. Magnuson, M. A., Quinn, P. G., and Granner, D. K. (1987) J. Biol. Chem. 262,14917-14920

14. Hoeffler, J. P., Meyer, T. E., Yun, Y., Jameson, J. L., and Habener, J. F. (1988) Science 2 4 2 , 1430-1433

15. Gonzalez, G. A., Yamamoto, K. K., Fischer W. H., Karr, D., Menzel, P., Biggs, W., 111, Vale, W. V., and Montmihy, M. R (1989) Nature 3 3 7 , 749-769

16. Quinn, P. G., and Granner, D. K. (1990) Mol. Cell. Biol. 10,3357-3364 17. Liu, J., Park, E. A., Gurney, A. L., Roesler, W. J., and Hanson, R. W.

18. Forest, C. F., O'Brien, R. M., Lucas, P. C., Magnuson, M. A,, and Granner,

19. Petersen D. D., Magnuson, M. A,, and Granner, D. K. (1988) Mol. Cell.

20. Miner, J. N., and Yamamoto, K. R. (1992) Genes & Deu. 6,2491-2501 21. Schmid W. Striihle U. Schiitz, G., Schmitt, J., and Stunnenberg, H.

22. Gametchu, B., and Harrison, R. W. (1984) Endocrimlog 114,274-279 23. Diamond, M. I., Miner, J. N., Yoshinaga, S. K., and $amamoto, K. R.

24. Lai, J.-S., and Herr, W. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6958-

. _ _ ."

(1991) J. Biol. Chem. 2 6 6 , 19095-19102

D. K. (1990) Mol. Endocrinol. 249,1302-1310

Biol. &96-104

(198d) EdBO J. 8: 226772263

(1990) Science 249,1266-1272 COC9

25. BerGrM. (1989) Cell 56,335-344 26. Jonat, C Rahmsdorf H. J., Park K.-K., Cato, A. C. B., Gebel, S., Ponta,

27. Schule, R., Ran arajan P I Kliewer, S., Ransone, L. J., Bolado, J., Yang, H., and Herrlich, P: (1990) Cell'62, 1189-1204

28. Yang-Yen H.-F., Chambard, J.-C., Sun, Y.-L., Smeal, T., Schmidt, T. J., N., Verma, I. h., and Eians, R. M. (1990) Cell 62,1217-1226

29. Nicholsoi R. C Made; S Na al, S., Leid, M., Rochette-Egly, C., and Drouin 'J. and Karin M. (1990) Cell 62,1205-1215

30. Gaub, M.-P., Bellard, M., Scheuer, I., Chambon, P., and Sassone-Corsi, P. Chambdn, P. (1990) EMBO J.%, 4443-4454

31. Schule R. Umesono K. Mangelsdorf D. J., Bolado, J., Pike, J. W., and (1990) Cell 63,1267-1276

32. Owen T: A., Bortell R., Yocum, S. A. Smock, S. L., Zhang, M. Abate C., Eva& R. M. (199d) Clll 61,497-502

Shhhouh V., Arinin N., Wri ht, k L , Van Wunen, A. J., Stein, J: L.,, Curran, 'f., Lian, J. b., and itein, G.'S. (1990) Proc. Natl. Acad. Scr.

33. Ozono, K., Liao, J., Kerner, S. A., Scott, R. A,, and Pike, J. W. (1990) J.

34. Granner, D. K. (1979) in Glucocorticoid Hormone Action (Baxter, J. D., and

U. S. A . 87,9990-9994

Biol. Chem. 2 6 5 , 21881-21888

Rousseau, G., eds) Vol. 12, pp. 593-609, Springer-Verlag, Heidelberg