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Division of Nephrology (M.A., J.W., A.B., D.P.), Department of Medicine, University of California, San Francisco, San Francisco, California 94143-1341; and Division of Medical Pharmacology (O.C.M.), Leiden/Amsterdam Center for Drug Research, 2300 RA Leiden, The Netherlands
Address all correspondence and requests for reprints to: David Pearce, Division of Nephrology, Department of Medicine, University of California, San Francisco, San Francisco, California 94143-1341. E-mail: pearced{at}medicine.ucsf.edu.
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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) enhances rather than diminishes GR activity driven by canonical GRE multimers (12); this is in contrast to transcription driven by a single canonical GRE, which is markedly diminished by the same mutations (5, 12). Second, two types of GR dimer mutants (dms) bind to multimerized GRE probes with affinity comparable to that of wild type (12). Third, a naturally occurring mutant of the androgen receptor (AR) that causes a similar destabilization of dimer contacts has reduced activity at only a subset of transactivated genes and causes a partial androgen resistance (Reifenstein’s) syndrome (13). The partial androgen resistance seen in patients with Reifenstein’s syndrome is reminiscent of the GR dim/dim mouse, a knock-in mouse model in which the GRwt was replaced by a mutant with destabilized DBD dimer interface (10). Interestingly, in animals homozygous for this mutation, the expression of some GR-regulated genes, such as tyrosine amino transferase, was reduced, whereas the expression of at least one GR-driven gene, phenylethanolamine N-methyltransferase (PNMT), was normal to increased (10). This is in contrast to complete GR knockout mice, which express no PNMT mRNA (1, 2, 10) in spite of the presence of adrenal medullary chromaffin cells (2). The survival of the dim/dim mouse was interpreted by the authors as supporting the idea that the ability of GR to bind DNA is unnecessary for survival; however, this conclusion was based on the assumption that destabilization of the DBD dimer interface would disrupt binding to all GREs, not just a subset.
The PNMT gene (coding for the enzyme that catalyzes the conversion of norepinephrine to epinephrine) has long been considered a model for rapid and robust gene response to corticosteroids and stress (14) and was originally reported to contain a single consensus near-palindromic GRE, 533 bp upstream of the transcription initiation site (15). In view of its normal expression in GR dim/dim mice, and of the evidence that GRdms could bind and transactivate at multiple synthetic GREs, we hypothesized that multiple GREs should be present in the PNMT promoter. Accordingly, it could represent a subset of GRE-containing promoters, which may be transactivated independently of receptor dimerization. We examined the PNMT 5'-regulatory region in more detail, directly addressing the question of binding and transcriptional activity of GR dimer mutants on this naturally occurring regulatory sequence.
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RESULTS |
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); dimer mutant activity was greater than wild type for all three dimer mutants. As a control for the dimer mutant’s impaired transactivation at promoters containing a single GRE, we compared wild-type and dimer mutant activity at the endogenous tyrosine amino transferase-luciferase (eTAT-Luc) reporter, which is driven by the regulatory 5'-flanking region of the tyrosine aminotransferase promoter, containing a single GRE. At this promoter, GRwt led to a reliable induction of transcription, whereas all three dimer mutants were impaired (Fig. 2B).
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ZF1, which has a point mutation in the first Zn-finger of the DBD that directly interferes with DNA binding (16), could not transactivate rPNMT-997/-466 reporter (data not shown).
Identification of Multiple GR Binding Sites in the PNMT 5'-Regulatory Region Using GR DBD
Our previous studies of synthetic promoters had established that the enhanced activity of dimer mutants was dependent on a minimum of two GREs (12). Cross-species comparison of PNMT 5'-regulatory sequences revealed four potential GREs fitting the consensus RGNACYnnnWGWNCY and multiple consensus half-sites upstream of GRE-1 (Fig. 3B). To delineate the actual sites of GR DBD binding, rPNMT-997/-466 was subjected to deoxyribonuclease (DNase I) footprint analysis using increasing concentrations of human GR DBD peptide (amino acids 407–556, gift of K. Yamamoto) (17). Five footprints were visible at 2 µg GR DBD peptide (400 pmol) (Fig. 3A). Two hypersensitive sites were apparent at 0.5 µg GR-DBD (5' of the footprint FP-I, encompassing GRE-1, and between footprinted regions FP-III and FP-IV, although the latter band disappeared at higher receptor concentrations, possibly due to in vitro protein saturation). This suggested a slightly higher binding affinity at these sites; however, all footprints appear at the same GR DBD concentration, indicating that the binding affinity of isolated GR DBD was comparable at all sites. The presence of multiple potential GR binding sites was consistent with the hypothesis that the PNMT 5'-regulatory region has multiple GREs, and notably, four of the five footprints encompassed regions that contained GREs. FP-IV overlaps a DNA segment shown recently in gel shift assays to bind GRwt (18).
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). Point mutation revealed that activation by GRwt and the GRdms A477T and R479D was abrogated when the near-consensus GRE within FP-IV (termed GRE-3), and the upstream, overlapping half-site within FP-IV were disrupted. Sequential truncations further revealed that the strong synergy of GRwt and GRdms was maintained by an 105-bp fragment spanning FP-III and FP-IV (-785/-681), containing two potential GREs, each flanked by a consensus half-site. FP-IV alone was minimally responsive to GRdms, and unresponsive to wt in the absence of FP-III (-802/-732). Reporters containing only sequence downstream of FP-III were not responsive to GRwt or GRdms. Hence, these data suggest that GRE-2 and GRE-3 (within FP-III and FP-IV, respectively) were necessary and sufficient for strong PNMT activation by GRwt or GRdms.
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, GRE-3 construct) that binds GR DBD fragments, and because GRdms can bind to DNA at a synthetic probe containing multiple GREs, it seemed likely that PNMT sequences containing multiple GREs that are activated by GRdms can bind GRdms as well. To address this directly, EMSAs were performed with full-length GRwt and GRdms on a DNA probe encompassing FP-III and FP-IV (-785/-681). GRwt and GRdms were expressed in vitro, as described in Materials and Methods, and expression was shown to be equal by Western blot (data not shown). Radiolabeled PNMT -785/-681 fragment was then incubated with either GRwt or two of the dimer-deficient mutants, R481D and A477T, in the presence of specific or nonspecific competitor, and run on a nondenaturing gel. As shown in Fig. 5, GRwt and both GRdms specifically bound to the 105-bp PNMT fragment -785/-681, although signal intensity for PNMT complexes with GR D481R or GR A477T was consistently less than that for complexes with GRwt, consistent with a lower affinity or higher off-binding rate under these in vitro conditions.
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) the transactivation brought about by the GRdms was the consequence of saturation of reporter DNA by receptor, at a receptor vector (500 ng)-reporter plasmid (25 ng) ratio of 20:1. To test this we compared the efficacy of GRwt and GR A477T under conditions in which the amount of receptor protein becomes rate limiting. As is shown in Fig. 6, both receptors mediate equally well hormone-induced transcription from receptor-reporter ratios of 1:4 up to 1:40. The similar efficacy of the mutant and wild-type receptors strongly suggest that DNA binding and transactivation by the dimer mutants are not due to receptor excess with consequent saturation of reporter expression. However, it is notable that the reporter inductions by GRwt and GRdms under nonsaturating conditions are comparable (Fig. 6). This is in contrast to Figs. 2 and 4, in which higher ratios of receptor to reporter were used, and in which the dimer mutants have greater activity than wild type. The cause of this remains uncertain, but it may reflect the balance between a somewhat lower DNA binding affinity (Fig. 5) and a greater potency to transactivate once bound to the DNA (Fig. 2A) of the mutant receptors. Alternatively, it is plausible that the dimer mutant and wild type bind the PNMT promoter with a similar affinity under physiological conditions. In any case, the striking difference between mutant receptor activity on PNMT vs. eTAT are still manifest, even at high reporter to receptor ratios.
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). Finally, both wild type and dimerization-deficient GRs bind with high specificity and affinity to this promoter fragment (Fig. 5). A limitation of our present data is the in vitro character of our DNA binding and transactivation studies. However, we believe that our data are strongly supported by their complete correspondence with the data from the dim/dim mouse, in which a GR dimer mutant regulates the PNMT promoter in a near-complete natural cellular and genomic context (10). This latter observation is in striking contrast with the GR knockout mouse, in which PNMT is not expressed (1). In light of these observations, it is premature to conclude that binding of the GR to DNA is unnecessary for survival (10, 20). However, it is important to emphasize that our data do not demonstrate that GR DNA binding is necessary for survival: the neonatal mortality of GR-null mice appears to be due to a defect in lung maturation unrelated to PNMT. Determining the importance of DNA binding to survival will require a GR gene replacement experiment (e.g. a knock-in mouse) using a receptor mutant that cannot bind DNA at all rather than one that can bind to a significant subset of regulatory elements, including PNMT. Our data suggest the possibility that key gene products involved in lung maturation are driven by compound GRE cassettes similar to those driving PNMT.
Role of Individual GREs in PNMT Activation
In the present study, we found that point mutations or truncations that disrupted GRE-2 or GRE-3 markedly decreased the PNMT response to both wild-type and dimer mutant receptors. In contrast, mutations that disrupted GRE-1 disturbed the function of pPNMT-997/-466 mildly if at all (Fig. 4). In addition, the pPNMT fragment -706/-520, containing GRE-1 and the upstream nonconsensus GRE in footprinted region FP-II, did not respond to GRwt or GRdms. A similar GRE dependence of GRwt was recently reported by Tai et al. (18), who studied the proximal 893 bp of PNMT 5'-flanking region in pheochromocytoma-derived RS1 cell (21). These authors also demonstrated that the region equivalent to the present FP-IV contributes to glucocorticoid responsiveness, and that this fragment can be cut in two ways, each yielding a GR-binding fragment (18). In addition, they found that a truncated promoter lacking FP-IV but containing FP-III, FP-II, and GRE-1 was modestly activated by GR, but that promoter constructs lacking both FP-III and FP-IV led to a loss of steroid responsiveness (18), as did our similar construct (-706/-520). In contrast, the earlier characterization of the PNMT promoter by Ross et al. (15) in primary adrenal medullary cells pointed to the DNA element, which we have termed GRE-1, as necessary and sufficient for glucocorticoid responsiveness. It is possible that GR bound to GRE-1 might interact with other transcription factors that are selectively expressed in adrenal medullary cells. The transcription factor activator protein 2, for example, has been shown to act synergistically with GRE-1 (22), and this activity may be absent in our system. However, it is important to note that the fold activation mediated by GRE-1 in this earlier report was quite low, substantially less than that seen in the present report or described by Tai et al., which was closer to the striking levels of induction seen for the endogenous gene (14).
Mechanism of Higher Order Complex Formation
The increase in receptor self-synergy at GRE multimers after disruption of the dimer interface has also been observed for the MR (12) and AR (23, 24); any proposed mechanism is likely conserved between members of the steroid receptor family. Importantly, dimer mutants of GR or MR lacking either their N- or C-terminal regions bind very poorly to TAT2 promoters and do not activate strongly at either PNMT or the artificial TAT2 or TAT3 promoters (data not shown and Refs. 12 and 23), suggesting that receptor sequences in both regions stabilize the formation of a higher order GR complex (referred to here as "concerted multimers" to reflect the lack of second zinc finger-mediated dimerization as a prerequisite for complex formation). Studies in yeast and mammalian cells show evidence for physical and functional interactions between the carboxy-and amino-terminal domains of the human AR (25, 26), and it is appealing to speculate that GR (wt or dms) complex formation on the PNMT 5'-regulatory region is stabilized through intermolecular N-C interactions. However, the N-terminal FxxLF and WxxLF motifs that mediate interaction with activation function 2 in AR (27) are not present in GR or MR. For the estrogen receptor and the type 2 nuclear receptors that bind direct repeats of hormone response element half-sites (in some cases at relatively large distances), the importance of a strong dimerization surface in the ligand binding domain has been demonstrated (28, 29). Higher order complex formation may depend on a similar interface in the ligand binding domain of wild-type or dimer mutants of GR and other type I nuclear receptors, particularly as direct repeats are inherently present in the multiple palindromic elements found in PNMT. The precise nature of the interactions that sustain the higher order GR complex on PNMT requires further characterization.
We first observed the up phenotype of GR dimer mutants on TAT3- and TAT2-Luc reporters, and have now observed it on the much more complex PNMT GR-responsive region. This multiple GRE effect was observed with reporters driven by both thymidine kinase, simian virus 40, and Drosophila alcohol dehydrogenase minimal promoters (Ref. 12 , and Pearce, D., unpublished observations). In contrast, the eTAT gene (unresponsive in dim/dim mice), its GR-responsive 5'-regulatory genomic fragment, the TAT1 reporter (harboring a single TAT gene-derived GRE), and the MMTV promoter long-terminal repeat are dependent on an intact dimer interface both for GR binding and transactivation (10, 12, 19). The minimum GRE configuration necessary for the proposed mechanism of transactivation through concerted multimers remains to be determined. There is the possibility that binding of additional transcription factors permits or inhibits the concerted-multimer type of transactivation: in the MMTV promoter several sequences other than GR-binding sites are necessary for GR-mediated transactivation (30). Egr-1 and AP-2 can synergize with the GR at the PNMT promoter (22); however, no binding sites for these factors are present in the FP-III+FP-IV fragment, which was bound and activated by both GRwt and the GRdms.
Candidate Promoters
It is unknown to what extent the dimer interface-independent mode of transactivation actually is used by the wild-type receptor in vivo. However, an increasing number of glucocorticoid-responsive mammalian genes have been reported that contain scattered GR consensus half-sites, and partial GREs: the 
-amylase 2 gene and the CYP3A5 member of the cytochrome P450 gene family illustrate regulatory regions that contain only two isolated consensus half-sites (separated by 32 and 160 bp, respectively), each of which are critical for gene expression (31, 32). It is not known whether isolated consensus half-sites bind GR monomers that have been otherwise recruited and stabilized; however, it is clear that a consensus half-site is required for GR-mediated transcriptional activation of these promoters. Published data [e.g. on the insulin receptor (33)] and inspection of the human genome data [e.g. hSGK (34) and hGILZ (35)] reveal that clearly there are promoters and 5'-flanking regions of a number of GR-activated genes that contain complex configurations of GREs and half-sites analogous to that of PNMT. Similarly to the PNMT gene, the 5'-regulatory region of the prostate-specific antigen gene contains a striking number of clustered nonconsensus androgen response elements that provide a base for cooperative assembly of a large AR nucleoprotein complex (36).
GR-Dependent Functions in dim/dim Mice
A number of important GR-dependent functions are disrupted in the GR dim/dim mouse, such as glucocorticoid effects on thymocyte apoptosis, erythroid progenitor proliferation, aspects of feedback signaling of the hypothalamo-pituitary-adrenal axis (10), as well as modulation of neuronal function in the hippocampus (11, 37). These effects probably depend on transactivation via GR at promoters with a relatively simple GRE configuration that require dimerization for GR DNA binding and regulation of gene transcription. According to the present view, the GR-dependent functions that are still intact in the dim/dim mouse could depend on nontranscriptional events (38), on transrepression (10), or on transactivation of genes with a more complex GRE configuration, as exemplified by PNMT (present results) and artificial GRE multimers (12). We propose that the protein-DNA and protein-protein interactions that support the formation of concerted multimers are essential for activation of genes driven by these types of regulatory regions, and we suggest that DNA binding by GR might indeed be essential to survival. In any case, our data establish that GRdms can bind specifically to a class of GREs and stimulate transcription, hence contradicting the conclusion that GRdms are globally deficient in DNA binding-dependent transactivation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Oligonucleotides and Plasmid Constructs
The wild-type PNMT promoter-luciferase reporter constructs for rat and mouse 5'-regulatory regions were obtained using standard recombinant DNA technologies. PNMT inserts were released from pCR2.1 with NheI and BglII and ligated into the pGL3 core-promoter luciferase-reporter plasmid (Promega Corp., Madison, WI). Mutations of GRE sequences were generated by three-way ligation of PCR products into the pGL3 promoter-vector. A PCR product was generated with one mutagenic primer directed at the region containing the GRE that contained transversions on positions 2 and 5 of each half-site and an added restriction site, and one primer directed at the pGL3-vector. The other PCR product was generated with a primer directed at the region immediately adjacent to (and containing the same restriction site as) the mutagenic primer, and a primer directed to pGL3-vector. The GRE-specific primers were as follows (GREs underlined; capital letters indicating mutations from the wild-type promoter). For GRE-1: gggaaa-aggccT-TaaAagagtTtcAt-ctcaaggaggatagag (rPNMT -549/-512; StuI), and gggaaa-Aggcct-ctgtcttggtggccctgggtactga (reverse complement of rPNMT -572/-543). For GRE-3: gggaaa-AtCGAT-tTcaAtctctTttAt-tacacgagtccggtgtccctg (-773/-738; ClaI) and gggaaa-ATCGaT-cattcttggtactgccacagccacac (reverse complement of -805/-779).
PGL3-promoter luciferase reporter plasmids containing PNMT truncations were constructed by PCR amplification using primers with NheI or BglII restrictions sites, which were then ligated into the PGL3 vector and cloned according to standard technique. Primers were designed with restriction enzyme overhangs for cloning into the pGL3-promoter vector. Upstream sequences [5'-ggaa-gctagc-tagagcaccaagcaggacctgaag (-944/-921, NheI); 5'-ggaa-gctagc-accaaaaagtgcgcatgcgctg (-832/-811, NheI); 5'-GGAA-gctagc-tgaactaaccatgcttgccggac (-706/-684, NheI); 5'-ggaa-gctagc-gcttgctaaaagcattagacccc (-643/-621, NheI)] were paired with the downstream sequence 5'-ggaa-agatct-tcagagaggacactctgttctggcctctg (-520/-550, BglII). Truncation -894/-739 (NheI, BglII) was synthesized with upstream primer 5'-ggaa-gctagc-acagggttgttgactgaggg and downstream primer 5'-ggaa-agatct-ggacaccggactcgtgtaa; truncation -785/-681 (NheI, BglII) with upstream primer 5'-ggaa-gctagc-aagaatgtgttctgcactctctg and downstream primer 5'-ggaa-agatct-tgcgctgcttatctgaggt. Fragment -802/-732 was created by ligation of the annealed oligonucleotides of sequence 5'-gcctcgctagcggctgtggcagtaccaagaatgtgttctgcactctctgttcttacacgagtccggtgttctgacctggagatctcatcc. All constructs were sequenced by D-rhodamine (Perkin-Elmer Corp., Norwalk, CT) to verify the presence of PNMT promoter fragments, and mutations therein.
The eTAT-Luc plasmid contains 3 kb of 5'-regulatory region of the rat tyrosine amino transferase gene driving luciferase and was a kind gift of K. Yamamoto (originally named pTAT-LUC; we have used the name eTAT to distinguish the endogenous regulatory region clearly from the synthetic TAT1 and TAT3-Luc reporters).
GR Mutants
Expression vectors for GRwt and the dimer mutant R479D were described previously (12). Briefly, point mutations were introduced into GR using PCR with the desired point mutagenic primer paired with a primer either upstream or downstream of a convenient restriction site, introducing silent restriction sites and the desired point mutations. Resulting PCR products were then introduced into the vector 6RGR (12). The dimer mutant GR A477T was generated by mutation of the codon for A477 from GCT to ACT using PCR with a primer that contained this point mutation.
Cell Culture and Transfections
Monkey kidney CV1-b cells (Cell Culture Facility, University of California, San Francisco) were grown at 5% CO2, 37 C in DMEM (Life Technologies, Inc.) supplemented with 5% fetal calf serum, penicillin G (100 U/ml), and streptomycin SO4 (100 µg/ml). One day before transfection, cells were plated in medium containing charcoal-stripped serum in six-well plates at 2 x 105 cells per well. PGL3 promoter reporter plasmids under control of the PNMT-regulatory region and its mutations were typically transfected at 25 ng plasmid DNA/transfection, as well as the control for transfection efficiency Rous sarcoma virus-ß-galactosidase plasmid 6RL (20 ng). Expression vectors for wild-type and mutant GR were transfected at 0.5 µg per transfection. In the case of the titration experiment in which the receptor-reporter ratio was varied, the reporter was used at 400 ng/well, and receptor was decreased from 100 to 10 ng/well. Transfection was done in 1 ml steroid-free Opti-Mem medium containing DNA complexed to lipofectamine (Life Technologies, Inc.). Five hours later, 1 ml of fresh DMEM containing 10% stripped fetal calf serum was added to each well without removing the transfection mixture and incubated for an additional 16–18 h. Subsequently, the cells were incubated in fresh medium containing 10-7 M corticosterone when appropriate. Cells were harvested 24 h later, and extracts were prepared and assayed for luciferase, ß-galactosidase activity, and protein content. All experiments were repeated at least twice and values were normalized to ß-galactosidase activity.
EMSAs
Full-length GRwt and GRdms were synthesized in vitro according to TNT Coupled Reticulocyte Lysate Systems (Promega Corp.). Protein was evaluated by Western blot according to standard procedures with anti-GR BuGR antibody as primary antibody (gift of Dr. K. Yamamoto). The 105-bp probe (-785/-681) was isolated by NheI/BglII digestion of the appropriate reporter plasmid followed by gel electrophoresis and QUAXII gel extraction (QIAGEN, Chatsworth, CA). The fragments were end labeled with [-32P]ATP by filling in overhangs with Klenow polymerase (Roche Clinical Laboratories, Indianapolis, IN). Binding reactions were performed with 50 mM NaCl; 20 mM Tris-HCl, pH 7.9; 1 mM EDTA; 10% (vol/vol) glycerol; 0.1% Nonidet P-40; 1.0 mM dithiothreitol; 1 µg of salmon testes DNA (Sigma, St. Louis, MO) and 10-7 M corticosterone. Approximately 80 µg of programmed reticulocyte lysate were preincubated in 20 µl of binding buffer with or without unlabeled competitor for 10 min at room temperature followed by addition of 104 to 105 cpm of 32P end-labeled probe (
20 fmol). The mixture was incubated for 10 min at room temperature. Supershifts were performed with BuGR antibody. Antibody was included in every reaction, to shift specifically GR receptor, which otherwise is obscured by an aspecific background signal present in the reticulocyte lysate. For the supershifts, 1 µg per reaction of BuGR antibody was added after the GR-DNA binding reaction and incubated for 15 min in ice. Where shown, specific (unlabeled oligo) or nonspecific competitor (appropriate size) was present at 500x or 1000x molar excess. The DNA-protein complexes were resolved on 4% 0.5x TBE (44.5 mM Tris, 44.5 mM boric acid, 1 mM EDTA, pH 8) nondenaturating polyacrylamide gel (37.5:1 acrylamide:bis, National Diagnostics, Atlanta, GA), subjected to electrophoresis at 250 V at 4 C, and autoradiography at -70 C.
DNase I Footprinting
DNase I footprinting was performed according to the Core Footprinting System (Promega Corp.). The 532-bp 5'-regulatory region (-996 to -466) was isolated and purified by gel electrophoresis and the QUAXII gel extraction kit (QIAGEN). The fragment was end labeled with [
-32P]rsqb]ATP using T4 polynucleotide kinase (Life Technologies, Inc). Asymmetric labeling of the 3'-end was achieved by MluI digestion which removed 12 bp from the 5'-end. Recombinant GR DBD peptide X566 (human GR amino acids 407–566, gift of Dr. K. Yamamoto) was incubated with approximately 105 cpm of probe in 50 µl of binding buffer [25 mM Tris-HCL (pH 8.0), 50 mM KCl, 6 mM MgCl2, 0.5 mM EDTA, 10% glycerol, and 0.5 mM dithiothreitol). After 10 min at 4 C, 50 µl of 5 mM CaCl2/10 mM MgCl2 solution was added and incubated at room temperature for 1 min. RQ1 ribonuclease-free DNase I (0.005 U) digestion was performed at room temperature for 2 min. Cleavage reactions were terminated by addition of 90 ml of warmed stop buffer (200 mM NaCl, 30 mM EDTA, 1% sodium dodecyl sulfate, and 100 µg/ml yeast RNA). Samples were extracted with phenol-chloroform and the products were precipitated with ethanol. Precipitates were dissolved in formamide loading buffer, analyzed on 6% polyacrylamide-urea sequencing gels, and visualized by exposure to XAR-5 film. Protected sequences were mapped using the Maxim-Gilbert sequencing technique.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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M.A. and O.C.M. contributed equally to this work.
Abbreviations: AR, Androgen receptor; DBD, DNA binding domain; DNase, deoxyribonuclease; eTAT-Luc, endogenous tyrosine amino transferase; FP I–IV, footprinted regions I–IV; GR, glucocorticoid receptor; GRdms, GR dimer mutants; GRE, glucocorticoid response element; GRwt, wild-type GR; MMTV, mouse mammary tumor virus; MR, mineralocorticoid receptor; PNMT, phenylethanolamine N-methyltransferase.
Received for publication September 3, 2002. Accepted for publication August 8, 2003.
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REFERENCES |
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-amylase 2 gene mediates response to the hormone. Mol Endocrinol 7:907–914[Abstract]
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