Characterization of DNA Binding and Retinoic Acid Binding Properties of Retinoic Acid Receptor

Home , Retinoic acid receptor

Proc. Natl. Acad. Sci. USA Vol. 88, pp. 3559-3563, May 1991 Physiology/Pharmacology Characterization of DNA binding and retinoic acid binding properties of retinoic acid receptor

NA YANG*t, ROLAND SCHLLEt, DAVID J. MANGELSDORFt, AND RONALD M. EVANSt *Department of Chemistry, University of California, San Diego, La Jolla, CA 92093; and tHoward Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, CA 92037 Contributed by Ronald M. Evans, December 28, 1990

ABSTRACT High-level expression of the full-length hu- RAR and the factor is localized in the DNA binding domain man retinoic acid receptor (RAR) a and the DNA binding of RAR. domain of the RAR in Escherichia coli was achieved by using Activation of RAR is known to be triggered by the asso- a T7 RNA polymerase-directed expression system. After in- ciation with its ligand RA. However, determination of the duction, full-length RAR protein was produced at an estimated accurate dissociation constant of RA with RAR from mam- level of 20% of the total bacterial proteins. Both intact RAR malian sources has been hampered because of the interfer- molecules and the DNA binding domain bind to the cognate ence of the cellular RA binding protein in mammalian tissues DNA response element with high specificity in the absence of (15). In contrast, the RAR produced in E. coli binds RA with retinoic acid. However, this binding is enhanced to a great high affinity, displaying a Kd of 2.1 x 10-1o M. extent upon the addition ofeukaryotic cell extracts. The factor responsible for this enhancement is heat-sensitive and forms a AND METHODS complex with RAR that binds to DNA and exhibits a distinct MATERIALS migration pattern in the gel-mobility-shift assay. The interac- Expression ofHuman (h) RARa and RARDBD in E. coli. The tion site ofthe factor with RAR is localized in the 70-amino acid cloning procedure offull-length hRARa cDNA was described DNA binding region of RAR. The hormone binding ability of in ref. 16. For expression of the DNA binding domain of the RARa protein was assayed by a charcoal absorption assay RAR, the cDNA sequence of RARa corresponding to amino and the RAR protein was found to bind to retinoic acid with a acids 87-156 (6) was PCR-amplified. An Nco I site and a Kdof 2.1 x 10'10M. BamHI site flanking the 5' and 3' ends, respectively, were put in for subsequent cloning into the pET-8c vector (14). The All-trans-retinoic acid (RA), a vitamin A derivative, is a resulting plasmids pET-8cRARDBD or pET-8cRAR for full- biologically active vertebrate morphogen. It has profound length RAR expression were then transformed into E. coli effects on cellular differentiation, pattern formation, and BL21(DE3)plysS. A single colony was picked and inoculated embryonic development. RA also has a striking effect on in LB medium with ampicillin (25 ,ug/ml) and chloramphen- regenerating limbs and has been implicated as a natural icol (25 /Lg/ml). The culture was grown at 370C until cell morphogen in chicken and frog embryogenesis (refs. 1-5 and density corresponding to an OD6w of 0.7 was reached. references therein). Functions of RA are presumed to be Isopropyl 83-D-thiogalactopyranoside (IPTG; Boehringer mediated in part by its nuclear receptor proteins that are Mannheim) was added to 0.4 mM to induce RAR protein. members ofthe steroid hormone receptor superfamily. Three Induction continued for 3 hr and cells were collected by species of RA receptor (RAR) cDNAs have been cloned and centrifugation. The cell pellet was then resuspended in 10o are referred to as a, ,, and y (refs. 6-10). However, despite of the original volume of the lysis buffer (20 mM Hepes, pH the fact that certain genes regulated by RAR have been 7.9/60 mM KCI/2 mM dithiothreitol). Cells were lysed by identified (refs. 11-13 and references therein), the molecular two cycles offreezing/thawing. Glycerol [20%o (vol/vol)] was events that occur between RA signaling and the end biologic then added. Centrifugation at 100,000 x g was performed to effects are largely unknown as yet. To understand how RAR separate soluble proteins from cell debris. The supernatant interacts with its cognate DNA response sequence and its was saved and used for subsequent DNA binding and hor- ligand RA as well as the mechanisms of its regulation, we mone binding analyses. have expressed the full-length human RARa protein and the Gel-Mobility-Shift Assay. DNA binding activity was as- DNA binding domain of RAR in Escherichia coli under the sayed by a gel-retardation assay. 32P-labeled DNA oligonu- control of T7 polymerase (14). After isopropyl ,B-D- cleotide probes (8 x 104 dpm; 1 ng) were incubated with 1 ,1 thiogalactopyranoside (IPTG) induction, up to 20% of the ofpET-8cRAR- or pET-8cRARDBD-transformed BL21(DE3)- bacterial proteins is full-length RAR protein. In vitro DNA plysS extracts in the binding buffer [6 mM KCI/22 mM binding studies show that the bacterially expressed full- Hepes, pH 7.9/0.2 mM dithiothreitol/5 mM spermidine/8% length RAR protein and the 70-amino acid DNA binding glycerol/2% (vol/vol) Ficoll/0. 1% Nonidet P-40]. Poly- domain bind specifically to a 27-mer oligonucleotide defined (dIdC) (1 ,ug) was present in each binding reaction mixture as as the RAR response element in the promoter region of the the nonspecific DNA competitor. Eukaryotic cell extracts RAR,B gene (refs. 11 and 13). However, the affinity of this were prepared as described in ref. 17. Total protein of cell binding is greatly enhanced by a heat-sensitive factor in extracts (5 ,ug) was used in the binding reaction. For heat eukaryotic cells. Upon addition of cellular extracts to the inactivation, cell extracts were treated at 65°C for 10 min gel-mobility-shift assay, the RAR-DNA complex is further before addition to the binding reaction. For the competition retarded. We conclude that in vivo, RAR protein requires assay, a 25-fold molar excess of unlabeled oligonucleotide another protein factor for high-affinity DNA binding. We was added simultaneously with the radioactive probe. Incu- show herein that at least one site of the interaction between bation was carried out on ice for 10 min. Separation of bound

The publication costs of this article were defrayed in part by page charge Abbreviations: RA, retinoic acid; RAR, RA receptor; IPTG, isopro- payment. This article must therefore be hereby marked "advertisement" pyl ,B-D-thiogalactopyranoside; h, human; ,lRE, /3-responsive ele- in accordance with 18 U.S.C. §1734 solely to indicate this fact. ment; GR, glucocorticoid receptor. 3559 Downloaded by guest on September 25, 2021 3560 Physiology/Pharmacology: Yang et al. Proc. Natl. Acad. Sci. USA 88 (1991) probes from free probes was by PAGE on a 5% gel in 0.5 x A SD 1 TBE (44 mM Tris/44 mM boric acid/0.5 mM EDTA, pH 8.0). Electrophoresis was performed at 40C. The gel was preelec- trophoresed for 1.5 hr at 200 V. The dried gel was autora- diographed with an intensifying screen at -70'C with Kodak XAR film. hRARa RA Binding. The charsorb method (18) was used to determine the hormone binding activity ofRAR. A desired amount of 3[H]RA was dried down to evaporate trace of ethanol and mixed thoroughly with 1 ul of RAR containing bacterial lysate and 79 /.l of mock BL21(DE3)plysS lysate in the RA binding buffer (0.12 M KCl/8 mM TrisHCl, pH 7.4/8% glycerol/4 mM dithiothreitol/0.24 mM phenylmethylsulfonyl fluoride). The final volume was 1 ml. To determine nonspecific binding, a 100-fold molar excess of unlabeled RA was mixed with radioactive RA before addition of the lysate. Incubation was carried out at 40C for 16 hr in the dark. Then 500 1.l of 3% (wt/vol) activated charcoal (Sigma) was added B at the end ofincubation, mixed well, and incubated at 40C for 1 2 3 15 min. Charcoal was activated according to ref. 18 and was w w s then brought to 3% in 0.15 M NaCl/0.1 M Na2HPO4/0.039 M IPTG - + + NaH2PO4/0.1% gelatin/0.015 M NaN3, pH 7.03. Charcoal- absorbed free RA was then separated from bound RA by centrifugation at 15,000 x g for 15 min. The supernatant was collected and radioactivity was measured in 20 ml ofEcolume (ICN Biochemicals). 97,400---

RESULTS 66,200-- -- Expression of hRARa and the DNA Binding Domain ofRAR in E. coli. To produce the RAR protein in E. coli, a full-length _W 40- RARcx hRAR coding sequence was inserted into the high-level 45,000-- _ _ expression plasmid pET-8c vector (Fig. 1A). As indicated in the diagram, the hRARa sequence starting from the initiation methionine was inserted into the Nco I-BamHI cloning site of the vector. The resulting plasmid allows the production of a full-length nonfusion RAR protein under the regulation of 31,000- the T7 promotor. Cleavage of the first methionine residue during bacterial synthesis makes this protein product one amino acid shorter than wild-type RAR. This was confirmed by microsequencing. pET-8cRARa plasmid was transformed 21 ,500--- _ into E. coli BL21(DE3)plysS, which harbors in its genome the T7 polymerase gene driven by an IPIG-inducible UV5 promotor. After a 3-hr IPTG induction, high-level expression of FIG. 1. Construction of pET-8cRAR and expression of RAR RAR protein with the predicted molecular mass of 54 kDa protein in E. coli. (A) A schematic presentation of plasmid pET- 8cRAR. hRARa coding sequence starting from the first methionine was observed (Fig. 1B, lanes 1 and 2). The estimated level of is presented as an open box. First and last amino acids of RAR are induced RAR protein was about 20% of the total bacterial indicated by numbers 1 and 462. Nco I, BamHI, and Asp718 cloning proteins (Fig. 1B, lane 2). Two freeze/thaw cycles were sites are indicated. The Shine-Dalgarno sequence (SD) is preceded sufficient to lyse bacteria cells to completion because of the by T7 promotor T7410-slO. (B) Expression of hRARa in E. coli. low-level constitutive expression of lysozyme in Before (lane 1) and after (lane 2) a 3-hr IPTG induction, cells in 20 BL21(DE3)plysS (14). RAR proteins were recovered in the ,ul of cell culture were lysed in loading buffer and proteins were supernatant fraction as shown in Fig. 1B, lane 3, which resolved on a 10%o polyacrylamide gel containing SDS and stained indicates that full-length RAR protein is highly soluble. The with Coomassie brilliant blue R. Lane 3 is the soluble fraction (20 ul) DNA binding domain of RARa was induced at a from a culture after 3 hr of IPTG induction. Molecular weight slightly markers are labeled on the left of the gel. RARa protein with a lower level compared to full-length RAR (data not shown). molecular mass of 54 kDa is indicated by the arrow on the right. About 60%o of the RARDBD polypeptide can be recovered in Lanes: W, whole cell; S, soluble fraction. +, IPTG induction; -, no the soluble fraction of the bacterial lysate. induction. DNA Binding Property of the RAR. We have analyzed the in vitro DNA binding activity of the RAR by a gel-mobility- with /3RE is competed by an excess amount ofunlabeled PRE shift assay. The cognate DNA sequence used in the assay is neither the a 27-mer oligonucleotide from the promotor region of the oligonucleotide (lanes 4-6), glucocorticoid recep- RARj3 gene. This sequence contains a perfect direct repeat of tor responsive element nor the inactive RAR response ele- the nucleotide sequence motif GTTCAC and has been de- ment derivative mtl (16) can compete the binding (lanes 8 and fined as a natural RAR response element, which is both 7, respectively). In vitro DNA binding activity is independent necessary and sufficient for mediating RA responsiveness of the presence or absence of RA. (11, 13). As shown in Fig. 2, RAR expressed in E. coli binds The DNA binding activity of RAR was assigned to a the 8-responsive element (I3RE) to give rise to a retarded 66-amino acid peptide sequence residing in the middle of the band (lane 3). This binding is highly specific for the RAR RAR molecule based on sequence homology of RAR with response element but not for other DNA sequences as shown other receptors of the family (6). To demonstrate that this by the competition experiment. Whereas binding of RAR region of the receptor is a functionally independent domain Downloaded by guest on September 25, 2021 Physiology/Pharmacology: Yang et al. Proc. Natl. Acad. Sci. USA 88 (1991) 3561

1 2 3 4 5 6 7 8 mock RAR RARDBD competitor - - - - ORE - mtl GRE cell extract -+! [7- +

Is _

1 2 3 4 5 6 FIG. 3. Eukaryotic cellular factor enhances RAR DNA binding and the factor interacts with the DNA binding domain of RAR. Gel retardation was done under the same conditions as in Fig. 2. Total proteins of COS cell extracts (5 Ag) were added to the binding reaction mixtures (+; lanes 2, 4, and 6). Lanes: 1 and 2, 2 Al of BL21(DE3)plysS lysate; 3 and 4, 2 p.1 of RAR-transformed bacterial FIG. 2. DNA binding properties of hRARa assayed by gel- lysate; 5 and 6,22A1 ofRARDBD-transformed bacterial lysate. Arrows mobility shift. A 32P-labeled oligonucleotide (1 ng) containing /RE on the left indicate the RAR-DNA complex (lane 3) and the RAR- was incubated with 2 ,u1 of a RARa-containing bacterial lysate in the DNA-cellular factor complex (lane 4). The arrow on the right absence of a responsive element (-, lane 3) or the presence of 5 ng, indicates the RARDBD-DNA-cellular factor complex. -, Cell extract 10 ng, or 25 ng of unlabeled /BRE (lanes 4-6, respectively) or 25 ng not added. of mtl or 25 ng ofglucocorticoid receptor responsive element (GRE) competitor oligonucleotides (lanes 7 or 8, respectively) for 10 min at DNA Binding Enhancing Factor Is Heat-Sensitive and In- 0WC. Lanes 1 and 2 are control experiments. Lane 1 has no protein teracts with the RAR DNA Binding Domain. To test the added. Lane 2 has 2 A.l of mock bacterial extract added. Mock bacterial extract is prepared from a BL21(DE3)plysS culture after a stability of the factor, we pretreated the cell extracts at 650C 3-hr IPTG induction. Poly(d-dC) (1 fug) was added to each reaction for 10 min. The DNA binding enhancing activity is com- mixture as a nonspecific DNA competitor. The DNA-protein com- pletely destroyed after this treatment (Fig. 4, lane 6). Most plex was then resolved from free probe on a 5% nondenaturing interestingly, when 5 pug of total proteins from a COS cell polyacrylamide gel. extract was added to RARDBD in this analysis, a much enhanced DNA binding activity was detected (Fig. 3, com- structure that has specific DNA binding activity, we ex- pare lane 6 with lane 5). The new complex ofthe RARDBD and pressed the polypeptide sequence from amino acid 87 to the DNA binding enhancing factor is very stable under amino acid 156 of RAR encompassing the DNA binding high-stringency conditions. domain in E. coli. As shown in Fig. 3, this 70-amino acid Based on these experiments, we conclude that a protein polypeptide binds to the PRE (lane 5). (Due to the tendency factor in eukaryotic cells is required for high-affinity DNA to overexposure of other lanes in this gel, lane 5 was much binding of the RAR protein. And at least one site of this underexposed.) interaction is located in the DNA binding domain of RAR. High-Affinity RAR DNA Binding Requires a Protein Factor RAR Protein Binds to RA with High Affinity. The charcoal Present in Eukaryotic Cell Extracts. Both intact RAR and the absorption hormone binding assay was used to analyze the DNA binding domain of RAR bind the DNA probe with high ligand binding property of RAR (18). The linear plot of the specificity. However, this binding can only be detected under saturation binding isotherm for RA-RAR binding is shown in low-stringency condition. The presence of KCI or prolonged Fig. 5A. Nonspecific binding determined by addition of a incubation will dissociate the DNA-protein complex com- 100-fold molar excess of unlabeled RA showed a linear pletely, indicating that the binding has a very high offrate and response. Specific binding of RAR protein was obtained by hence a very low association constant. We therefore specu- subtracting values for nonspecific binding from total binding. lated that a nuclear factor might exist to promote or improve No specific binding was detected in a mock-transformed RAR binding to DNA. To address this question, cell extracts BL21 bacteria cell lysate. A dissociation constant of 2.1 x from eukaryotic sources were added to the in vitro binding 10-10 M was determined by Scatchard analysis (Fig. 5B). reaction mixture. Interestingly, in the presence of 5 ,ug of total proteins from monkey kidney COS cell extracts, RAR displayed a much stronger DNA binding activity (Fig. 3, DISCUSSION compare lane 4 with lane 3). Other mammalian cell extracts The key event in the process of RAR activation and subse- tested including F9 cells and HeLa cells showed the same quent gene transcription is the association of RAR with RA. activity. Drosophila melanogaster Schneider cell extracts The efficient binding of bacterially expressed RAR to RA is also conferred this DNA binding enhancing activity (Fig. 4, significant for several reasons. First, the binding kinetics compare lane 5 with lane 4). In the presence and absence of have been difficult to measure in mammalian cells because of cell extracts, the RAR-DNA complex showed different mo- the relatively low levels of RAR compared to the high bilities on the gel (Figs. 3 and 4). These further retarded bands concentrations of the endogenous cellular RA binding pro- suggest that RAR, DNA, and a third factor formed a new tein. This is further confounded by the high affinity of the complex. This complex was very stable'under high salt and cellular RA binding protein for RA (Kd = 7-42 nM) (19). Thus high-stringency conditions. these properties conspired to preclude identification of the Downloaded by guest on September 25, 2021 3562 Physiology/Pharmacology: Yang et al. Proc. Natl. Acad. Sci. USA 88 (1991) control RAR A I I 10000 .

x x~ -x -x G) a) a) CD 8000 -

6000.

V 4000. c I 0co 2000 .

1.2 3[H]R.A. (nM) B

0.30 -

0.20- It 1 2 3 4 5 6 0co 0.10 - FIG. 4. DNA binding enhancing activity is present in Schneider cells and the factor is heat-sensitive. Gel retardation was done under 2.1 x10 'oM the same condition as in Fig. 2. An untransformed bacterial lysate K~d- (lanes 1-3) and a RAR-transformed bacterial lysate (lanes 4-6) were .I . . .. I . I . 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 incubated with cell extracts. Lanes: 1 and 4, no protein (-); 2 and 5, 4 jug of proteins of Schneider cell extracts (extr.); 3 and 6, 4 ,.g of 3[1IR.A. (nM) proteins ofSchneider cell extracts pretreated at 650C for 10 min [extr. (H.I.)]. Arrows indicate the migration of the RAR-DNA complex in FIG. 5. Saturation curve and Scatchard analysis. (A) RA binding the absence (lower arrow) or presence (upper arrow) of Schneider was assayed by the charcoal absorption assay. Total bacterial cell extracts. extracts (1 ,l) containing RAR were incubated with [3H]RA for 16 hr at 40C in the dark in 1 ml of binding buffer. Nonspecific binding was endogenous RAR protein prior to the cloning of its cDNA. determined by addition of 100-fold molar excess of unlabeled RA. The expression studies confirm that Free RA was separated from bound RA by charcoal absorption. No bacterially synthesized specific binding was seen in mock-transformed BL21(DE3)plysS RARa possesses an intrinsic affinity for RA (Kd = 2.1 x 10-10 extracts. o, Nonspecific binding activity; o, total binding activity; *, M) that is close to its half-maximal values of stimulating RAR-specific binding activity. (B) Scatchard analysis of [3HJRA cellular differentiation and transcriptional activation. In ad- binding in extract prepared from pET-8cRAR-transformed IPTG- dition, it has been suggested that glucocorticoid receptor induced BL21 cells. Each point was assayed in duplicate. The line (GR) hormone binding activity is modulated by another was best-fitted by a Cricket graph program. The Kd value was protein factor hsp90. When expressed in E. coli in the calculated as 2.1 x 10-10 M. absence of hsp90 GR protein products bind dexamethasone DNA binding and hormone binding activities. We have also with a Kd value about two magnitudes lower than the GR identified a eukaryotic cellular protein factor whose presence protein expressed in eukaryotic cells. In contrast, our data greatly facilitates RAR-DNA binding. Expression of the showed that no posttranslational modification or other eu- functional hRARa protein provides us with the possibility of karyotic cellular factors are required for high-affinity RA binding. This is consistent with the experimental observation studying the biochemical and physical chemical properties of hRAR molecules as well as the that unlike GR and progesterone receptor (PR), RAR is free molecular mechanisms of of association with hsp90 in the cell. transcriptional activation mediated by RAR. The DNA binding domain of nuclear receptors has been 1. Brockes, J. P. (1989) Neuron 2, 1285-1294. defined as a 66-amino acid peptide sequence located in the 2. Thaller, C. & Eichele, G. (1987) Nature (London) 327,625-628. middle of the primary sequence of the receptor molecules, 3. Roberts, A. B. & Sporn, M. B. (1984) in The Retinoids, eds. based on the fact that this region is highly conserved in all Sporn, M. B., Roberts, A. B. & Goodman, D. S. (Academic, members ofthe family. We now demonstrate that this central Orlando, FL), Vol. 2, pp. 209-286. core of the receptor molecule when expressed in E. coli 4. Maden, M., Ong, D. E., Summerbell, D. & Chytil, F. (1988) functions as an independent domain fully capable of binding Nature (London) 335, 733-735. DNA in a sequence-specific fashion. We have also shown 5. Durston, A. J., Timmermans, J. P. M., Hage, W. J., Hendriks, that this peptide sequence forms a complex with a cellular H. F. J., de Vries, N. J., Heideveld, M. & Nieuwkoop, P. D. protein factor that exhibits remarkably improved DNA bind- (1989) Nature (London) 340, 140-144. ing kinetics, suggesting that in vivo this sequence 6. Giguere, V., Ong, E. S., Segui, P. & Evans, R. M. (1987) interacts Nature with another factor as a prerequisite for (London) 330, 624-629. the RAR-DNA 7. Petkovich, M., Brand, N. J., Krust, A. & Chambon, P. (1987) binding. Nature (London) 330, 444-450. In conclusion, we have developed a high-expression sys- 8. Benbrook, D., Lernhardt, E. & Pfahl, M. (1988) Nature (Lon- tem for producing large quantities of nuclear receptor pro- don) 333, 669-672. teins in E. coli. Bacterially expressed receptors exhibit full 9. Brand, N., Petkovich, M., Krust, A., Chambon, P., de The, H., Downloaded by guest on September 25, 2021 Physiology/Pharmacology: Yang et al. Proc. Nati. Acad. Sci. USA 88 (1991) 3563

Marchio, A., Tiollais, D. & Dejean, A. (1988) Nature (London) 15. Chytil, F. & Ong, D. E. (1984) in The Retinoids, eds. Sporn, 332, 850-853. M. B., Roberts, A. B. & Goodman, D. S. (Academic, Orlando, 10. Krust, A., Kastner, P. H., Petkavich, M., Zelent, A. & Cham- FL), Vol. 2, pp. 89-123. bon, P. (1989) Proc. Nadl. Acad. Sci. USA 86, 5310-5314. 16. Schule, R., Mangeldorf, D. J., Umesono, K., Borado, J. & 11. de The, H., Vivanco-Ruiz, M., Tiollais, P., Stunnenberg, H. & Evans, R. M. (1990) Cell 61, 497-504. Dejean, A. (1990) Nature (London) 343, 177-180. 17. Damm, K., Thompson, C. C. & Evans, R. M. (1989) Nature 12. Vasios, G. W., Gold, J. D., Petkovich, M., Chambon, P. & (London) 339, 583-597. Gudas, L. J. (1989) Proc. NatI. Acad. Sci. USA 86, 9099-9103. 18. Dokoh, S., Pike, J. W., Chandler, J. S., Mancini, J. M. & 13. Sucov, H. M., Murikami, K. K. & Evans, R. M. (1990) Proc. Haussler, M. R. (1981) Anal. Biochem. 116, 221-222. Nadl. Acad. Sci. USA 87, 5392-53%. 19. Haussler, M. R., Donaldson, C. A., Kelly, M. A., Mangels- 14. Studier, W. F., Rosenberg, A. H. & Dunn, J. J. (1990) Meth- dorf, D. J., Bowden, G. T., Meinke, W. J., Meyskens, F. L. & ods Enzymol. 185, 60-89. Sidell, N. (1984) Biochim. Biophys. Acta 803, 54-62. Downloaded by guest on September 25, 2021