©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression Screening Reveals an Orphan Receptor Chick Ovalbumin Upstream Promoter Transcription Factor I as a Regulator of Neurite/Substrate-Cell Contacts and Cell Aggregation (*)

Henry Connor , Howard Nornes , Toomas Neuman (§)

From the (1)Department of Anatomy and Neurobiology, Colorado State University, Fort Collins, Colorado 80523

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

A rat homologue of chick ovalbumin upstream promoter transcription factor I (COUP-TF I) was isolated using an expression cloning method developed to iso-late neurite outgrowth inhibitors. Overexpression of COUP-TF I in 3T3 fibroblasts resulted in reduction of stable contact formation between neurites and transfected cells. Additionally, COUP-TF I enhanced retinoic acid response element-dependent reporter gene expression in 3T3 fibroblasts, indicating that COUP-TF I can modulate transcriptional activation in these cells. Our data suggest that COUP-TF transcription factors are involved in the regulation of cell surface molecules during neurogenesis.


INTRODUCTION

Nuclear hormone receptors, which function as ligand-dependent transcription factors, have a crucial role in establishing initial cellular diversity in the nervous system. Among the best characterized members of the superfamily of nuclear hormone receptors are the receptors for retinoids, thyroid hormones, steroid hormones, and glucocorticoids, whose importance during neurogenesis is well described (for review, see Beato(1989); Evans and Arriza(1989); McEwen et al.(1991); and Linney(1992)). Hormone receptors activate or repress gene transcription through binding to cis-acting hormone response elements (HRE)()(Green and Chambon, 1988; Beato, 1989). Besides the ligand-dependent transcription factors, the hormone receptor superfamily comprises several orphan receptors for which ligands are not known (for review, see Evans(1988) and Green and Chambon(1988)). Orphan receptors, chicken ovalbumin upstream promoter transcription factor (COUP-TF) homologues, have been isolated from Drosophila (Mlodzik et al., 1990), sea urchin (Chan et al., 1992), zebrafish (Fjose et al., 1993), Xenopus (Matharu and Sweeney, 1992), chick (Lutz et al., 1994), and mammals (Miyajima et al., 1988; Wang et al., 1989; Richie et al., 1990; Wang et al., 1991; Ladias and Karathanasis, 1991). One possible function of COUP-TF transcription factors is to regulate the activity of ligand-activated hormone receptors through heterodimer formation or competition for specific response elements (Cooney et al., 1992, 1993; Tran et al., 1992).

Analyses of nuclear hormone receptors in various systems clearly demonstrate that regulation of gene expression by these transcription factors depends on the presence of different ligands and also on the interactions with activators and repressors. The role of COUP-TF orphan receptors in neurogenesis is virtually unknown. In Drosophila, the expression of COUP-TF homologue seven-up is required for development of specific subset of photoreceptor neurons during eye development (Mlodzik et al., 1990). In chick (Lutz et al., 1994) and mouse (Jonk et al., 1994; Lu et al., 1994; Qiu et al., 1994), COUP-TFs are expressed in a complex spatial and temporal pattern during development of the nervous system.

The central nervous system of most adult vertebrates is inhibitory to axonal growth. During development, inhibitory regions regulate the formation of neuronal pathways and connections (Tosney and Landmesser, 1984; Davies et al., 1990). Several factors responsible for neurite outgrowth inhibition have been characterized (Caroni and Schwab, 1988; Cox et al., 1990; Davies et al., 1990; Raper and Kapfhammer, 1990; Luo et al., 1993). We developed a screening system for cloning inhibitory molecules after transfection of a cDNA library into ``substrate'' cells, which are subsequently cocultured with neuronal cells and screened for functional expression of neurite outgrowth inhibition. This approach allows isolation of cDNAs for surface molecules, which cause direct inhibition of neurite outgrowth, as well as cDNAs for factors involved in regulating the expression of molecules, which inhibit or facilitate neurite outgrowth. This screening system enabled us to isolate a rat homologue of orphan receptor COUP-TF I, whose overexpression results in decreased contact stability between neurites and substrate cells. These data suggest the possible involvement of COUP-TF I in the regulation of neurite contacts and cell aggregation.


MATERIALS AND METHODS

Cell Culture

Mouse NIH-3T3 fibroblasts and neuroblastoma glioma hybrid NG108-15 (Hamprecht, 1977) cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Sigma). Neuronal differentiation of NG108-15 cells was achieved by culture in serum-free Dulbecco's modified Eagle's medium for 72 h.

To establish cell lines overexpressing COUP-TF I, cDNA was cloned into the expression vector pRcCMV (Invitrogen) using HindIII linkers and transfected into 3T3 cells followed by selection with G418 (600 µg/ml, Life Technologies, Inc.) for 14-21 days.

Generation of Subtraction cDNA Library

Poly(A) RNA was isolated (FastTrack, Invitrogen) from brains and spinal cords of embryonic day 17, postnatal day 5, and adult rats and also from chronically injured spinal cords. 24 µg of Poly(A) RNA from postnatal day 5, adult, and injured spinal cord (8 µg from each) was used to synthesize first strand cDNA using oligo(dT) primer with a NotI restriction site at the 5`-end (5`-CTAGATCGCGAGCGGCCGCCCTTTTTTTTTTTTTTTTT) andSuperScript RNaseH-reverse transcriptase (200 units/µg of Poly(A) RNA, Life Technologies, Inc.). The first strand synthesis reaction was labeled using [P]dCTP (5 µCi, >3000 Ci/mmol, Amersham Corp.) to facilitate tracing of the DNA. First strand cDNA was hybridized to embryonic day 17 poly(A) RNA (200 µg) in sealed ampules (total volume, 100 µl, buffer 0.5 M sodium phosphate, pH 6.8, 300 mM NaCl, 2 mM EDTA, and 0.2% SDS) for 18 h at 70 °C. The hybridization mix was diluted to a final molarity of 0.08 M sodium phosphate, loaded onto a DNA grade hydroxylapatite column (4 ml (volume), Bio-Rad), and washed extensively with 0.08 M sodium phosphate buffer (pH 6.8). Single-stranded cDNAs were eluted in 10 ml of 0.15 M sodium phosphate buffer. Column fractions (0.5 ml) exhibiting radioactivity above background were pooled, and the cDNAs were concentrated by butanol extraction followed by chromatography in STE buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA) on a Sephadex G-25 column (Pharmacia Biotech Inc.). Single-stranded cDNAs were mixed with 100 µg of poly(A) RNA from embryonic day 17 for the second cycle of hybridization. After the second cycle, first strand cDNAs were hybridized with 5 µg of the original Poly(A) RNA isolated from postnatal day 5, adult, and injured rats nervous systems, and the resulting DNA/RNA hybrids were used as template for second strand synthesis with RNaseH and Escherichia coli DNA polymerase I (Life Technologies, Inc.). Blunt ends were created with T4 DNA polymerase, and the HindIII adapter (5`-AGCTTGGCACGAG-3`, 3`-ACCGTGCTC-5`) was ligated to the cDNA. Longer cDNAs (>700 bp) were isolated by digestion with NotI followed by size selection on a Sephacryl S 400 column (Pharmacia) and were subsequently cloned between the HindIII and NotI restriction sites of the expression vector pRcCMV (Invitrogen). The library was divided into 20 aliquots and used to transform E. coli DH5 cells (MAX Efficiency, Life Technologies, Inc.). Each aliquot yielded 5-8 10 colonies, which were combined and grown for large scale plasmid isolation (plasmid maxi kit, Qiagen). The cDNA expression library has 2 10 independent clones with an average insert size of 1.5 kilobases (range, 0.6-3.7 kilobases).

Isolation of Cell Clones and cDNA

Transfection of the cDNA library into fibroblasts was performed by the calcium phosphate coprecipitation technique using 20 µg of DNA/100-mm tissue culture plates (Falcon) at a cell density of 2 10 cells per plate with an incubation time of 15-16 h. Each aliquot of the cDNA library (n = 20) was used to transfect cells in 20 plates. The cDNA library was transfected into NIH-3T3 fibroblast cells. Selection of transfected cells was performed with 600 µg/ml G418 (Life Technologies, Inc.) for 14-21 days prior to testing for neurite outgrowth inhibition. Screening for neurite outgrowth inhibition involved coculture of transfected fibroblasts with the neuroblastoma-glioma hybrid cell line NG108-15 differentiated for 72 h in serum-free culture medium prior to coculture. Differentiated NG108-15 cells rapidly established an extensive and robust network of contacts with control NIH-3T3 cells, indicating that fibroblasts provide a good substrate for contact formation. Inhibitory clones were identified as those that had fewer or no contacts with surrounding neurites. Inhibitory clones were isolated and propagated.

Genomic DNA was prepared from isolated clones (TurboGen, Invitrogen) and used as a template for amplification of cDNAs by polymerase chain reaction. Primers for cDNA amplification corresponding to flanking sequences in the pRcCMV vector (5`-primer 5`-AGCTCTCTGGCTAACTAGAGAAC, 3`-primer 5`-AGCGAGCTCTAGCATTTAGGTGA) were prepared, and 35 cycles of polymerase chain reaction were performed using the following conditions: 92 °C, 1.2 min; 58 °C, 2 min; and 72 °C, 4 min. Amplified DNAs were cloned into EcoRV site of Bluescript plasmid (Stratagene) for sequencing. Isolated cDNAs with vector (pRcCMV) sequences were subcloned into pRcCMV expression vector between HindIII and NotI sites and retested for neurite inhibition.

Northern Blot Analyses

Total RNA was isolated using acid guanidinium/phenol/chloroform extraction procedure (Chomczynski and Sacchi, 1987). The RNA (25 µg/lane) was fractionated on 1.2% agarose-formaldehyde gel and transferred to a nylon membrane (Hybond N, Amersham). Full-length COUP-TF I was radiolabeled (P) using Multiprime DNA labeling system (Amersham) and used as a probe. The blots were washed at high stringency (0.2 SSC, 65 °C) and exposed to x-ray film for 3 days. Transferred RNAs were monitored by methylene blue staining of the filters before hybridization.

Analyses of Contact Stability between Transfected Fibroblasts and Neurites of NG108-15 Cells

Fibroblasts were plated in 12-well dishes at a density of 2 10 cells/well 72 h prior to coculture. Differentiated NG108-15 cells were added at a density of 2 10 cells per well. 2 h after coculture initiation, fibroblast colonies were identified, and the number of contacts between fibroblasts and NG108-15 cells were recorded at three time points: 1) starting time, t = 0; 2) 0.5 h later, t = 0.5; and 3) 2.5 h after the starting time, t = 2.5. The number of contacts was used to determine the direction and magnitude of any change in the dynamics of cell-cell interaction. Analysis of the data was performed with one-tailed t test.

Transfections and CAT Assays

Cells (3 10) in 60-mm dishes were cotransfected overnight with a combination of plasmid DNA (total 15 µg) by the calcium phosphate coprecipitation method. The DNA mix consisted of expression plasmid (pRcCMV or pRc-COUP-TF I, 9 µg), CAT reporter gene construct (DR-1, -RARE, CRBP I, 3 µg), and pRcCMV-lacZ (3 µg). The pRcCMV-lacZ construct was used to facilitate normalization of the CAT activity to transfection efficiency. Transfections were performed in triplicate. Transfection medium was replaced after 18 h with a control media or media supplemented with 10M retinoic acid. Cells were harvested 48 h later and processed for CAT and lacZ assays according to the methods of Sambrook et al. (1989). Protein concentration was determined with a protein assay reagent (Bio-Rad) using bovine serum albumin as a standard. Samples from the CAT assays were resolved by thin-layer chromatography, visualized on x-ray film, and quantitated by liquid scintillation counting.

For construction of reporter plasmids, oligonucleotides containing corresponding HREs were synthesized, trimerized, and cloned into the EcoRV site of Bluescript II KS (Stratagene). Orientation of the oligonucleotides was determined by sequencing. The Bluescript vectors containing oligonucleotides were digested (HindIII/XbaI), and the fragments were separated on a 10% polyacrylamide gel in TBE buffer, electroeluted, and ligated to the HindIII/XbaI site of pBLCAT2 (Luckow and Schütz, 1987).

The following oligonucleotides, based on DR-1 (Kadowaki et al., 1992), -RARE (Tran et al., 1992), and CRBP I (Tran et al., 1992), were synthesized: DR-1, 5`- GGCTTCAGGTCAGAGGTCAGAGA and 5`- GGTCTCTGACCTCTGACCTGAAG; -RARE, 5`-GGTGTAGGGTTCACCGAAAGTTCACTCA and 5`-GGTGAGTGAACTTTCGGTGAACCCTACA; and CRBP I, 5`-CCATCCAGGTCAAAAAGTCAGGA and 5`-GGTCCTGACTTTTTGACCTGGAT. The HRE sequences are underlined.

Preparation of Whole Cell Extracts and Electrophoretic Mobility Shift Assay (EMSA)

These methods were performed at 4 °C as previously described (Scholer et al., 1989). Briefly, the extraction buffer contained 20 mM Hepes, pH 7.8, 450 mM NaCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, 25% glycerol, and protease inhibitors phenylmethylsulfonyl fluoride (0.5 mM), leupeptin (0.5 µg/ml), pepstatin (0.7 µg/ml), aprotinin (1 µg/ml), and bestatin (40 µg/ml). Sonicated extracts were cleared by centrifugation for 5 min. Binding conditions for the EMSA were 10 mM Hepes, pH 7.8, 1 mM spermidine, 5 mM MgCl, 50 mM KCl, 0.5 mM dithiothreitol, 9% glycerol, 0.8 µg of poly(dI-dC), 50,000 cpm of the P-labeled oligonucleotide, and 10 µg of cell extract. After incubation for 15 min at 25 °C, DNA-protein complexes were separated on a 5% polyacrylamide gel, followed by autoradiography. Three different batches of extracts were used in EMSA analyses, and no batch differences were observed.


RESULTS

Cloning of COUP-TF I

Expression screening of the subtraction cDNA library was performed to isolate genes that inhibit neurite outgrowth. The cDNA library in pRcCMV expression vector was transfected into NIH-3T3 fibroblasts, and, after 3 weeks of antibiotic selection, several clones that had less contacts with neurites were isolated. These clones were propagated, and the transfected cDNAs were isolated using polymerase chain reaction and retested for neurite outgrowth inhibition. Sequence analysis revealed that one cDNA is the rat homologue of human orphan receptor COUP-TF I lacking 40 amino acids of the transactivation domain localized near the amino terminus of the DNA binding domain (COUP-TF I). Full-length COUP-TF I cDNA was isolated by high stringency screening of the brain cDNA library cloned into ZAPII vector (Stratagene). The rCOUP-TF I cDNA encodes a protein with a predicted molecular mass of 45.8 kilodaltons (Genbank accession number, U10995) and is 98% identical to the human COUP-TF I at the amino acid level with 95% identity at the nucleotide level.

COUP-TF I Interacts with HREs in Fibroblasts

Expression of COUP-TF I was analyzed by Northern blot using six COUP-TF I and three pRcCMV vector-transfected clones. Vector-transfected and -untransfected fibroblasts did not express detectable levels of COUP-TF I (Fig. 1A, lanes1, 9, and 10). Three transfected clones expressed introduced COUP-TF I at high levels (Fig. 1A, lanes6, 7, and 9), two clones expressed at moderate levels (Fig. 1A, lanes3 and 5), while one clone did not express COUP-TF I at a detectable level (Fig. 1A, lane4).


Figure 1: Expression and activity of COUP-TF I in transfected fibroblasts. A, Northern blot analyses of COUP-TF I expression in control 3T3 fibroblasts (lane1) and fibroblast clones transfected with COUP-TF I cDNA cloned into pRcCMV expression vector (lanes2-8) or pRcCMV vector only (lanes9 and 10). Expression of COUP-TF I was high in clones 7-9 (lanes6-8, respectively) and varied from undetectable to moderate in clones 2-5 (lanes2-5, respectively). B, EMSA analysis of control fibroblasts (lane 1) and fibroblasts transfected with pRcCMV expression vector (lane2) and COUP-TF I cDNA in pRcCMV (lane3). Cell extracts were analyzed by EMSA using oligonucleotides DR-1, -RARE, and CRBP I. As a control for binding specificity, 100 times molar excess of corresponding unlabeled oligonucleotides were added to the binding reaction (lane4). C, the transient CAT activity of DR-1-TK-CAT (DR-1), -RARE-TK-CAT (-RARE), and CRBP I-TK-CAT (CRBP) reporter constructs in the presence of pRcCMV (CMV) or COUP-TF I-expressing plasmid pRcCMV-COUP-TF I (COUP). Subsets of each transfection group were treated with 10M retinoic acid (RA). All cells were cotransfected with pRcCMV/lacZ constructs to facilitate normalization of CAT gene expression to transfection efficiency. All treatments were done in triplicate, and the numbers represent values of two experiments. Values are normalized based upon transfection efficiency and are expressed as relative to the results obtained by transfection of pRcCMV-transfected cells with corresponding reporter plasmid for which activities were set at 1. Results were analyzed by a one-tailed t test.



Since COUP-TF I can form inactive complexes with retinoid X receptors (RXR) and binds to HREs from different genes as a homodimer to repress the hormone response (Cooney et al., 1992, 1993; Tran et al., 1992), we used EMSA analysis to examine whether the overexpression of COUP-TF I in fibroblasts affects formation of HRE binding complexes. Since the effects of COUP-TF I depend on the HRE sequence, we analyzed two natural and one synthetic HRE: -RARE, a direct repeat with a 5-bp spacer that is activated by RAR and is not inhibited by COUP-TF I (Tran et al., 1992); CRBP I-RARE, a direct repeat with a 2-bp spacer that is optimally activated by RARRXR heterodimers but not by RXR homodimers (Hermann et al., 1992; Zhang et al., 1992); and DR-1, a direct repeat that contains a 1-bp spacer and has a high affinity for COUP-TF I (Kadowaki et al., 1992) and RXRs (Mangelsdorf et al., 1990, 1991). Our results demonstrated that the pattern of protein-DNA complexes is changed in COUP-TF I overexpressing cells compared with controls (Fig. 1B). A unique DNA-protein complex (Fig. 1B, arrow) was detected in extracts made from COUP-TF I overexpressing cells. Antibodies (developed in our laboratory) against COUP-TF I supershifted the complex (data not shown), verifying the presence of COUP-TF I in the complex.

The role of COUP-TF I as an activator or inhibitor of transcription in 3T3 cells was examined by transient CAT assays using the -RARE, CRBP I, and DR-1 oligonucleotides coupled to the TK promoter of the CAT reporter gene. Results from cotransfections of reporter constructs and pRcCMV or COUP-TF I expression vectors are shown in Fig. 1C. Cotransfection of COUP-TF I significantly induced (p < 0.05) -RARE containing reporter construct activity (Fig. 1C). Addition of retinoic acid-induced expression (p < 0.01) of the -RARE reporter gene construct in cells cotransfected with the pRcCMV control expression vector to levels similar (p > 0.05) to cells cotransfected with COUP-TF I in the presence or absence of retinoic acid. No significant effects of COUP-TF I or retinoic acid treatment on reporter gene expression of other constructs was detected. In these experiments, COUP-TF I functioned as a positive regulator of -RARE-coupled reporter gene expression in 3T3 fibroblasts.

COUP-TF I Overexpression Changes Contact Stability between Transfected Fibroblasts and Neurites

The growth rate of neurites from NG108-15 cells is rapid and robust, which makes them a good model for studying cell-cell interaction and contact stability. Analyses of neurite contact stability were performed with fibroblast clones overexpressing COUP-TF I. Fibroblasts transfected with the pRcCMV vector only and naive fibroblasts were used as controls. There is a general increase in the number of contacts over the course of the experimental period. This increase was not different for naive control fibroblasts or fibroblasts transfected with the expression vector (205 ± 64% versus 160 ± 37%, respectively, p > 0.05). However, fibroblasts transfected with COUP-TF I (clones 5, 8, and 9) exhibited a significantly (p < 0.05) lower increase in contact number (51 ± 14%, 18 ± 20%, 38 ± 27%, respectively, Fig. 2) indicative of lower contact stability. While differences in level of expression of COUP-TF I were apparent in clones 5, 8, and 9 (Fig. 1A, lanes5, 7, and 9, respectively), contact stability was not dependent upon the level of expression of COUP-TF I cDNA (p > 0.05). Neuroblastoma-glioma NG108-15 cells have rapidly growing neurites with large growth cones and filopodia. After contact with control fibroblasts, growth cones maintain their form (Fig. 3, CMVpanel). In contrast, the processes of NG108-15 cells exhibit retraction or become very small after contact with COUP-TF I-expressing cells, indicative of growth cone collapse and contact instability (Fig. 3, COUPpanel). The increased number of contacts in these cultures comes from other NG108-15 cells in the vicinity of the fibroblasts. Since the differentiated NG108-15 cells observed have an equal probability to establish and maintain contacts with fibroblasts, the test determines the stability of cell-cell contact.


Figure 2: Cells expressing COUP-TF I exhibit lower (p < 0.05) contact stability when cocultured with NG108-15 cells. Contact stability was assayed in three groups: 1) naive fibroblasts (control); 2) cells transfected only with pRcCMV vector (CMV 3); and 3) three clones transfected with COUP-TF I cDNA (COUP 5, COUP 8, and COUP 9). The number of contacts between cell types at time 0 and 2.5 h was used to calculate the percentage change in the number of contacts for each colony of cells observed. The bars represent the average percentage change in the number of contacts observed for each group and represent the data from three separate experiments. The number of observations (n) for each group is given.




Figure 3: Interactions of neurites with control and COUP-TF I overexpressing 3T3 fibroblasts. 2 h after the addition of NG108-15 cells (t = 0), fibroblast clones were identified and photographed at various times during the culture period (given below each panel). Fibroblasts transfected with the pRcCMV control vector (F in upperCMVpanel) provide a good substrate for support of processes from NG108-15 cells (N). The processes are robust, and contacts (arrows) are persistent during prolonged culture. In contrast, cells overexpressing COUP-TF I cDNA (F in lowerCOUPpanel) do not sustain significant attachment of processes from NG108-15 cells. During the culture period, contacts (arrows) are not maintained.




DISCUSSION

Members of the steroid hormone receptor superfamily interact in a complex manner and stimulate or repress gene expression, depending upon the type and number of factors present and also the context of the gene promoter (Cooney et al., 1992, 1993). We have shown that overexpression of the orphan receptor, COUP-TF I, can alter cell-cell interaction in fibroblasts and block neuronal differentiation of PCC7 cells (Neuman et al., 1995). Expression of COUP-TF I in fibroblasts is correlated with detectable alterations of normal cell behaviors, presumably associated with alteration or modification of the expression of cell surface proteins. The exact nature of this change is unknown since no differences were detected in comparisons of isolated membrane proteins from control and COUP-TF I-expressing cells, nor was there any indication of involvement of secretory factors.()Discerning the mechanism by which COUP-TF I altered surface properties was further hampered due to a lack of characterization of fibroblasts with regard to members of the steroid hormone receptor superfamily, which are endogenously expressed and represent potential heterodimerization partners. However, data from transient CAT assays indicated that COUP-TF I induced transcriptional activity of -RARE-coupled reporter gene constructs. Together, these data are consistent with a model in which COUP-TF I interacts with other nuclear hormone receptors, causing changes in gene expression. Several scenarios are possible, including COUP-TF I homodimers binding to retinoic acid response elements, or COUP-TF I/retinoic acid receptor heterodimer formation (Tran et al., 1992) with subsequent alteration of gene expression. Retinoic acid alters expression of genes that promote cell growth and attachment, i.e. laminin B1 (Vasios et al., 1991), and is also important in the regulation of hox genes (Linney, 1992), which have been shown to interact with promoters of cell adhesion molecules, i.e. N-CAM and cytotactin (Jones et al., 1992a, 1992b, 1993). Our data are consistent with a model in which perturbation of a variety of processes, including differentiation, aggregation, and cell-cell interaction, is regulated by expression of COUP-TF I.


FOOTNOTES

*
This work was supported by the Spinal Cord Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U10995.

§
To whom correspondence should be addressed: Dept. of Anatomy and Neurobiology, Colorado State University, Fort Collins, CO 80523. Tel.: 303-491-5791; Fax: 303-491-7907; E-mail: toomas@lamar.colostate.edu.

The abbreviations used are: HRE, hormone response element; COUP-TF, chick ovalbumin upstream promoter transcription factor; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; RARE, retinoic acid response element; bp, base pair(s); RAR, retinoid acid receptor; RXR, retinoid X receptor.

H. Connor, H. Nornes, and T. Neuman, unpublished data.


REFERENCES
  1. Beato, M.(1989) Cell56, 335-344 [Medline] [Order article via Infotrieve]
  2. Caroni, P., and Schwab, M. E.(1988) J. Cell Biol.106, 1281-1288 [Abstract]
  3. Chan, S.-M., Xy, N., Niemeyer, C. C., Bone, J. R., and Flytzanis, C. N. (1992) Proc. Natl. Acad. Sci. U. S. A.89, 10568-10572 [Abstract]
  4. Chomczynski, P., and Sacchi, N.(1987) Anal. Biochem.162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  5. Cooney, A. J., Tsai, S. Y., O'Malley, B. W., and Tsai, M.-J.(1992) Mol. Cell. Biol.12, 4153-4163 [Abstract]
  6. Cooney, A. J., Leng, X., Tsai, S. Y., O'Malley, B. W., and Tsai, M.-J. (1993) J. Biol. Chem.268, 4152-4160 [Abstract/Free Full Text]
  7. Cox, E. C., Müller, B., and Bonhoeffer, F.(1990) Neuron4, 31-37 [Medline] [Order article via Infotrieve]
  8. Davies, J. A., Cook, G. M. W., Stern, C. D., and Keynes, R. J.(1990) Neuron4, 11-20 [Medline] [Order article via Infotrieve]
  9. Evans, R. M.(1988) Science240, 889-895 [Medline] [Order article via Infotrieve]
  10. Evans, R. M., and Arriza, J. L.(1989) Neuron2, 1105-1112 [Medline] [Order article via Infotrieve]
  11. Fjose, A., Nornes, S., Weber, U., and Mlodzik, M.(1993) EMBO J.12, 1403-1414 [Abstract]
  12. Green, S., and Chambon, P.(1988) Genetics4, 309-314
  13. Hamprecht, B.(1977) Int. Rev. Cytol.49, 99-170 [Medline] [Order article via Infotrieve]
  14. Herman. T., Hoffmann, B., Zhang, X.-K., Tran, P., and Pfahl, M. (1992) Mol. Endocrinol.6, 1153-1162 [Abstract]
  15. Jones, F. S., Chalepakis, G., Gruss, P., and Edelman, G. M. (1992a) Proc. Natl. Acad. Sci. U. S. A.89, 2091-2095 [Abstract]
  16. Jones, F. S., Prediger, E. A., Bittner, D. A., De Robertis, E. M., and Edelman, G. M. (1992b) Proc. Natl. Acad. Sci. U. S. A.89, 2086-2090 [Abstract]
  17. Jones, F. S., Holst, B. D., Minowa, O., De Robertis, E. M., and Edelman, G. M.(1993) Proc. Natl. Acad. Sci. U. S. A.90, 6557-6561 [Abstract]
  18. Jonk, L. J. C., de Jonge, M. E. J., Pals, C. E. G. M., Wissink, S., Vervaart, J. M. A., Schoorlemmer, J., and Kruijer, W.(1994) Mech. Dev.47, 81-97 [CrossRef][Medline] [Order article via Infotrieve]
  19. Kadowaki, Y., Toyoshima, K., and Yamamoto, T.(1992) Biochem. Biophys. Res. Commun.183, 492-498 [Medline] [Order article via Infotrieve]
  20. Ladias, J. A. A., and Karathanasis, S. K.(1991) Science251, 561-565 [Medline] [Order article via Infotrieve]
  21. Linney, E.(1992) Curr. Top. Dev. Biol.27, 309-350 [Medline] [Order article via Infotrieve]
  22. Lu, X. P., Salbert, G., and Pfahl, M.(1994) Mol. Endocrinol.8, 1774-1788 [Abstract]
  23. Luckow, B., and Schütz, G.(1987) Nucleic Acids Res.15, 5490 [Medline] [Order article via Infotrieve]
  24. Luo, Y., Raible, D., and Raper, J. A.(1993) Cell75, 217-227 [Medline] [Order article via Infotrieve]
  25. Lutz, B., Kuratani, S., Cooney, A. J., Wawersik, S., Tsai, S. Y., Eichele, G., and Tsai, M.-J.(1994) Development120, 25-36 [Abstract/Free Full Text]
  26. Mangelsdorf, D. J., Ong, E. S., Dyck, J. A., and Evans, R. M.(1990) Nature345, 224-229 [CrossRef][Medline] [Order article via Infotrieve]
  27. Mangelsdorf, D. J., Borgmeyer, U., Heyman, R. A., Zhou, J. Y., Ong, E. S., Oro, A. E., Kakizuka, A., and Evans, R. M.(1992) Genes & Dev.6, 329-344
  28. Matharu, P. J., and Sweeney, G. E.(1992) Biochim. Biophys. Acta1129, 331-334 [Medline] [Order article via Infotrieve]
  29. McEwen, B. S., Coirini, H., Danielsson, A., Frankfurt, M., Gould, E., Mendelson, S., Schumacher, M., Segarra, A., and Woolley, C.(1991) J. Steroid Biochem. Mol. Biol.40, 1-14 [CrossRef]
  30. Miyajima, N., Kadowaki, Y., Fukushige, S., Shimizu, S., Semba, K., Yamanashi, Y., Matsubara, K., Toyoshima, K., and Yamamoto, T.(1988) Nucleic Acids Res.16, 11057-11074 [Abstract]
  31. Mlodzik, M., Hiromi, Y., Weber, U., Goodman, C. S., and Rubin, G. M. (1990) Cell60, 211-224 [Medline] [Order article via Infotrieve]
  32. Neuman, K., Soosaar, A., Nornes, H. O., and Neuman, T.(1995) J. Neurosci. Res.41, 39-48 [Medline] [Order article via Infotrieve]
  33. Qiu, Y., Cooney, A. J., Kuratani, S., DeMayo, F. J., Tsai, S. Y., and Tsai, M.-J.(1994) Proc. Natl. Acad. Sci. U. S. A.91, 4451-4455 [Abstract]
  34. Raper, J. A., and Kapfhammer, J. P.(1990) Neuron4, 21-29 [Medline] [Order article via Infotrieve]
  35. Richie, H. H., Wang, L.-H., Tsai, S., O'Malley, B. W., and Tsai, M.-J. (1990) Nucleic Acids Res.18, 6857-6862 [Abstract]
  36. Sambrook J., Fritsch, E. F., and Maniatis, T.(1989) in Molecular Cloning: A Laboratory Manual, 2nd Ed., pp. 16.60-16.67, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  37. Scholer, H. R., Hatzopoulos, A. K., Balling, R., Suzuki, N., and Gruss, P.(1989) EMBO J.8, 2543-2550 [Abstract]
  38. Tosney, K., and Landmesser, L. T.(1984) J. Neurosci.4, 2518-2527 [Abstract]
  39. Tran, P., Zhang, X.-K., Salbert, G., Hermann, T., Lehmann, J. M., and Pfahl, M.(1992) Mol. Cell. Biol.12, 4666-4676 [Abstract]
  40. Vasios, G., Mader, S., Gold, J. D., Leid, M., Lutz, Y., Gaub, M.-P., Chambon, P., and Gudas, L.(1991) EMBO J.10, 1149-1158 [Abstract]
  41. Wang, L.-H., Tsai, S. Y., Cook, R. G., Beattie, W. G., Tsai, M.-J., and O'Malley, B. W.(1989) Nature340, 163-166 [CrossRef][Medline] [Order article via Infotrieve]
  42. Wang, L.-H., Tsai, S. Y., O'Malley, B. W., and Tsai, M.-J.(1991) Gene Expr.1, 207-216 [Medline] [Order article via Infotrieve]
  43. Zhang, X., Hoffmann, B., Tran, P., Graupner, G., and Pfahl, M.(1992) Nature355, 441-446 [CrossRef][Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.