Transcriptiona! Activation by Nur77, a Growth Factor-lnducible Member of the Steroid Hormone Receptor Superfamily

Ian J. Davis, Thomas G. Hazel, and Lester F. Lau' Department of Genetics University of Illinois College of Medicine Chicago, Illinois 60612

upon binding to its cognate ligand, interacts with specific DNA sequences associated with its target genes. nur77 is of interest not only because it is the only known member of the steroid receptor gene family that is rapidly and transiently induced by growth and differentiation signals, but also because its encoded protein may play a role in neuronal function (9,16). Analysis of the potential activities of Nur77 as a transcription factor presents a unique challenge, since neither the specific DNA sequence to which Nur77 binds nor the identity of its cognate ligand is known. To understand the function of Nur77, we have replaced its putative DNA-binding domain with the DNA-binding domain of either the E. coli LexA repressor or the yeast Gal4 transactivating protein, leaving both the Nur77 amino-terminal and carboxyl-terminal domains intact. This DNA-binding domain replacement strategy has been successfully applied for mechanistic studies on the glucocorticoid and estrogen receptors (17,18). We show that the Nur77-LexA and Nur77-Gal4 chimeras specifically transactivate a reporter gene under the control of either the LexA operator or the Gal4 DNAbinding site, respectively. Furthermore, the ability of these chimeric proteins to activate transcription does not require exogenously added ligands.

nur77 is a growth factor-inducible immediate early gene that encodes a protein with extensive sequence homology to members of the steroid/thyroid hormone receptor superfamily. By analogy to steroid receptors, the Nur77 protein is thought to act as a ligand-dependent transcription factor that regulates the genomic response to growth factors. Using chimeric gene constructs, we show that Nur77 can indeed function as a transcription activator. Furthermore, Nur77 chimeras can activate transcription in the absence of an exogenously added ligand. (Molecular Endocrinology 5: 854-859, 1991)

INTRODUCTION

The induction of cell growth or differentiation by polypeptide growth factors is accompanied by the rapid activation of a set of genes whose transcription does not require de novo protein synthesis (1-4). Among these immediate early genes are those that encode known or putative transcription factors, including members of the Fos and Jun families, Myc, Rel, and several zinc-finger containing proteins (5-7). These nuclear proteins are thought to activate and/or repress sets of genes, resulting in biological responses specific to the stimulating growth factor. One such immediate early nuclear protein, Nur77 (also known as NGFI-B, N10, TIS1, and NAK-1) (6, 8-11), appears by sequence homology to be a member of the steroid/thyroid hormone receptor superfamily (12-14). Members of this superfamily are typically composed of a nonconserved amino-terminal domain that plays a role in transcriptional regulation, a highly conserved central region containing two DNA binding zinc-finger motifs, and a moderately conserved carboxyl-terminal domain that binds ligand and is involved in dimerization (12-15) (Fig. 1A). By analogy to known steroid receptors, Nur77 is hypothesized to function as a transcription factor that,

RESULTS AND DISCUSSION Nur77 Chimeras Function as Transcription Activators To test whether Nur77 can function as a transcription activator, we have constructed fusion genes that express chimeric proteins consisting of Nur77 fused to the DNA-binding domains of known transcription regulators. By homology to other steroid receptors, the putative DNA-binding domain of Nur77 spans amino acids (aa) 270-335 (8) (Fig. 1A). We have replaced this region (aa 269-340) with sequences that encode the DNA-binding domains of either the E. coli LexA repressor or the yeast transcription factor Gal4 (Fig. 1 A); both DNA-binding domains recognize DNA in a sequence-

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Transcriptional Activation by Nur77

specific manner but lack the ability to activate transcription. However, fusion proteins containing these DNAbinding regions linked to transactivation domains are capable of activating transcription from a promoter that includes the cognate DNA-binding sequence (17, 19). Furthermore, when either the Gal4 or LexA DNA-binding domain is substituted for the DNA-binding region of a steroid hormone receptor, the chimeric protein maintains the native receptor's ligand dependence for activity (17,18). The positioning of the LexA and Gal4 DNAbinding domains within the Nur77 chimeras preserved the overall domain structure of native Nur77 and closely maintained its original size (Fig. 1A). Each fusion gene construct was cloned into an expression vector (pSG5) under the control of the simian virus-40 (SV40) early promoter (20). To test the transactivation properties of the Nur77 chimeras, these fusion genes were cotransfected into CV1 monkey kidney cells with reporter plasmids that contain various promoter elements cloned up-stream of the bacterial chloramphenicol acetyl transferase (CAT) gene (Fig. 1B). The LexA reporter (XBCO) contains the 26-basepair (bp) LexA operator sequence cloned upstream of the /3-globin promoter (17). For a negative control, we used a reporter (OBCO) that lacks the LexAbinding site, but is otherwise identical to XBCO. The Gal4 reporter (17MX2tkCAT) contains two perfect palindromic 17-nucleotide Gal4 DNA-binding sites cloned up-stream of the herpes virus thymidine kinase gene promoter (21). Cycling CV1 cells were cotransfected with a plasmid encoding a Nur77 chimera, a reporter plasmid, and a plasmid expressing 0-galactosidase under the control of the phosphoglycerate kinase promoter (pPGK-j8Gal) (22) as an internal control for transfection efficiency. After transfection, cell extracts were normalized for transfection efficiency and analyzed for CAT activity. Transfected cells express approximately equal levels of Nur77 or Nur77 chimeric proteins, as monitored by immunoprecipitation using affinity-purified polyclonal anti-Nur77 antibodies (16) (data not shown). As shown in Fig. 2, when either the XBCO or 17MX2tkCAT reporter was cotransfected into CV1 cells with the Nur77LexA or Nur77-Gal4 fusion gene, respectively, CAT activity was enhanced greater than 25-fold over cotransfection with the parental pSG5 vector. This enhancement was abrogated when the LexA or Gal4 DNA-binding site on the reporter plasmid was eliminated. Transfection of the unaltered full-length nurll cDNA did not induce CAT activity from either reporter plasmid. These results indicate that when linked to the DNA-binding domain of either LexA or Gal4, the Nur77 protein activates transcription specifically from a promoter with the cognate DNA-binding site. That the Nur77 fusion proteins act on the transcriptional level was confirmed by RNase protection analysis of the reporter plasmid transcripts (data not shown). Transcriptional Activation Is Independent of Exogenously Added Ligand Godowski et al. (17) have shown that when the DNAbinding domain of the glucocorticoid receptor was sub-

855

stituted with that of LexA (LxAZ), dexamethasonedependent transcription from XBCO can be observed in a cotransfection assay. In parallel experiments we show that, indeed, the addition of 1 HM dexamethasone induced CAT activity about 10-fold in a cotransfection of LxAZ and XBCO (Fig. 2), indicating that the glucocorticoid receptor is ligand-dependent under our assay conditions. Unlike the glucocorticoid receptor, the Nur77 chimera-dependent activation appears to occur without requiring an exogenously added ligand. To test whether the addition of a known steroid/thyroid hormone would enhance the activity of Nur77, we added /3-estradiol (1 MM)> dexamethasone (1 HM), testosterone (1 HM), and T4 (3,5,3',5'-tetraiodothyronine; 1 MM) to the cotransfection assays, as described above. None of these compounds quantitatively altered the transactivation activity of Nur77 chimeras, suggesting that Nur77 activity is independent of these purified exogenous ligands (data not shown). Since it has been recognized that the phenol red in tissue culture medium and small organic molecules in serum are capable of activating some steroid receptors (23), we cultured and transfected CV1 cells in medium lacking phenol red and containing charcoal-stripped serum. Under these conditions, Nur77-Gal4 still activated CAT transcription from 17MX2tkCAT (Fig. 3), indicating that neither phenol red nor small molecules in serum that are adsorbed by charcoal were responsible for activating Nur77. Since it has been observed with other steroid hormone receptors that removal of the ligand-binding domain alters the receptor activity (24), it was of interest to test whether deletion of the putative ligand-binding domain of Nur77 would affect transactivation. For this purpose, a construct that truncates the Nur77-Gal4 chimera four aa after the Gal4 DNA-binding domain was prepared by introduction of a frameshift mutation, resulting in Nur77-Gal4Trunc (Fig. 1A). This construct, when cotranfected with 17MX2tkCAT into CV1 cells, was able to induce CAT activity. Furthermore, the level of induction was nearly identical to that of the full-length Nur77-Gal4 fusion (Fig. 3). Taken together, these results indicate that Nur77 can activate transcription without an exogenously added ligand, and that the presence of the carboxyl-terminal domain does not significantly alter its transactivation activity. However, we cannot rule out the possibility that the Nur77 chimeric proteins are altered such that a ligand is no longer required for activity. This appears unlikely, since numerous chimeric gene fusion experiments have demonstrated functional independence of the ligand-binding domain of steroid receptors (17, 25). Transcriptional Activation by Nur77 Chimeras Is Constitutive in Both 3T3 Fibroblasts and PC12 Cells Like most immediate early genes, the levels of nur77 mRNA and protein are very low in quiescent or unstimulated cells. Stimulation by various agents results in a rapid and transient increase in nur77 mRNA and protein;

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MOL ENDO-1991 856

270

aa 1

335

1 I 8V40 [Putative Tranacrlotlonl DNA I artyPromoterl Activating Domain |2 70-338

Nur77-LexA

Nur77

I

Nur77-Gal4Trunc

V—

I

341 {

I

Nur77

aa 1

I

SV40 pA

601 Nur77

268

la 1 Nur77-Qal4

341

268

aa 1

601 Putatlv. Ligand Binding Domain

1 W4

I

601 Nur77

268

Nur77

B

0*14 1-74

\Nur77\\\\\ Stop TAA

XBCO

OBCO

17MX2tkCAT 17MX2

CAT

Fig. 1. Constructs for Transactivation Assays A, Expression constructs were each cloned under the control of the SV40 early promoter in the vector pSG5 (see text). The Nur77 construct expresses the full-length 601-aa protein, whereas the Nur77-LexA and Nur77-Gal4 constructs express the otherwise full-length Nur77 protein with aa 269-340 substituted by the DNA-binding domain of either LexA or Gal4, as diagrammed. The Nur77-Gal4Trunc construct is similar to Nur77-Gal4, except that a frame shift mutation was introduced to truncate the protein four aa after the Gal4 sequence. B, Reporter plasmids contain the bacterial CAT gene under the control of test promoters. XBCO (17) contains one copy of the LexA DNA-binding site up-stream of the /3-globin promoter; OBCO (17) is identical, except that the LexA-binding site has been deleted. 17MX2tkCAT (21) contains two copies of the GAL4-binding site up-stream of the herpes virus thymidine kinase promoter.

both have short half-lives (2,10,16, 26). In mouse 3T3 fibroblasts, nur77 is activated by serum or purified serum growth factors (1, 2, 10). In the rat pheochromocytoma cell line PC12, nur77 is activated by the differentiation agent nerve growth factor (NGF), by the mitogen epidermal growth factor, and to an even greater extent by membrane-depolarizing conditions, such as an elevated extracellular concentration of KCI (4, 26). If the transactivation activity of Nur77 is dependent on a specific ligand, this ligand must be present during the short window of time when the Nur77 protein is synthesized. One hypothesis is that conditions that activate nur77 also produce the ligand necessary to activate Nur77 as a transcription factor. For instance, a second messenger generated by extracellular stimulation may act as a ligand for Nur77. To test this possibility, NIH 3T3 cells were cotransfected with Nur77-Gal4 and 17MX2tkCAT, and cells were serum starved. Transfected cells were then stimulated with 20% serum for 2 h, and cellular extracts were made from both serum-starved and serum-stimulated transfected cells. Nur77-Gal4 transactivated CAT expression to similar levels in both starved and serum-stimulated 3T3 cells, whereas cotransfection with the parental pSG5 plasmid alone had no effect (Fig. 4). Similar results were observed with the Nur77LexA chimera cotransfected with XBCO (data not shown). In addition, when PC12 cells were cotrans-

fected with Nur77-Gal4 and 17MX2tkCAT, Nur77-Gal4 transactivated CAT activity to a similar level in both untreated cycling cells and cells depolarized with KCI or treated with NGF for 2 h (Fig. 4). Therefore, conditions that activate nur77 gene transcription do not appear to alter the ability of Nur77 chimeras to act as transcription activators. However, we cannot exclude the possibility that the DNA-binding domain of Nur77, which is absent in these chimeras, may be posttranslationally modified upon stimulation by various agents, resulting in altered activities. Taken together, these data show that the Nur77LexA and Nur77-Gal4 chimeras are able to activate transcription without the addition of an exogenous ligand, even in cells that have not been stimulated to synthesize Nur77. These results suggest that either the specific ligand for Nur77 is always present within the cell, or that Nur77 does not require a ligand for its activity. In contrast to other steroid receptors, Nur77 is synthesized transiently upon stimulation and is quickly degraded (16, 26). Thus, it is thus not surprising that cellular conditions would allow Nur77 to function upon its synthesis. There is now a large number of "orphan" receptors that are structurally related to steroid/thyroid hormone receptors for which there are no known ligands (13, 14). It is possible that Nur77 and other orphan receptors may not require a specific ligand for activity (27). If this

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Transcriptional Activation by Nur77

Nur77 Nur77LxA Nur77Gal4 LxAZ XBCO OBCO 17MX2tkCAT pSG5

+

+

+

+

+ 1UM Dex

%Conversion

1.4

1

38 0.9

1

CAT

0.6 26.5 1.8 2.3 20.5

Fig. 2. Transcriptional Activation by Nur77 Chimeras A, CV1 cells were transfected with 5 ^g Nur77 or glucocorticoid receptor chimera expression constructs, 1 ^g 17MX2tkCAT, or 2 fig XBCO or OBCO along with 4 ^g pPGK-/?Gal. Each transfection was adjusted with the pSG5 plasmid to 15 ^g DNA/10-mm dish. Where indicated, dexamethasone was added to a final concentration of 1 HM 48 h before harvesting. Extracts from cells were normalized for transfection efficiency and analyzed for CAT activity by TLC (see Materials and Methods). The sample labeled CAT represents a control reaction performed with purified CAT (Sigma). The percent conversion of chloramphenicol to the acetylated forms is shown.

is the case, the sequence conservation in the carboxylterminal domain among these proteins might be related to its role in receptor dimerization, interaction with other proteins, and transactivation, rather than ligand binding (15,28). The availability of the Nur77-LexA and Nur77-Gal4 chimeras will allow further structure/function analysis of Nur77, while the DNA sequence to which it binds is not known. Moreover, separation of functional domains has been extremely useful in localizing the transactivation domains of other steroid receptors. For example, a low level of transactivation activity that resides within the DNA-binding domain of the glucocorticoid receptor has complicated analysis of other transactivation domains (29). The use of these Nur77 chimeras will avoid this potential complication. Mutational studies to localize the Nur77 transactivation domain(s) are now underway.

MATERIALS AND METHODS Cell Culture CV1 cells were grown in Dulbecco's Modified Eagle's Medium (DME) containing 10% fetal bovine serum (Biologos, Naperville, IL) and supplemented with 100 U/ml penicillin and 100 Mg/ml streptomycin. NIH 3T3 cells were cultured in DME containing 10% calf serum (Hyclone, Logan, UT) with penicillin

and streptomycin. PC12 cells were grown on poly-L-lysinecoated plates in DME supplemented with 10% fetal bovine serum, 5% heat-inactivated horse serum (Sigma, St. Louis, MO), and penicillin and streptomycin. Where indicated, CV1 cells were grown in DME lacking phenol red supplemented with 10% fetal bovine serum that had been treated with charcoal. Nur77 chimera expression constructs To generate 5' and 3' deletions, the nur77 cDNA was restricted with Haell at position 1016 (nucleotide numbers are according to the cDNA sequence, as in Ref. 8) and treated with Ba/31 nuclease (30). The resulting DNA was repaired with Klenow polymerase, ligated to BamHI linkers, and cloned into pGEM3 (Promega, Madison, Wl). The end points of deletion clones were determined by sequence analysis. Two fragments, containing nucleotides 1-915 (encoding aa 1-268) and 11332467 (encoding aa 341-601) of the nur77 cDNA were joined at the BamHI site to create a nur77 cDNA lacking the DNAbinding domain. Heterologous DNA-binding domains were then cloned into the BamHI site of the nur77 internal deletion. The LexA fragment was isolated from a BamHI-Xftol digest of LxAZ (17) (a gift of P. Godowski), end filled with Klenow polymerase, and blunt end ligated between the nur77 5' and 3' deletions. The Gal4 fragment was isolated from pAG4 (31) (a gift of M. Ptashne) by polymerase chain reaction-mediated amplification (30) of the region encoding aa 1-74 (5'-primer, 5'-CTGGATCCGATGAAGCTACTGTCTTCTATC; 3'-primer, 5'-CCGGATCCCTCGAGGAAAAATCAGTAGAAATAGC). The 3'-primer incorporates a frame shift mutation truncating the fusion gene four aa after the BamHI site. This Gal4 fragment was cloned between the nur77 5' and 3' deletions to create Nur77-Gal4Trunc (Fig. 1A). To create the full-length Nur77-

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Gal4 fusion gene, Nur77-Gal4Trunc was cleaved at the unique Xho\ site adjacent to the BamH\ site, end filled with Klenow, and religated. This shifted the reading frame to encode the Nur77 putative ligand-binding domain, creating Nur77-Gal4 (Fig. 1 A). Constructs were confirmed by direct DNA sequence analysis and cloned into the EcoRI site of pSG5 (20), an expression vector using the SV40 early promoter. Construction of Reporter Plasmids XBCO (a gift from P. Godowski) and 17MX2tkCAT (a gift from P. Chambon) are as previously described (17, 21). XBCO contains the 26-bp LexA operator sequence flanked by Xho\ ends cloned into a plasmid containing the rabbit /3-globin promoter at position -125 relative to the transcription start site. OBCO was constructed by restricting XBCO with Xftol, followed by religation, which precisely removes the LexAbinding site. 17MX2tkCAT contains two 17-mer perfect palindromic repeats of the Gal4 DNA-binding site cloned 105 bp up-stream of the transcription start site of the herpes virus thymidine kinase gene. Both promoters drive the bacterial CAT gene, followed by the SV40 polyadenylation sequence.

Nur77 Nur77Gal4 Nur77Gal4Trunc 17MX2tkCAT PR %Conversion

0.6

PR

1

7.7

12.2

PR 7.2

CAT

13.1

Transfections and Enzymatic Assays

Fig. 3. Transcriptional Activation in Phenol Red-Free and Charcoal-Stripped Medium CV1 cells were cultured and transfected in phenol red-free medium containing charcoal-stripped serum. Sixteen hours after the addition of precipitate, cells were washed with PBS and refed with either the same medium or medium containing phenol red and untreated serum (PR). CAT activity was measured in cell extracts normalized for transfection efficiencies. The sample labeled CAT is the enzymatic control. The percent conversion of chloramphenicol to acetylated forms is shown.

*











CV1 and 3T3 cells were transfected by using the calcium phosphate precipitation procedure previously described (30). Briefly, 3 x 105 cells were seeded onto a 100-mm dish about 24 h before transfection. Calcium phosphate/DNA precipitates were added to cells and incubated at 37 C at 10% CO2 for 16 h, followed by refeeding and a further incubation for an additional 48 h before harvest. PC 12 transfections were carried out as previously described (32) with a 15% glycerol shock in DME for 3 min 5 h after the calcium phosphate/DNA precipitate was added to cells. Cells were refed with normal growth medium and incubated for 60 h before preparation of cell extracts. Where indicated dexamethasone was added to a final concentration of 1 ^M, NGF (2.5S; Collaborative Research, Waltham, MA) to 50 ng/ml, and KCI to 60 rriM. All transfections were carried out with 4 ng of a control plasmid (pPGK-/?Gal) that expresses /3-galactosidase from the phosphoglycerate kinase promoter (22) (a gift from P. Soriano). Cell extracts were analyzed for 0-galactosidase activity, as previously described (33), to normalize for transfection efficiencies, and normalized amounts of cellular extract were assayed for CAT activity as previously described (34). Conversion of chloramphenicol to acetylated forms was assayed by TLC and quantitated on a Betagen (Waltham, MA) Betascope 603.

Acknowledgments We are grateful to P. Chambon, P. J. Godowski, M. Ptashne, and P. Soriano for generous gifts of plasmids.

Received March 8, 1991. Revision received April 11,1991. Accepted April 11, 1991.

pSG5 Nur77Gal4 17MX2tkCAT i

%Conversion

0.7

KCI NGF

- KCI NGF Q S Q l i — PC12 — 3T31 0.7 26 38 26 0.6 0.5 69

S

CAT

73

Fig. 4. Constitutive Activity of Nur77 Chimeras in NIH 3T3 and PC12Cells NIH 3T3 and PC12 cells were cotransfected with the indicated plasmids as described in Fig. 2, except that in PC12 cells 10 ng expression plasmid were used. Transfected cells were subsequently serum-starved (Q) and restimulated with serum (S) for 2 h, or treated with NGF (50 ng/ml) or KCI (60 ITIM) for 2 h. Cell extracts normalized for transfection efficiencies were analyzed for CAT activity, and the percent conversion to acetylated forms is shown.

Address requests for reprints to: Dr. Lester F. Lau, Department of Genetics, University of Illinois College of Medicine, 808 South Wood Street, Chicago, Illinois 60612. This work was supported by USPHS Grant R01-CA-46565. * Pew Scholar. Supported by the American Cancer Society Junior Faculty Research Award.

REFERENCES 1. Lau LF, Nathans D 1985 Identification of a set of genes expressed during the G0/G1 transition of cultured mouse cells. EMBOJ 4:3145-3151

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Transcriptional Activation by Nur77

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Transcriptional activation by Nur77, a growth factor-inducible member of the steroid hormone receptor superfamily.

nur77 is a growth factor-inducible immediate early gene that encodes a protein with extensive sequence homology to members of the steroid/thyroid horm...
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