Analysis of the DNA-binding Site for Xenopus Glucocorticoid Receptor Accessory Factor
CRITICAL NUCLEOTIDES FOR BINDING SPECIFICITY IN VITRO AND FOR AMPLIFICATION OF STEROID-INDUCED FIBRINOGEN GENE TRANSCRIPTION*

Min Li, Xiongwen Ye, Robert N. WoodwardDagger , Cindy Zhu, LaNita A. Nichols, and Lené J. Holland§

From the Department of Physiology, University of Missouri School of Medicine, Columbia, Missouri 65212

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

In addition to the glucocorticoid receptor, DNA-binding proteins called accessory factors play a role in hormone activation of many glucocorticoid-responsive genes. Hormonal regulation of the gamma -fibrinogen subunit gene from the frog Xenopus laevis requires a novel DNA sequence that binds a liver nuclear protein called Xenopus glucocorticoid receptor accessory factor (XGRAF). Here we demonstrate that the recognition site for XGRAF encompasses GAGTTAA at positions -175 to -169 relative to the start site of transcription. This sequence is not closely related to the binding sites for known transcription factors. The two guanosines make close contact with XGRAF, as shown by the methylation interference assay. Single-point mutagenesis of every nucleotide in the 9-base pair region from positions -177 to -169 showed an excellent correlation between ability to bind XGRAF in vitro and ability to amplify hormone-induced transcription from DNA transfected into Xenopus primary hepatocytes. Conversely, XGRAF had little or no effect on basal transcription of the gamma -fibrinogen gene. Maximal hormonal induction also requires three half-glucocorticoid response elements (half-GREs) homologous to the downstream half of the consensus GRE. Interestingly, the XGRAF-binding site is immediately adjacent to the most important half-GRE. This close proximity suggests a new mechanism for activation of a gene lacking a conventional full GRE.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Steroid hormones, which include glucocorticoids and mineralocorticoids from the adrenal cortex and estrogens, progestins, and androgens from the gonads, regulate a vast array of physiological processes that are essential for development, differentiation, growth, metabolism, homeostasis, behavior, and reproduction in vertebrate organisms. In the classical model of steroid hormone action (1), the steroid ligands bind to specific intracellular protein receptors in target cells. The hormone-receptor complexes interact with particular short nucleotide sequences in the chromosomal DNA and modulate transcription of nearby genes. This model, however, cannot account fully for the complex tissue-specific and gene-specific actions of hormones. Transcriptional induction by steroids is influenced by many factors, such as local chromatin structure (2), stages of the cell cycle (3), cellular morphology or differentiation state (4, 5), specific hormone ligand (6), and physiological state (7). Differential hormone responsiveness depends in part on the availability of other transcriptional regulatory proteins including coactivators and corepressors, which do not themselves bind to DNA (8), and accessory factors, which are DNA-binding proteins (9). For glucocorticoid-regulated genes, several accessory factors have been identified (2, 10-17), but the mechanisms by which these proteins potentiate hormonal activation of transcription are not known.

To understand the role of accessory DNA-binding proteins in determining responsiveness to a steroid hormone signal, we are investigating glucocorticoid induction of fibrinogen gene expression in the liver. Fibrinogen is the precursor of fibrin, the major structural protein of a blood clot, and its synthesis is regulated by adrenal steroids both in basal homeostasis and following physiological stresses such as infections, inflammation, surgery, burns, etc. (18, 19). Using primary liver cells from the frog Xenopus laevis as a model system (20), we have demonstrated that glucocorticoids stimulate transcription of the three separate genes coding for the fibrinogen subunits, termed Aalpha , Bbeta , and gamma  (21).

Identification of the specific DNA sequences that mediate steroid regulation of the Xenopus fibrinogen genes was accomplished by linking the 5'-flanking DNA of these genes to the firefly luciferase reporter gene and transfecting the DNA into primary Xenopus hepatocytes. Hormonal activation of the Bbeta gene occurs through a single element (22) with a close match to the consensus glucocorticoid response element (GRE),1 GGTACAnnnTGTTCT (23). Full stimulation of the gamma  gene, on the other hand, requires three closely spaced weak binding sites for the glucocorticoid receptor (GR) between nucleotides -168 and -135 relative to the start site of transcription (24). These sites have homology only to the downstream portion of the consensus GRE and therefore are referred to as half-GREs. In addition, steroid responsiveness is affected by bases within the sequence AAGAGTTAA at positions -177 to -169, immediately adjacent to the 5'-most half-GRE (24). This tract is unrelated to the conventional GRE and does not match other known transcription factor-binding sites. A protein present in Xenopus liver nuclei binds to this DNA sequence in vitro. Experiments with DNA containing blocks of mutations within and around bases -177 to -169 showed that the DNA-protein interaction correlates with hormonal activation of transcription. Thus, we named the nuclear protein Xenopus glucocorticoid receptor accessory factor (XGRAF). Compared with other known GR accessory factors, the location of the XGRAF-binding site is unusual since it replaces the sequence that would normally constitute the upstream half of a GRE.

In this work, we describe the detailed mutational analysis of the 9-bp region to which XGRAF binds. This investigation located the 5'- and 3'-boundaries of the recognition sequence and revealed which nucleotides are most critical for DNA binding in vitro and for glucocorticoid-stimulated transcription of transfected DNA in Xenopus primary hepatocytes.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Introduction of Point Mutations into Reporter Gene Constructs-- All possible single-point mutations in the potential XGRAF-binding site in the gamma  gene upstream region were obtained by the following general strategy. 1) The gamma  gene sequence from positions -187 to +41 was synthesized by the polymerase chain reaction (PCR) using a downstream primer with wild-type sequence and an upstream primer consisting of a mixed population of oligonucleotides with mutations in the XGRAF-binding region from positions -177 to -169. 2) The PCR products were inserted into a luciferase reporter vector, and the DNA was cloned by transformation into bacteria. 3) The nucleotide sequence of the gamma  DNA in individual clones was determined to ascertain which nucleotide(s) of the XGRAF region had been mutated.

The upstream primer, 5'-GGGGTACCAGACAGAAAAGAGTTAATGTTCCCTCTTATGTTC-3', was synthesized (Genemed Biotechnologies, Inc.) in a single reaction, with the reservoir for each nucleotide in the potential binding site (underlined) deliberately contaminated to a concentration of 2.5% with each of the three nondesignated bases. PCR products from this primer yielded 15 of the 27 possible single-point mutants. A second population of mixed primers was synthesized (Genosys Biotechnologies), with contamination at the desired positions to optimal levels based on the formula described by Derbyshire et al. (25), yielding several additional mutants. The few remaining mutants were synthesized in PCRs with specific individual primers.

The PCR amplification was carried out with the pLLgamma -187 construct (24) as the template DNA (which contains gamma  gene DNA from positions -187 to +41), the upstream primers described above, a downstream primer within the vector, and Pfu polymerase (Stratagene) following the protocol from the manufacturer. The PCR products were digested upstream with KpnI and adjacent to position +41 downstream with HindIII, purified through 2% low-melting-temperature agarose gels (26), and cloned into KpnI- and HindIII-digested pLuc-Link 2.0 (27). Transformation into Escherichia coli DH5alpha was as described (24). The nucleotide sequences of the gamma  DNA and of the junctions with vector DNA were confirmed for each mutant. Plasmid DNA was purified over anion-exchange resin and, for transfection, over a cesium chloride gradient (24).

Gel Shift Assays-- Both the probe and the competitors for the gel shift assays contained the gamma  sequence extending from positions -187 to -115 and were generated by PCR using Pfu polymerase. In addition, the molecules included 19 bases of vector sequence upstream of position -187 and a MfeI restriction enzyme site downstream of position -115. Wild-type pLLgamma -187 was used as the template for making the probe, and the point mutation constructs described above were the templates for the competitors. The PCR products were purified through 2% low-melting-temperature agarose gels (26), and the probe was 5'-end-labeled (28).

Nuclear extracts were made from X. laevis primary hepatocytes after 4 days in culture exactly as described (24), except that the concentration of HEPES-KOH in buffer C+ was 20 mM. The gel shift assays were carried out as described (24) in a final volume of 15 µl with 0.5 ng of radioactive probe (7000-51,000 cpm) and either no specific competitor or a 0.5-500-fold molar excess of specific competitor. Native 5% polyacrylamide gels (24) were run at 350 V for 6-7 h at 4 °C, dried at 80 °C for 2 h, exposed to XAR film (Eastman Kodak Co.) at -80 °C with one Lightning Plus intensifying screen (Dupont), and exposed to a PhosphorImager screen (Molecular Dynamics, Inc.). The data from the phosphorimaging scan were analyzed with ImageQuant 3.3 software (Molecular Dynamics, Inc.).

Data Analysis for Gel Shift Assays-- The ability of XGRAF to bind gamma  DNA with mutations in the putative XGRAF-binding region was determined by competition gel shift assays, which contained a constant amount of radioactively labeled wild-type gamma  DNA and either no competitor or various concentrations of DNA with a single-point mutation. An example of the gel shift assay with mutant A-171 as the competitor is shown in Fig. 1A. Radioactivity in the shifted DNA·XGRAF complex in each lane was quantitated from the phosphorimaging scan. The total amount of XGRAF in the complex with radioactive DNA in the absence of any competitor (Fig. 1A, lane 2) was defined as 1.0, and the amount of XGRAF in the complex with radioactive DNA in the presence of a 100-fold molar excess of wild-type competitor was defined as zero (Fig. 1A, lane 1). The quantity of XGRAF remaining in the complex with radioactive DNA in the presence of mutated competitor (Fig. 1A, lanes 3-17) was expressed as a fraction of 1.0. The difference between the total and the fraction still bound to the radioactive probe equaled the fraction of XGRAF bound to the competitor DNA.


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Fig. 1.   Quantitation of binding of XGRAF to gamma  DNA with a single-point mutation at position -171. A, gel shift assay. The binding of XGRAF in Xenopus liver nuclear extract to a DNA fragment spanning positions -187 to -115 of the gamma -fibrinogen subunit gene upstream region was analyzed. The reactions contained the wild-type (WT) DNA sequence as the radioactive probe and the following competitors: lane 1, 100-fold molar excess of wild-type DNA; lane 2, no competitor; lanes 3-17, three replicates (labeled sets A, B, and C) of gamma  DNA from positions -187 to -115 with the single-point mutation (Mut) A-171 at 50-, 100-, 200-, 300-, and 500-fold molar excesses. B, Scatchard analysis. The data from A were plotted according to Equation 3 (see "Experimental Procedures"). Bound XGRAF on the x axis corresponds to [DNA·XGRAF], and Bound XGRAF/Competitor DNA on the y axis corresponds to [DNA·XGRAF]/[DNA]t in Equation 3. Bound XGRAF is expressed as a fraction of total XGRAF, and competitor DNA is expressed as the -fold excess over the radioactive probe.

The equilibrium binding equation describes the interaction between XGRAF and DNA (Equation 1),
K<SUB>d</SUB>=[<UP>DNA</UP>][<UP>XGRAF</UP>]/[<UP>DNA·XGRAF</UP>] (Eq. 1)
where [XGRAF] indicates free XGRAF protein concentration, [DNA] represents free competitor DNA concentration, and [DNA·XGRAF] represents concentration of XGRAF complexed with competitor DNA. Since competitor DNA is in excess over XGRAF, the concentration of total competitor DNA, [DNA]t, can be substituted for the concentration of free competitor DNA. Also, [XGRAF] can be expressed as [XGRAF]t minus [DNA·XGRAF], and the binding equation can be rearranged to the Scatchard equation (Ref. 29) (Equation 2),
f/[<UP>DNA</UP>]<SUB>t</SUB>=((<UP>−</UP>1/K<SUB>d</SUB>)×f)+1/K<SUB>d</SUB> (Eq. 2)
where f is the ratio of bound XGRAF to total XGRAF, [DNA·XGRAF]/[XGRAF]t. As explained above, [XGRAF]t has been defined as 1.0. Since we are not calculating an absolute value for the equilibrium constant, Kd is substituted with the term C50, which represents the -fold excess of competitor required to displace 50% of XGRAF from the probe DNA, and Equation 2 is simplified to Equation 3.
 [<UP>DNA·XGRAF</UP>]/[<UP>DNA</UP>]<SUB>t</SUB>=((<UP>−</UP>1/C<SUB>50</SUB>)×[<UP>DNA·XGRAF</UP>])+1/C<SUB>50</SUB> (Eq. 3)
[DNA·XGRAF] is expressed as a fraction of [XGRAF]t. [DNA]t is expressed in units of -fold excess of unlabeled competitor DNA over radioactively labeled DNA. In Fig. 1B, the data from Fig. 1A were plotted according to Equation 3, and C50 was calculated as the reciprocal of the y intercept. For each of the 27 single-point mutations, C50 was determined in this way in three independent experiments. Values for [DNA·XGRAF] below 0.1 or above 0.9 were not included in the Scatchard plots. Essentially the same results were obtained when C50 was calculated as the negative reciprocal of the slope.

Methylation Interference Footprinting-- DNA probes containing gamma  gene upstream sequence from positions -232 to -6, 32P-labeled on the sense strand, and from positions -232 to -115, 32P-labeled on the antisense strand, were produced by PCR with 0.1 µM primers and Taq enzyme (24). The PCR products were purified through a native 6% polyacrylamide gel (26) followed by organic extraction. The end-labeled DNA fragments (~1 × 106 cpm/pmol) were partially methylated at the guanine moieties by incubation for 2-3 min with dimethyl sulfate without carrier (30); in some cases underwent organic extraction; were precipitated twice with ethanol; and were dissolved in 10 mM Tris-HCl and 0.1 mM EDTA (pH 8.0). For preparative binding, the reaction volumes described above for the gel shift assay were scaled up 15-30-fold, with ~300,000 cpm of DNA probe, and electrophoresis was carried out at 250 V for 2.5-3.5 h at 4 °C. Wet gels were exposed to XAR film at 4 °C overnight; portions of the gel containing bound and unbound DNA were excised; and DNA was eluted by shaking overnight once or twice at 37 °C in 0.2 M NaCl, 10 mM Tris-HCl (pH 7.5), and 0.1 mM EDTA. The eluted DNA was purified over a NACS column (Life Technologies, Inc.) according to the manufacturer's instructions, and 8-16 µg of carrier yeast RNA was added. After ethanol precipitation, piperidine cleavage was performed (31), and piperidine was removed (24). The entire recovered bound DNA fraction and an approximately equal amount of radioactivity of the unbound DNA were analyzed by gel electrophoresis as described previously for methylation protection footprinting (24). The gels were exposed to XAR film at -80 °C with one Lightning Plus intensifying screen.

Transfection and Assays for Reporter Gene Activity-- Isolation of hepatocytes from three adult female X. laevis frogs and transfection were as described previously (24), except that 10 µg of control plasmid pCMV-beta gal (32) was used, and electroporation was conducted at a setting of 130 V. The cells were divided equally and incubated with or without 10-7 M dexamethasone and 10-9 M triiodothyronine in 12-well Primaria plates (Falcon) with 1 × 106 cells in 4 ml of medium. After 44-48 h, cell extracts were prepared, and the activities of beta -galactosidase (using the Galactolight method) and of luciferase were assayed as described (24).

Data Analysis for Transfection Assays-- Luciferase values were normalized to the beta -galactosidase activity in each transfection. -Fold hormonal induction was calculated by dividing the amount of luciferase activity/beta -galactosidase activity in hormone-treated cells by the amount of luciferase activity/beta -galactosidase activity in untreated cells. The data for each mutant are expressed relative to wild-type DNA as follows. XGRAF activity with wild-type DNA is defined as FWT - FD, where FWT represents the -fold increase in transcription in response to hormone treatment for the wild-type construct pLLgamma -187 and FD represents the -fold increase in transcription in response to hormone treatment for the mutation D construct, which has multiple mutations in the XGRAF site. Mutation D (-175ACT-173) abolished the XGRAF-binding site, so the remaining -fold response is due solely to the half-GREs (24). For each single-point mutant, the percent of wild-type XGRAF activity was calculated as ((FM - FD)/(FWT - FD)) × 100, where FM is the -fold increase in transcription in response to hormone treatment for each mutant. In a total of eight experiments, the average FWT was 5.3 ± 0.6 (mean ± S.E.), and the average FD was 2.2 ± 0.2 (mean ± S.E.). Each mutant was tested in three independent experiments.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Identification of Specific Guanosines Involved in XGRAF Binding-- Previously, we mapped the DNA elements between 177 and 135 bp upstream of the transcription start site that are important for glucocorticoid regulation of the Xenopus gamma -fibrinogen subunit gene (24). Three sites designated half-GRE1 (positions -168 to -163), half-GRE2 (positions -156 to -151), and half-GRE3 (positions -140 to -135) in Fig. 2C have a close match to the downstream half of the consensus GRE, bind to the DNA-binding domain of GR in vitro, and contribute to steroid-induced transcription in primary hepatocytes. Half-GRE1 is the most critical of the three GREs for hormonal induction. In addition, the tract from positions -177 to -169 is necessary for full hormone responsiveness even though it does not match the consensus GRE. The nuclear protein XGRAF binds to this region of the DNA (Fig. 2C).


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Fig. 2.   Identification of guanosines in the gamma -fibrinogen subunit gene upstream regulatory region that contact XGRAF. A, sense strand methylation interference. The assay used gamma  DNA from positions -232 to -6 synthesized by PCR with an end-labeled sense strand primer. Lane G+A, DNA sequencing ladder; lane B, protein-bound DNA; lane F, free DNA. Numbers indicate genomic sequence positions. Arrowheads mark the bands with reduced intensity in the protein-bound DNA fraction compared with free DNA (lane B versus lane F). B, antisense strand methylation interference. The conditions were the same as described in A, except that the probe was gamma  DNA from positions -232 to -115, 32P-labeled on the antisense strand. C, the nucleotide sequence of the gamma  upstream region from positions -187 to -130. Arrowheads at positions -184, -175, and -173 indicate those guanosines at which methylation inhibited binding of XGRAF. The squares above the sequence indicate 10-bp intervals. All the elements that contribute to glucocorticoid responsiveness of the gamma  gene are indicated below the sequence: the XGRAF-binding region between positions -177 and -169 and the three half-GREs (positions -168 to -163, -156 to -151, and -140 to -135).

To identify bases that have direct contacts with XGRAF, we used the methylation interference assay, which disrupts protein binding by methylation of critical guanines within a recognition site. A radioactively labeled DNA fragment including nucleotides -177 to -169 of the gamma  gene upstream region was partially methylated in vitro. The DNA was incubated with Xenopus liver nuclear extract, and both protein-bound DNA and free DNA were isolated by preparative native gel electrophoresis. The DNA was cleaved at all methylated guanosines, and the two populations of DNA were compared for relative abundance of fragments ending at particular positions (Fig. 2). When the sense strand was radioactively labeled (sequence shown in Fig. 2C), the two DNA fragments ending at positions -175 and -173 were significantly reduced in intensity in the protein-bound DNA fraction (Fig. 2A, lane B) as compared with the free DNA fraction (Fig. 2A, lane F), indicating that methylation of these guanines interfered with binding of a protein in the nuclear extract. Thus, bases G-175 and G-173 are important contact points for XGRAF. The intensity of the fragment ending at G-184 was also reduced. This nucleotide is not considered to be part of the core XGRAF-binding site since the intervening bases from positions -178 to -181 are not required for XGRAF binding or function (24). When the antisense strand was radioactively labeled, no differences in intensities of bands were observed (Fig. 2B, compare lanes F and B). Although no guanosines are present on the antisense strand within the putative XGRAF-binding site between positions -177 and -169, this experiment confirmed that the binding site does not extend upstream to position -182 or downstream to position -164, where the nearest guanosines are located.

Effect of Point Mutations on XGRAF Binding Ability-- The relative contribution of each nucleotide to the specific interaction between XGRAF and the gamma  DNA was assessed by saturation mutagenesis of the site from positions -177 to -169, generating all 27 single-point mutants in the 9-bp sequence. The effect of the mutations on DNA binding in vitro was analyzed by the gel shift assay, using the mutated DNA sequences as competitors for binding of XGRAF to radioactively labeled wild-type DNA, as described under "Experimental Procedures." Binding ability is expressed as C50, the -fold excess of competitor required to displace half of XGRAF from the wild-type probe. For each mutant, the C50 value was calculated in three independent experiments, and the results are presented as the mean ± S.E. of the three determinations (Fig. 3 and Table I). The wild-type DNA had a C50 value of 1.7-fold excess.


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Fig. 3.   Relative ability of single-point mutants of the gamma  DNA from positions -177 to -169 to bind XGRAF. Using the gel shift assay and Scatchard analysis described under "Experimental Procedures" and exemplified in Fig. 1, the ability of each mutant to bind to XGRAF was quantitated. The wild-type (WT) nucleotides at positions -177 to -169 and their corresponding single-point mutations (mut) are indicated along the y axis. C50 on the x axis represents the -fold excess of mutant DNA required as competitor to displace half of XGRAF from the radioactively labeled wild-type DNA probe. The data are shown as the mean ± S.E. of values determined in three independent experiments. For each mutant, three independently produced preparations of competitor DNA and at least two different batches of nuclear extract were used (except for mutant A-172, for which the data were derived from two experiments with two different preparations of competitor and one nuclear extract).

                              
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Table I
Relative ability of the 27 single-point mutants at positions -177 to -169 of the gamma  gene to bind XGRAF, to enhance glucocorticoid induction of transcription, and to regulate basal transcription

At positions -177, -176, and -174, all of the mutants bound strongly to XGRAF, with low C50 values of 1.7-3.8-fold excess. Therefore, these positions are not critical determinants for binding specificity since any nucleotide can be substituted without significantly affecting the interaction with XGRAF. Similarly, changing G-175 to either C or T was not deleterious to binding. However, when this position was mutated to A, the binding ability was reduced, with a C50 of 36-fold excess. It is interesting that the introduction of A at position -175 creates a stretch of six adenosines, which may interfere with binding due to structural alterations rather than the specific nucleotide substitution (33).

In contrast, nearly all changes in the bases from positions -173 to -169 substantially impaired XGRAF binding ability (Fig. 3 and Table I), with C50 values from 20-fold excess for mutant C-171 to 323-fold excess for mutant C-173. The only exception was mutant C-169, which retained relatively strong XGRAF binding (C50 = 6.3-fold excess).

Effect of Point Mutations on Glucocorticoid Responsiveness-- We also examined the effects of the point mutations in the XGRAF-binding site on glucocorticoid induction of transcription. The mutated gamma  gene upstream region was inserted into a luciferase reporter vector, and the constructs were transfected into primary Xenopus hepatocytes. Transfected cells were divided for plus or minus glucocorticoid treatment for 2 days, and lysates were analyzed for luciferase activity. The total -fold increase in luciferase levels in hormone-treated cells reflects the role of not only the XGRAF site, but also the three half-GREs in the upstream regulatory region of the gamma  gene (Fig. 2C). As described under "Experimental Procedures," the effect of the mutations only on the XGRAF contribution to the hormonal stimulation was assessed by comparing each single-point mutant with wild-type DNA, representing 100% induction, and with a triple mutant in the XGRAF-binding site, which eliminated XGRAF binding and therefore represented 0% activity of XGRAF in the induction. The triple mutant, which changed -175GAG-173 to -175ACT-173, has been shown previously to reduce glucocorticoid responsiveness through effects on the XGRAF-binding site rather than the GRE (24). Retention of at least 60% of XGRAF activity was considered normal function, whereas activity below 47% was defined as impaired.

As shown in Fig. 4 and Table I, full XGRAF activity ranging from 79 to 135% of the value obtained with wild-type DNA was achieved for all single-nucleotide substitutions at positions -177, -176, and -174. Hence, these positions are not essential for conferring XGRAF function on the gamma  gene. At position -175, XGRAF activity was reduced to 33% when the site was mutated to A, but 85 and 107% of wild-type function were attained with the T and C substitutions, respectively. Thus, the only functionally deleterious mutation from positions -177 to -174 is the G to A transition at position -175.


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Fig. 4.   Relative ability of single-point mutants of the gamma  DNA from positions -177 to -169 to enhance glucocorticoid induction of transcription. Transfection of Xenopus primary hepatocytes with constructs containing mutations in the XGRAF-binding region of the gamma  DNA and maintenance of the cells with or without hormone treatment are described under "Experimental Procedures." The wild-type (WT) nucleotides at positions -177 to -169 and their corresponding single-point mutations (mut) are indicated along the y axis. The data are reported as XGRAF activity, which is defined as the portion of the hormonal induction attributable to XGRAF (rather than GR) for each mutant compared with that of wild-type DNA (see "Experimental Procedures"). The values are the mean ± S.E. of three independent experiments. The value obtained in each experiment was the average of usually triplicate, and in a few cases duplicate, measurements.

Conversely, almost all mutations at positions -173 to -169 significantly decreased XGRAF activity to between 11 and 46% of function with wild-type DNA (Fig. 4 and Table I). The most dramatic departure from this general pattern is the G to C transversion at position -173, which improved the glucocorticoid induction, apparently to 453% of normal XGRAF activity. The other two exceptions are mutants G-172 and C-171, which allowed 60 and 71% of wild-type activity, respectively.

Effect of Point Mutations on Basal Transcription-- The level of transcription of the single-point mutants in the absence of hormone treatment ranged from 48 to 142% of that of wild-type DNA (Table I). Each data point for basal expression was obtained from an independent transfection event, whereas changes due to hormone treatment were assessed on a single cell population that was divided after the transfection.

    DISCUSSION
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Procedures
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Discussion
References

Correlation between XGRAF Binding to DNA in Vitro and Stimulation of Transcription-- In Table I, the ability of DNA with single-point mutations at positions -177 to -169 to bind to XGRAF in the gel shift assay is classified as strong (+) if half-maximal competition was achieved with <7-fold molar excess of mutated competitor over wild-type probe or as weak (-) if 20-fold or greater excess competitor was needed. Similarly, in Table I, the ability of DNA with each single-point mutation to enhance glucocorticoid-induced transcription in transfected primary hepatocytes is labeled as high (+) if at least 60% of wild-type XGRAF activity was retained or low (-) if 46% or less of wild-type XGRAF activity was observed. With only four exceptions, which will be discussed below, high activity in the transfection assay correlated with strong binding ability in the gel shift assay, whereas low functional activity corresponded with weak binding of XGRAF to the mutated DNA in vitro. This correlation can be seen clearly in a plot of XGRAF activity as a function of C50 (Fig. 5). The excellent agreement between binding and function firmly supports the conclusion that XGRAF, defined by its interaction with DNA in vitro, is the same protein that plays a role in hormonal stimulation of gene transcription in vivo.


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Fig. 5.   Correlation between the ability of the single-point mutants in region -177 to -169 to amplify hormone-induced transcription and to bind XGRAF. The portion of the glucocorticoid response attributable to XGRAF, expressed as XGRAF activity with mutant DNA as a percentage of that with wild-type DNA, is plotted as a function of the ability of the DNA to bind XGRAF in vitro, expressed as C50. See "Experimental Procedures" and "Discussion" for details.

The most striking discrepancy between physical association with XGRAF and ability to amplify glucocorticoid responsiveness was seen with mutant C-173, for which the hormonal induction was much higher than for any other construct, while ability to bind XGRAF was the weakest (Table I). This mutant was not included in Fig. 5 because its functional activity was much greater than that of the other mutants. Our method of computation attributed the stimulation of transcription to XGRAF, but we believe that the effect in this case was actually due to GR. The C-173 mutation generated the following sequence: -177AAGACTnnnTGTTCC-163, with two matches to the upstream half of the consensus GRE (GGTACAnnnTGTTCT) in addition to the five out of six matches to the downstream half. We have shown previously that the C at position -173 is essential for GR to interact with this site as a dimer and for hormonal stimulation greater than 10-fold (24). Therefore, the strong glucocorticoid response seen with the single-point mutation to C-173 could be explained by strong GR binding that eliminated the role of XGRAF in the induction. A comparable effect was not expected with any of the other nucleotides because even when all the bases except -173 were changed to match the consensus GRE, no increase in GR dimer binding or hormonal induction was observed (24).

Two other mutants, G-172 and C-171, were also classified in Table I as having weak binding while retaining the capacity to stimulate transcription. The levels of XGRAF transcriptional activity were, however, moderate at 60 and 71%, respectively, the lowest values that were still considered positive. The binding abilities (C50 values of 24- and 20-fold excess), although defined as weak, were intermediate between the strongest and weakest. In Fig. 5, the data for these mutants are represented by the two points in the center of the plot, which lie close to the line and therefore show good correlation between binding and function.

Mutant C-169 had a striking disjunction between strength of binding to DNA and ability to amplify glucocorticoid action. XGRAF bound quite well to the C-169 mutant since only a 6.3-fold molar excess of this DNA was required to displace half of XGRAF from the wild-type probe in the gel shift assay (Table I). Nonetheless, hormonal stimulation was very poor, with only 25% of wild-type XGRAF activity. The distinction between the C-169 mutant and all other constructs is evident from the anomalous position of the C-169 data in the lower left portion of Fig. 5. These results cannot be explained by changes in the interaction of GR with the DNA since the mutation lies within the 3-bp region between the two half-sites of the GRE, which is not critical for GR binding and function (23). Although the C-169 mutant binds fairly tightly to XGRAF in vitro, we hypothesize that it is incapable of conferring a functionally important conformational change on the protein that would occur upon binding to the natural recognition sequence.

Another important question is whether XGRAF had general effects on transcription independent of its amplification of GR action. The level of basal transcription for each of the single-point mutants is shown in Table I. Basal transcription was inherently more variable than -fold hormonal induction in the transfection assay because each data point was derived from an independently transfected sample. When transcriptional activity in the absence of hormone treatment was plotted versus binding ability (Fig. 6), only a slight correlation was observed. Therefore, we conclude that XGRAF may have a small effect on general transcription of the gamma  subunit gene, but that the major function of XGRAF is to enhance glucocorticoid induction in response to GR. This specificity is in contrast to many other glucocorticoid receptor accessory factors (such as nuclear factor-1; activator protein-1; cAMP response element-binding protein; hepatocyte nuclear factor-1, -3, and -4; and chicken ovalbumin upstream promoter transcription factor), which also stimulate basal transcription (2, 10-17).


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Fig. 6.   Correlation between the ability of the single-point mutants in region -177 to -169 to stimulate basal transcription and to bind XGRAF. Basal transcription, the luciferase activity/beta -galactosidase activity in untreated cells transfected with mutant DNA expressed as a percentage of that with wild-type DNA, is plotted as a function of the ability of the DNA to bind XGRAF in vitro, expressed as C50. See "Experimental Procedures" and "Discussion" for details.

Consensus Sequence for the XGRAF Recognition Site-- Based on the physical and functional data presented here, a consensus sequence can be derived for the XGRAF-binding site that reflects the most favorable nucleotide at each position. Bases -177 and -176 are no longer considered part of the recognition sequence since no substitutions at these positions affected XGRAF binding or activity. For nucleotides -175 to -169, the consensus sequence is BNGTTAA (B = C, G, or T; N = A, C, G, or T). Even with this more well defined site, we found no striking matches to recognition sequences in the transcription factor site data base TRANSFAC 3.2 (34) using the TESS searching program (36).2 Thus, XGRAF appears to have a novel sequence specificity for binding to DNA.

Models for Interaction of XGRAF and GR with Contiguous or Overlapping Binding Sites-- Previously, we presented four possible models for the interaction of XGRAF and GR with DNA at closely juxtaposed sites (24). Model 1 depicted simultaneous binding of XGRAF to its site at nucleotides -175 to -169 and GR binding as a dimer at nucleotides -177 to -163, which would constitute a full-length GRE. To bind these sites concurrently, XGRAF must contact the DNA in the minor groove since GR is known to occupy the major groove (35). However, the methylation interference experiment (Fig. 2) established that XGRAF also binds in the major groove since modification of the N-7 positions of guanines, which are accessible only in the major groove, interfered with binding. Therefore, Model 1 is not a likely mechanism for interaction of GR and XGRAF with their respective sites.

Model 2 proposed an interaction between XGRAF and a monomer of GR. Model 3 depicted a trimeric complex consisting of one molecule of XGRAF and a dimer of GR, with GR contacting only the downstream half of the GRE. Both of these scenarios are possible but must take into account that the XGRAF- and GR-binding sites are directly contiguous. In Model 4, binding of XGRAF and GR was sequential rather than simultaneous, which would obviate problems of steric hindrance for two protein molecules binding to abutted recognition sites. Experiments are in progress to distinguish between these mechanisms.

Classically, the presence of a receptor defined a tissue as being a target for a steroid hormone, and the presence of a high affinity receptor-binding site on the DNA was a prerequisite for a responsive gene. It is becoming increasingly clear that steroid hormone action is dramatically influenced by many other aspects of the local cellular environment and the structure of the gene regulatory region. Accessory DNA-binding proteins such as XGRAF can play as important a role as the receptor in determining the extent of hormonal induction. The fact that diverse cellular responses rely on unique combinations of accessory DNA-binding proteins, coactivators, corepressors, and other factors makes it possible for multiple control mechanisms to regulate genes differentially in the same cell in response to a single hormonal stimulus.

    ACKNOWLEDGEMENTS

We gratefully acknowledge insightful discussions with Drs. Mark Milanick and Mark Hannink and helpful comments on the manuscript from Brian Morin.

    FOOTNOTES

* This work was supported by NHLBI Grant RO1-HL39095 and Research Career Development Award HL02934 (to L. J. H.) and Post-doctoral and Pre-doctoral Training Grant HL07094 (to M. L. and R. N. W.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Gladstone Inst. of Cardiovascular Disease, University of California, San Francisco, CA 94141.

§ To whom correspondence should be addressed: Dept. of Physiology, MA415 Medical Sciences Bldg., University of Missouri School of Medicine, Columbia, MO 65212. Tel.: 573-882-5373; Fax: 573-884-4276; E-mail: physljh{at}muccmail.missouri.edu.

1 The abbreviations used are: GRE, glucocorticoid response element; GR, glucocorticoid receptor; XGRAF, Xenopus glucocorticoid receptor accessory factor; bp, base pair(s); PCR, polymerase chain reaction.

2 Program is available on the World Wide Web (URL: agave.humgen.upenn.edu/tess/index.html).

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
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