Novel Accessory Factor-Binding Site Required for Glucocorticoid Regulation of the {gamma}-Fibrinogen Subunit Gene from Xenopus laevis

Robert N. Woodward1, Min Li and Lené J. Holland

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Glucocorticoids induce gene expression by binding to an intracellular receptor that interacts with genomic DNA and stimulates transcription of specific genes. The consensus DNA-binding site for the glucocorticoid receptor, called a glucocorticoid response element (GRE), is GGTACAnnnTGTTCT. In the classical model, binding of the receptor as a dimer to the two halves of the GRE is required for activation of transcription. For some glucocorticoid-regulated genes, additional DNA-binding proteins called accessory factors are necessary for hormonal responsiveness. We have identified a new factor required for glucocorticoid-induced expression of the {gamma}-fibrinogen subunit gene from the frog Xenopus laevis. Transfection of cloned DNA fragments into primary Xenopus hepatocytes showed that the DNA between 163 and 187 bp upstream of the transcription initiation site is essential for hormonal activation. A single complex forms when this small region of DNA is incubated in vitro with Xenopus liver nuclear proteins. The protein recognition site has been narrowed to AAGAGTTAA, a sequence not previously described as a transcription factor-binding site. We have named the protein(s) bound to this sequence Xenopus glucocorticoid receptor accessory factor (XGRAF). In addition to the XGRAF-binding site, glucocorticoid regulation of the {gamma}-fibrinogen gene requires at least three nearby GREs, each of which is a poor match to the consensus GRE. The position of the binding site for XGRAF overlaps the putative upstream half of the most important GRE. Models are presented to show possible ways that the novel accessory factor and the glucocorticoid receptor could act through closely juxtaposed sites on the DNA.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Steroid hormones regulate numerous critical processes during development and in differentiated cells. Glucocorticoid hormones from the adrenal gland stimulate metabolic processes and contribute to the control of inflammatory responses. In target cells, glucocorticoids bind to the glucocorticoid receptor (GR), allowing the hormone-receptor complex to translocate to the nucleus. GR binds to a specific DNA element and activates gene transcription (1). Although some genes may require only GR as the transcriptional mediator of glucocorticoid responsiveness (2), many other glucocorticoid-regulated genes, including phosphoenolpyruvate carboxykinase, tyrosine aminotransferase (TAT), mouse mammary tumor virus (MMTV), and proliferin require additional DNA-binding proteins called accessory factors (3, 4, 5, 6). Some accessory factor proteins that have been identified include chicken ovalbumin upstream promoter-transcription factor (COUP-TF), hepatocyte nuclear factor (HNF)-4, HNF-3, nuclear factor 1 (NF-1), and activator protein-1 (AP-1) (3, 4, 5, 6, 7, 8). Presumably different complexes of GR and accessory factors are necessary for precise control of specific sets of genes.

The DNA sequence bound by GR is called a glucocorticoid response element (GRE). The consensus sequence for the GRE, GGTACAnnnTGTTCT, was derived from functional binding elements in multiple genes (1). GR can bind in vitro as a monomer to the downstream half of the GRE (TGTTCT), but transcriptional activation requires that GR interact with the full GRE as a dimer (9).

Glucocorticoids regulate hepatic production of several proteins as part of the acute phase response, a reaction to tissue injury, infection, or inflammation (10, 11). One of these proteins is fibrinogen, the precursor to fibrin, which is the primary structural protein of a blood clot. We have used a liver cell culture system from Xenopus laevis to examine mechanisms of transcriptional regulation of the genes coding for fibrinogen. This model is used because the fibrinogen genes from Xenopus have a strong steroid hormone response (12, 13).

Fibrinogen is secreted from the liver as a hexameric molecule comprising two each of three different subunits, A{alpha}, Bß, and {gamma}. In Xenopus primary hepatocytes, glucocorticoid treatment coordinately stimulates synthesis of the subunit mRNAs (12) through an increase in transcription of each of the three genes (13). For the Bß-subunit gene, a single GRE is located upstream of the transcription initiation site (14). Mutation of this GRE eliminates the ability of the Bß-fibrinogen gene upstream regulatory region to respond to glucocorticoids. In the present report we examine glucocorticoid regulation of the gene coding for the {gamma}-subunit of X. laevis fibrinogen.2 Rather than a single GRE as in the Bß-subunit gene, glucocorticoid regulation of the {gamma}-fibrinogen subunit gene requires accessory factor binding to a novel DNA element and GR binding at multiple GREs.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Isolation of Genomic Clones for the {gamma}-Subunit of Xenopus Fibrinogen and Determination of the DNA Sequence
Three genomic clones of the X. laevis {gamma}-fibrinogen subunit gene were isolated that together encompass more than 21 kb of DNA. The genomic DNA sequence from base -1537 to base +52 is presented in Fig. 1Go. This DNA includes the transcription initiation site, identified by primer extension (data not shown) as an A (+1 in Fig. 1Go) after a C, conforming to the consensus sequence derived for transcription initiation sites (15).



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Figure 1. Nucleotide Sequence of the DNA Upstream of the Xenopus {gamma}-Fibrinogen Subunit Gene

The sequence of bases -1537 to +52 is shown. (The sequence from bases -1537 to +1210 has been entered in Genbank.) The TATA box is marked by underlining. The transcription initiation site is indicated as +1 and the sequence continues to +52, which includes the first codon of the protein-coding sequence (40).

 
Identification of a DNA Region Required for the Response to Glucocorticoids
To determine which DNA sequences were necessary for hormone regulation, the upstream region of the {gamma}-fibrinogen subunit gene was connected to the luciferase reporter gene in the plasmid pLuc-Link 2.0 (pLL). The plasmid constructs were transfected into Xenopus primary hepatocytes, which allowed the evaluation of the hormone effect in a more physiological system than provided by transformed cell lines. Luciferase levels were measured after 44–52 h in culture either without hormone treatment or with 10-7 M dexamethasone. The plasmid containing 5000 bp of the {gamma}-subunit gene upstream sequence responded to glucocorticoid treatment with an approximately 3.5-fold increase in transcription (Fig. 2Go). This response was less than the 5- to 15-fold stimulation observed for the {gamma}-fibrinogen gene in run-on assays (13). The discrepancy between the two assays is probably due to differences in cell physiology. The run-on assays were performed in cells maintained in culture for several days before hormone treatment, during which time the cells become more responsive to glucocorticoids (our unpublished observations). The transfected hepatocytes were cultured only 2 days because the electroporation must be performed on freshly isolated cells in suspension. Nonetheless, the 3.5-fold hormonal stimulation observed in the transfection experiment is a physiologically significant induction and permits analysis of unique mechanisms responsible for regulation of the Xenopus {gamma}-fibrinogen gene by glucocorticoids.



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Figure 2. Glucocorticoid Regulation of Transcription by the {gamma}-Subunit Gene Upstream DNA

Primary hepatocytes were transfected with plasmids containing selected lengths of the {gamma}-subunit gene upstream region, then incubated either without hormone treatment or with 10-7 M dexamethasone. The data are reported as the fold increase in transcription due to hormone treatment, calculated for each construct by dividing the amount of luciferase activity in hormone-treated cells by the amount of luciferase activity in untreated cells. As a control for transfection efficiency, the luciferase values were normalized to the ß-galactosidase activity from the cotransfected reporter plasmid pCMVßgal. The labels below the bars are the 5'-boundary of the DNA from the {gamma}-subunit gene. Each bar is the mean + SEM of four to 35 transfections (n, shown in white numbers) from two to 13 different preparations of primary cells. For constructs where n = 2 the data are the mean + range. For -2900 only one determination was made. In the absence of hormone treatment, the basal levels of luciferase/ß-galactosidase activity were as follows for each construct: -5000, 87 ± 9.6 (range); -2900, 43; -1158, 136 ± 26 (SEM); -603, 144 ± 10 (range); -369, 125 ± 38 (SEM); -232, 100; -187, 113 ± 8.5 (SEM); -163, 84 ± 8.5 (SEM); -129, 90 ± 13 (SEM); -105, 91 ± 6.0 (SEM). The data are expressed as a percentage of the value for the -232 construct, except the data for the -369 and -1158 constructs were normalized to the value for the -369 construct in one experiment that did not include the -232 construct.

 
Progressive deletion of DNA from the 5'-end of the {gamma}-construct showed that the 3.5-fold induction was maintained with as few as 187 bp of upstream sequence (Fig. 2Go). With further deletion to position -163, the glucocorticoid response was severely decreased to only 1.4-fold. Thus, the 24 bases encompassed by nucleotides -187 to -164 were essential for glucocorticoid induction. The basal level of transcription in the absence of hormone treatment was approximately the same in all the deletion constructs containing from 105-5000 bp of upstream sequence (see legend to Fig. 2Go). In particular, basal activity did not change significantly when the hormone-responsive region from -187 to -164 was removed.

The specific bases important for hormone regulation of {gamma}-fibrinogen gene expression were defined more precisely using site-specific mutations (Fig. 3AGo) made in the -232 deletion construct (pLL{gamma}-232). A transfection construct with mutations in bases -181 to -178 had a glucocorticoid response similar to wild type (Fig. 3BGo, mut A). Mutation D, with base changes from -175 to -173, significantly decreased the hormone induction (1.7-fold; Fig. 3BGo, mut D). A more extensive mutation, from -182 to -173, impaired glucocorticoid induction to the same extent (Fig. 3BGo, mut B). Mutation of bases from -168 to -166 decreased the hormone response to only 1.3-fold (Fig. 3BGo, mut I). All three of these mutations (D, B, and I) affect the sequence of a potential GRE from -177 to -163, AAGAGTnnnTGTTCC, designated GRE1 (see Fig. 7Go). The downstream half of GRE1 has a strong match (five of six positions) to the GRE consensus sequence. Mutation I, which changed bases in the downstream half of GRE1, probably caused loss of function by disrupting the GRE. The role of GRE1 and other GREs in glucocorticoid regulation of the {gamma}-gene will be discussed below. Mutations B and D changed nucleotides in the upstream half of GRE1, but this region was already a poor match to the consensus GRE. Therefore it is unlikely that the decreased glucocorticoid response from the constructs with mutations B and D was due to disruption of the GR-binding site. These observations raised the possibility that a non-GR transcription factor binds to these bases and is necessary for glucocorticoid regulation.



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Figure 3. Effect of Site-Specific Mutations on the Glucocorticoid Response of the {gamma}-Fibrinogen Subunit Gene Upstream Region

A, Mutations to the DNA sequence in the hormone-responsive region. The mutations (A, B, D, and I) are shown below the sequence. Lowercase letters are bases that differ from the wild type sequence. The 24-bp hormone-responsive region from -187 through -164 is labeled. B, Effect of mutations on glucocorticoid responsiveness. The mutations were generated in the context of the pLL{gamma}-232 construct. Transfection assays with these mutated constructs were performed as in Fig. 2Go. Labels below the bars indicate which mutation is present in the construct. Each bar is the mean + SEM of five to 20 transfections (n, white numbers) from two to six different preparations of primary cells. The data for the -129 deletion construct is the mean + SEM for six determinations. In the absence of hormone treatment, the basal levels of luciferase/ß-galactosidase activity were as follows for each construct (expressed as a percentage of the value for the -232 construct ± SEM): -232, 100; mut A, 104 ± 30; mut B, 69 ± 15; mut D, 89 ± 7.4; mut I, 101 ± 6.5; -129, 88 ± 5.4.

 


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Figure 7. Comparison of Potential GREs to the Consensus Sequence

Each of the putative GREs is aligned with the consensus GRE from Beato (1). The bases on shaded background are matches to the consensus sequence.

 
Identification of a DNA-Binding Activity in Nuclear Extracts
We sought to determine whether a transcription factor other than GR bound the hormone-responsive region of the {gamma}-gene in vitro. We used a crude extract from nuclei of Xenopus hepatocytes that is enriched for transcription factors but is not expected to contain a significant amount of GR (our unpublished observations). When this extract was incubated with a DNA fragment of the {gamma}-gene from -200 to -115, a single DNA-protein complex was detected in the gel shift assay (Fig. 4Go, lane 2). Addition of 100-fold molar excess of nonlabeled double-stranded oligonucleotide (33 bp from -189 to -157) completely eliminated binding (Fig. 4Go, lane 3). Thus, hepatocyte nuclear protein(s) bound to a 33-bp DNA fragment that encompassed the glucocorticoid-responsive region.



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Figure 4. Gel Shift Assay to Identify the Nuclear Protein-Binding Site

Assays were performed with 32P-labeled {gamma}-DNA (from position -200 to -115; 31 fmol, 2.8 x 104 cpm per reaction). Lane 1, DNA, no nuclear extract. Lanes 2–6, DNA plus 3 µg nuclear extract without (lane 2) or with (lanes 3–6) 100-fold molar excess of unlabeled DNA competitors. Competitors: lane 3, {gamma} (-189 to -157), the 33-bp oligonucleotide from -189 to -157 that includes the glucocorticoid-responsive region ({gamma}GRR); lane 4, {gamma} (-189 to -157) with mutation A; lane 5, {gamma} (-189 to -157) with mutation D; lane 6, {gamma} (-189 to -157) with mutation I. F, Free DNA; B, DNA bound by nuclear protein.

 
Definition of the Nuclear Protein-Binding Site
The position of the protein-binding site was defined with DNA competitors containing blocks of mutated bases (Fig. 3AGo). An oligonucleotide with changes in bases -181 through -178 (mutation A) competed for binding (Fig. 4Go, lane 4) as effectively as wild type sequence (Fig. 4Go, lane 3), showing that these four bases were not required for binding. Mutation D (bases -175 to -173) destroyed the ability of the oligonucleotide to compete for nuclear protein binding (Fig. 4Go, lane 5), demonstrating the importance of the bases at these positions for the DNA-protein interaction. Mutation I (bases -168 to -166) did not inhibit the ability of the oligonucleotide to compete (Fig. 4Go, lane 6), indicating that the bases required for binding of the nuclear protein complex did not include -168 to -166. Together these experiments show that the binding site for the nuclear factor could extend from -177 to -169.

Since mutation I disrupted the potential GR-binding site in this region (GRE1), but did not affect formation of the protein-DNA complex detected in the gel shift assay, it is unlikely that the protein in the nuclear extract was GR. We confirmed that this nuclear protein-DNA binding did not have the same sequence specificity as a GR-GRE interaction by competition experiments. Excess oligonucleotide containing either the GRE from the Xenopus Bß-fibrinogen subunit gene or the palindromic GRE (14) did not compete for nuclear protein binding to the wild type DNA (Fig. 5AGo, lanes 4 and 5). Thus the protein bound to the wild type sequence with a much higher affinity than to a strong GR-binding site, identifying it as a protein distinct from GR.



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Figure 5. Gel Shift Assays to Characterize the Hepatocyte Nuclear Protein Bound to the Hormone-Responsive Region

A, Competition with GR and HNF-1 binding sites. Assays were performed with 32P-labeled {gamma}-DNA (from position -200 to -115; 31 fmol, 6000 cpm per reaction). Lane 1, DNA, no nuclear extract. Lanes 2–7, DNA plus 3 µg nuclear extract without (lane 2) or with (lanes 3–7) 100-fold molar excess of unlabeled DNA competitors. Competitors: lane 3, 33-bp oligonucleotide from -189 to -157 that includes the glucocorticoid-responsive region ({gamma}GRR); lane 4, GRE from the Xenopus Bß-fibrinogen subunit gene; lane 5, palindromic GRE; lane 6, HNF-1-binding site from the Xenopus Bß-subunit gene; lane 7, the HNF-1 sequence from the rat Bß-fibrinogen gene. All GRE and HNF-1 competitor sequences were described by Roberts et al. (14). F, Free DNA; B, DNA bound by nuclear protein. B, Analysis of protein interaction with the AP-1-binding site. Assays were performed with 9.2 fmol (6500–9300 cpm) of probe in each reaction. The probe in lanes 1–6 was {gamma} (-189 to -157). Lane 1, DNA, no nuclear extract. Lanes 2–6, DNA plus 3 µg nuclear extract without (lane 2) or with (lanes 3–6) competitors. Competitors: lanes 3 and 4, 20- or 100-fold molar excess of {gamma} (-189 to -157); lanes 5 and 6, 20- or 100-fold molar excess AP-1 double-stranded 33-bp oligonucleotide. This oligonucleotide is the {gamma}-sequence from -189 to -157 with two base changes to create an AP-1 site (see text). The probe in lanes 7–12 was the same AP-1 double-stranded oligonucleotide. Lane 7, DNA, no nuclear extract. Lanes 8–12, DNA plus 3 µg nuclear extract without (lane 8) or with (lanes 9–12) competitors. Competitors: lanes 9 and 10, 20- or 100-fold molar excess of unlabeled AP-1 oligonucleotide; lanes 11 and 12, 20- or 100-fold excess of {gamma} (-189 to -157). F, Free DNA; B, {gamma}GRR DNA bound by nuclear protein.

 
Although the nuclear protein did not bind to a GRE, the binding could be due to some other previously described transcription factor site. We analyzed the sequence of the {gamma}-DNA from -189 to -157 for matches to protein-binding elements by searching the TFD and TRANSFAC databases. No striking similarities to known transcription factor-binding sites were identified, despite allowing two mismatches in the search. One possibly meaningful match was from -173 to -168 (GTTAAT), a perfect match to one half of the palindromic binding site for the liver-specific transcription factor, HNF-1. Binding sites for HNF-1 were used as competitors for nuclear protein binding in the gel shift assay. Neither the HNF-1 site from the Xenopus Bß-fibrinogen gene nor a rat Bß-fibrinogen HNF-1 binding site (14) was able to compete, demonstrating that the complex was not due to a protein bound specifically to the HNF-1 site (Fig. 5AGo, lanes 6 and 7).

A second potentially important sequence was from -176 to -170. This sequence is a five of seven match to the binding site for the ubiquitous transcription factor AP-1 (TGAGTCA) (16). Therefore, we determined whether the protein-binding site was bound by AP-1 in the Xenopus nuclear extract. When the 33-mer oligonucleotide (bases -189 to -157) containing the nuclear protein-binding site was used as a probe in the gel shift assay, a single band appeared (Fig. 5BGo, lane 2). This binding was competed by both 20- and 100-fold excess of the same nonlabeled oligonucleotide (Fig. 5BGo, lanes 3 and 4). A second competitor was used that contained the AP-1-binding site, which was made in the {gamma}-189 to -157 oligonucleotide by changing position -176 from A to T and position -171 from T to C. The AP-1 oligonucleotide provided little competition at 20-fold, but some competition at 100-fold (Fig. 5BGo, lanes 5 and 6), indicating that the Xenopus nuclear protein bound with much lower affinity to an AP-1 site than to the {gamma}-fibrinogen gene glucocorticoid-regulatory region. To establish further that the protein bound to the {gamma}-fibrinogen hormone-responsive region was not AP-1, the oligonucleotide that contains the AP-1 site was labeled and used as a probe in the gel shift assay. The AP-1-specific probe was bound by several proteins in the nuclear extract, none of which migrated at the same position as the protein that bound to the wild type {gamma}-fibrinogen DNA (Fig. 5BGo, compare lane 8 to lane 2). Proteins bound to the AP-1 site were competed by 20- and 100-fold self-competition (Fig. 5BGo, lanes 9 and 10), but were not competed by 20- or 100-fold excess of the wild type {gamma}-fibrinogen DNA (Fig. 5BGo, lanes 11 and 12). Thus, proteins in Xenopus nuclear extract that did bind an AP-1 site did not bind the {gamma}-fibrinogen sequence from -189 to -157.

The functionally important element from -177 to -169 appears to be a novel transcription factor-binding site required for glucocorticoid regulation of the {gamma}-fibrinogen gene. We have named the protein(s) bound to this site Xenopus glucocorticoid receptor accessory factor (XGRAF).

GR-Binding Sites in the {gamma}-Subunit Gene Upstream Region
Previously described accessory factors usually regulate transcription in conjunction with nearby GREs (17). The region around the XGRAF-binding site and downstream of the site was analyzed for possible GREs by identifying GR-binding sites with the methylation protection assay. The DNA upstream of -187 was not analyzed since the 5' deletion constructs indicated that no elements beyond -187 were required for hormone induction (Fig. 2Go). Since the nuclear extract was not likely to contain a significant amount of active GR, the protein used for these assays was bacterially expressed GR DNA-binding domain (GR-DBD), which is composed of amino acids 440–525 of the rat GR (20). Only three amino acids near the C terminus of this section differ from the comparable portion of X. laevis GR (21). The GR-DBD exists as a monomer (22) and can bind either as a monomer to half-site GREs or cooperatively as a dimer to GREs that have a high match to the full consensus sequence (22, 23).

When GR-DBD was bound to the {gamma}-upstream DNA, six guanines were protected from methylation: on the sense strand at positions -167, -155, and -139 and on the antisense strand at -164, -163, and -136 (Fig. 6Go, A and B). This protection pattern identified three GR-binding sites that may be part of several potential GREs (Fig. 6CGo and Fig. 7Go). The protected guanines at -167, -164, and -163 are within a binding site that could be the downstream half of a GRE from -177 to -163 (GRE1, Fig. 6CGo). The guanines at -139 and -136 are in a second binding site that could be the downstream half of a GRE that extends from -149 to -135 (GRE3, Fig. 6CGo). GR-DBD binding to GRE1 and GRE3 was only to the downstream halves, which match the consensus well. No binding to the poor upstream halves occurred. The protected guanine at -155 identified a third binding site that could be part of either GRE2a from -165 to -151, or GRE2b from -156 to -142 (Fig. 6CGo). The guanine at -152 on the antisense strand is also part of a consensus match for GRE2a and GRE2b, but was not protected (Fig. 6BGo). Therefore, the GRE2 area may have lower affinity for GR than does either GRE1 or GRE3.



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Figure 6. Methylation Protection of the {gamma}-Fibrinogen Gene Upstream Regulatory Region by Recombinant Rat GR-DBD

A, Sense strand methylation protection. The assays used 10 fmol (1.7 x 104 cpm) of a DNA fragment from -200 to -6 made using PCR with an end-labeled primer for the sense strand. G + A, DNA sequencing ladder; F, free DNA; B, protein-bound DNA. Numbers are genomic sequence position. Arrowheads mark the bases protected from methylation by the GR-DBD. B, Antisense strand methylation protection. Same conditions as panel A except the -200 to -42 DNA fragment (2.1 x 106 cpm) was made using an end-labeled primer for the antisense strand. C, The nucleotide sequence of the {gamma}-upstream region from -179 to -133 with the bases protected from methylation by GR-DBD indicated with arrowheads. The four potential GREs are shown as pairs of boxes below the sequence, with dark shading to indicate matches to the GRE consensus sequence.

 
Identification of Functionally Important GREs
In addition to GR-binding ability, we determined which of the four potential GREs were functionally important. The GREs were analyzed with site-specific mutations in the context of the pLL{gamma}-232 construct (Fig. 8Go).



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Figure 8. Four Poor Matches to the GRE Consensus Sequence in the -187 to -130 Region

Each of the potential GREs identified by methylation protection is shown in its position below the {gamma} upstream DNA sequence. Matches to the GRE consensus are underlined. For each GRE the site-specific mutations that affect its sequence are shown under the GRE. The lowercase letters indicate bases changed from wild type.

 
The importance of GRE1 was investigated with mutation I, a site-specific mutation containing three changes to the downstream half (bases -168 to -166). The mutated construct had a substantially lower response to glucocorticoid treatment than wild type (Fig. 9Go, mut I=1.3-fold). This is lower than the response of the construct with mutation D (1.7-fold), a mutation that eliminated XGRAF binding (Fig. 4Go). Mutation I affected only GRE1, with no consequence either on the XGRAF-binding site or on the matches to the consensus sequence of the other potential GREs. Thus the large decrease in the hormone response with mutation I showed specifically that the downstream half of GRE1 was required for the glucocorticoid induction.



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Figure 9. Effect on Transcription of Site-Specific Mutations to the Potential GREs

The mutations shown in Fig. 8Go and described in the text were introduced into DNA constructs that were used in the transfection assays as in Fig. 2Go. All of the mutations are in pLL{gamma}-232 except mutation I which is in pLL{gamma}-187. (No difference is expected with different parent constructs because several mutations were examined in both pLL{gamma}-232 and pLL{gamma}-187 and the same results were obtained.) The mean + SEM is shown for each construct. White numbers are the number of transfections (six to 25), from two to eight different preparations of primary cells. For the -129 deletion construct n = 9. In the absence of hormone treatment, the basal levels of luciferase/ß-galactosidase activity were as follows for each construct (expressed as a percentage of the value for the -232 construct ± SEM): -232, 100; mut D, 89 ± 7.4; mut I, 101 ± 6.5; mut J, 100 ± 7.4; mut L, 124 ± 12; -129, 89 ± 5.4.

 
The functional importance of the GRE2 area was examined with mutation J. (Mutation J also affected GRE3, which is discussed later.) GRE2a was changed from a five of 12 match to a four of 12 match, including disruption of one of the most conserved bases of a GRE (24). Based on comparison to the consensus GRE, this mutation should eliminate any possible function of GRE2a. Mutation J changed GRE2b from an eight of 12 match to the consensus to a five of 12 match, retaining only three of the most conserved bases (24). The mutated construct had a decreased glucocorticoid responsiveness of 2.1-fold (Fig. 9Go, mut J), indicating that either GRE2a or GRE2b was required for the full hormone response.

As mentioned above, GRE3 was mutated in its upstream half by mutation J, but the changes increased its match to the consensus by one base so that GRE3 became a nine of 12 match to the consensus sequence with a perfect downstream half. Therefore the decreased hormone response caused by mutation J (Fig. 9Go, mut J) cannot be explained by changes in the match of GRE3 to the consensus. The importance of GRE3 was analyzed with mutation L, which converted the downstream half of GRE3 from a perfect match to only a one of six match. Overall, the mutated GRE3 had only a three of 12 match to the consensus sequence. Since mutation of these bases resulted in a 2-fold glucocorticoid response (Fig. 9Go, mut L), GRE3 was necessary for the {gamma}-subunit gene to respond completely to glucocorticoids.

Analysis of the Region Encompassing Both the XGRAF-Binding Site and GRE1
The XGRAF-binding site extends from -177 to -169 (Fig. 4Go) and GRE1 is from -177 to -163 (Fig. 8Go). If glucocorticoid induction requires binding of GR as a dimer to GRE1, then one monomer of GR must interact with some of the same DNA sequence as XGRAF. We resolved which protein was important for function at this position by examining both protein binding and the hormone response with mutated binding sites.

Both mutation B and mutation D changed bases in this potential overlap region and reduced hormone responsiveness (Fig. 3Go). For mutation B the reduction occurred despite the fact that GRE1 was made into a better match to the consensus GRE. We expanded on this result with a new series of site mutations that incrementally increased the match of the upstream half of GRE1 to the canonical GRE (Fig. 10AGo). Mutation E matched three of six bases in the upstream half. Mutation F matched the upstream half at four bases, mutation G matched at five bases, and mutation C had a perfect match of its upstream half to the GRE consensus. Each of these DNAs contained the near perfect downstream half of GRE1. In addition, each mutation changed bases in the XGRAF-binding site.



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Figure 10. Effect of Site-Specific Mutations on GR-DBD or XGRAF Binding in Gel Shift Assays

A, Sequences of the site-specific mutations in the XGRAF-binding site and the upstream half of GRE1. Overlining indicates the bases of the potential binding site for XGRAF. Lowercase letters indicate bases that are changed from wild type. Underlining shows bases that match the consensus sequence for the GRE. B, XGRAF binding. The labeled probe was from -200 to -115 of the {gamma}-gene (62 fmol, 2500 cpm per reaction). Lane 1, DNA, no nuclear extract. Lanes 2–7, DNA plus 3 µg nuclear extract without (lane 2) or with (lanes 3–7) 100-fold molar excess of unlabeled DNA competitors. Competitors: lane 3, {gamma} (-189 to -157) ({gamma}GRR); lane 4, {gamma} (-189 to -157) with mutation E; lane 5, {gamma} (-189 to -157) with mutation F; lane 6, {gamma} (-189 to -157) with mutation G; lane 7, {gamma} (-189 to -157) with mutation C. F, Free DNA; B, nuclear protein bound DNA. C, GR-DBD binding. Lanes 1, 3, 5, 7, and 9, DNA only (9.2 fmol, 3.2–5 x 104 cpm per reaction). Lanes 2, 4, 6, 8, and 10, DNA incubated with GR-DBD. DNA probes: lanes 1 and 2, {gamma} (-189 to -157), the 33-bp double-stranded oligonucleotide of the {gamma}-sequence that includes the glucocorticoid-responsive region; lanes 3 and 4, {gamma} (-189 to -157) with mutation E; lanes 5 and 6, {gamma} (-189 to -157) with mutation F; lanes 7 and 8, {gamma} (-189 to -157) with mutation G; lanes 9 and 10, {gamma} (-189 to -157) with mutation C. F, Free DNA; I, monomer of GR-DBD bound to DNA; II, dimer of GR-DBD bound to DNA.

 
Initially we examined the ability of these mutated sequences to form a complex with XGRAF. Protein binding to the wild type {gamma}-fibrinogen DNA (Fig. 10BGo, lane 2) was competed by an oligonucleotide containing the XGRAF-binding site (Fig. 10BGo, lane 3). The four mutated oligonucleotides that each had bases changed in the XGRAF-binding site (Fig. 10AGo) were not able to compete for binding of the accessory factor (Fig. 10BGo, lanes 4–7). Therefore, none of the mutated sequences were able to bind XGRAF.

Since these mutations increased the match of GRE1 to the consensus, we examined the ability of GR-DBD to bind the mutated sequences. In gel shift assays the wild type DNA was weakly bound by only a monomer of GR-DBD (Fig. 10CGo, lane 2). Probes containing mutations E, F, and G were also bound by monomer of the protein, despite increasing matches to the GRE consensus sequence (Fig. 10CGo, lanes 4, 6, and 8). In each case the monomer probably bound to the wild type downstream half of GRE1 that had five of six bases matching the consensus GRE. The DNA probe that contained mutation C, with 11 of 12 bases that match consensus, was bound by dimer of GR-DBD (Fig. 10CGo, lane 10). Therefore, none of the mutations changed the ability of the DNA to bind GR-DBD except mutation C, which created a stronger binding site.

The results of the binding experiments were compared with the effects of the mutations on hormone inducibility. The wild type -232 deletion construct had a 3-fold response to glucocorticoid treatment (Fig. 11Go). The construct containing mutation C had a 10.5-fold response (Fig. 11Go). This large hormonal induction, as well as the binding of GR-DBD as a dimer to the DNA with mutation C (Fig. 10CGo), are both probably due to introduction of a strong GRE that overrides the wild type mechanism. Constructs with mutations E, F, and G had decreased hormone responses compared with the wild type (Fig. 11Go). Since each of the E, F, and G mutations did not alter GR binding (Fig. 10CGo), but did disrupt XGRAF binding (Fig. 10BGo), the loss of function correlated with loss of XGRAF binding.



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Figure 11. Transcriptional Effect of Site-Specific Mutations to the Overlapping XGRAF-Binding Site and GRE1

Transfections were performed as in Fig. 2Go. The labels below the bars are the mutations in pLL{gamma}-232 defined in Fig. 10AGo. For each construct the bar is the mean + SEM of five to seven transfections (n, white numbers) from two or three different preparations of primary cells. In the absence of hormone treatment, the basal levels of luciferase/ß-galactosidase activity were as follows for each construct (expressed as a percentage of the value for the -232 construct ± SEM): -232, 100; mut E, 84 ± 10; mut F, 59 ± 12; mut G, 90 ± 5.2; mut C, 74 ± 14.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have shown that glucocorticoid regulation of the Xenopus laevis {gamma}-fibrinogen gene requires multiple GREs and an accessory factor binding site, all within the first 187 bp of the promoter. No additional glucocorticoid-regulatory regions were found up to 5000 bp upstream (Fig. 2Go), and no sequences with a strong match to the consensus GRE were found within 1210 bases downstream (Genbank U66896). The accessory factor site is a novel transcription factor recognition sequence and is bound by a Xenopus liver nuclear protein called XGRAF. The position of the XGRAF-binding element suggests that it may be involved in a unique glucocorticoid-regulatory mechanism since it overlaps the upstream half of the most critical GRE.

Fibrinogen Gene Regulation by Glucocorticoids
Our analyses of the Xenopus Bß- and {gamma}-fibrinogen genes are the only descriptions of specific elements required for glucocorticoid regulation of fibrinogen genes from any animal. Although glucocorticoids play a role in regulation of fibrinogen gene expression in other species (25, 26, 27), the molecular mechanisms underlying the induction have not been identified. Each of the rat and human fibrinogen subunits for which the glucocorticoid response has been examined has a general region of 150 to 1400 bases that confers glucocorticoid regulation on the gene (28, 29, 30). However, no significant matches to the consensus GRE were described for any of the genes in either species. Thus, these genes may have complex glucocorticoid-regulatory systems similar to the Xenopus {gamma}-fibrinogen gene.

Arrangement and Quality of GREs
We have identified several GREs within the Xenopus {gamma}-fibrinogen gene that are bound by GR in vitro and are required for the functional response to glucocorticoids in vivo. When examined as full GREs, the match between the sites in the {gamma}-gene and the 12 base consensus sequence ranges from five to eight bases. These GREs could also be viewed as three tandem half-sites (downstream halves of GRE1, GRE2a, and GRE3) that are similar to an arrangement of half-site direct repeats reported to have a glucocorticoid-responsive function in the MMTV gene (31). This isolated region from the MMTV gene is the first description of glucocorticoid induction mediated through half-site GREs arranged as direct repeats.

Most glucocorticoid-regulated genes have one or more sites with a high match to the canonical GRE. Limited precedent does, however, exist for genes with functional GREs that have few matches to the consensus sequence. In the human alcohol dehydrogenase gene, ADH2, three GREs were identified by DNase I footprinting within a region required for glucocorticoid responsiveness (32). The best of these GREs has seven bases that match the 12-base consensus sequence, with only one of the four guanine moieties critical for binding (33). For the rat atrial natriuretic peptide gene, two positions were identified as GR-binding sites in a glucocorticoid-responsive region (34). Of these, the one with the highest similarity to the consensus sequence is only a seven of 12 match, with two of the four critical guanines.

Accessory Factors for Glucocorticoid Regulation
Several genes have been shown to require additional DNA-binding proteins for complete regulation by glucocorticoids. The rat tryptophan oxygenase gene is regulated through two GREs, but in addition requires a CACCC box-binding element (35, 36). Regulation of the rat phosphoenolpyruvate carboxykinase gene requires two DNA sites that are bound by accessory factors in addition to two GREs. Multiple proteins, including COUP-TF, HNF-4, and HNF-3, bind to these sites and are required for the glucocorticoid response (3, 7). The rat TAT gene has two glucocorticoid response units that involve HNF-3 and an Ets family protein as accessory factors (4, 8, 37). The mouse proliferin gene contains a composite GRE that includes an AP-1 site. Jun:Jun homodimers are required for glucocorticoid induction, while Fos:Jun heterodimers inhibit the hormone response (6). Glucocorticoid stimulation of the stably transfected MMTV promoter requires a binding site for the NF-1 accessory factor (5). All of these previously reported accessory factor-binding sites are distinct from the XGRAF-binding site upstream of the X. laevis {gamma}-fibrinogen subunit gene.

Models for XGRAF Interaction with the GR
An interesting aspect of the mechanism of glucocorticoid regulation of the {gamma}-fibrinogen gene is that the XGRAF-binding site overlaps the putative upstream half of GRE1. We propose several possibilities as to how these two factors can both be required at the same binding position (Fig. 12Go). The first model suggests that the two factors can bind to the DNA simultaneously. This arrangement might be possible if the two proteins bind opposite sides of the DNA helix. Since the DNA-binding domain of GR interacts with DNA in the major groove (20), the minor groove could be accessible for XGRAF binding.



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Figure 12. Models Proposed for XGRAF and GR Activation of Transcription in Response to Glucocorticoids

The white boxes represent GREs, the shaded box is the XGRAF-binding site, the ovals are GR, and the triangle is XGRAF. On the right, GR is shown bound to either GRE2a/b or to GRE3. On the left, various arrangements of XGRAF and either monomer or dimer of GR bound to GRE1 are depicted. See text for details.

 
The second model proposes that XGRAF forms a heterodimer with GR. The spacing of the binding sites for these two proteins is appropriate for such a close interaction between GR and an accessory factor. The only protein GR has been reported to heterodimerize with is the mineralocorticoid receptor, a related protein that binds the same DNA recognition site (38). Although heterodimerization could be required for function, it is probably not required for DNA binding in vitro because nuclear extract prepared from cells deprived of glucocorticoids for several days still showed significant XGRAF binding (our unpublished data).

The third model is that XGRAF binding displaces one part of the GR homodimer. In support of the feasibility of this model, the crystal structure of the GR-DBD was initially determined with a GRE containing one perfect half and one suboptimal half (20). The GR bound to this site as a homodimer through specific binding to the perfect half of the GRE and nonspecific binding to the other half of the site. XGRAF could stabilize the binding of GR homodimer in the absence of a GRE that has a high match to the consensus.

The fourth model requires sequential binding of GR and XGRAF. Two examples of this mechanism have been described. For the TAT gene the binding site for the accessory factor HNF-3 is in the same position as one of the GREs, and HNF-3 binding to this site is glucocorticoid-dependent (4, 8). In MMTV, glucocorticoid activation of transcription requires the accessory factor NF-1, for which binding to DNA is dependent on GR disruption of chromatin structure (5, 39).

The potential overlap between the XGRAF site and a poor GRE in the Xenopus {gamma}-fibrinogen gene may be part of a novel mechanism for glucocorticoid action. Experiments are in progress to characterize further the XGRAF protein and distinguish between the proposed models of its action with GR. XGRAF or similar proteins may be involved in glucocorticoid regulation of other genes, not only in Xenopus but also in other species.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Isolation of Genomic DNA and Determination of Nucleotide Sequence
Genomic DNA for the {gamma} fibrinogen subunit gene was cloned from a X. laevis HD1 genomic library in {lambda}gem11 provided by Dr. Donald Brown of the Carnegie Institute. The 5'-portion of the {gamma}-subunit cDNA clone Xl{gamma}3 (40) was used to screen the library essentially as described (14) except that the bacterial strain was ER1647 (New England Biolabs, Beverly, MA). The genomic DNA was digested with restriction enzymes and subcloned into pBluescript SK- (Stratagene, La Jolla, CA). The transcription initiation site was identified by primer extension (41).

The DNA sequence from -1537 to +1210 of both strands was determined on an Applied Biosystems Inc. (ABI, Foster City, CA) 373A automated sequencer using dye-termination methods as recommended by ABI. Sequencing was done at the Molecular Biology Program DNA Core Facility at the University of Missouri. Sequence information was analyzed for homology to known transcription factor-binding sites with the latest available versions of three different databases. The SITES table of TRANSFAC 2.5 (December 1995) (42) and the TFD database (release 7.5 SITES, March 1996) (43) were examined with the Findpatterns program of the Wisconsin Package, Version 8.0 (Genetics Computer Group, Madison, WI). The MATRIX table of TRANSFAC 3.0 (August 1996) (42) was searched with the program MatInspector 2.0 (44).

Reporter Gene Construction
Deletion Mutagenesis
The {gamma}-fibrinogen gene deletion constructs -5000, -2900, -1158, -603, -369, and -232 were made by restriction enzyme digestion of genomic DNA subclones followed by ligation into the luciferase reporter vector, pLuc-Link 2.0 (45). Often ligation of a short adapter sequence or preliminary subcloning into pBluescript (pBS) was required. The -105 deletion was created using Exonuclease III digestion, and deletion constructs -187, -163, and -129 were prepared by PCR. Primer oligonucleotides for PCR were obtained either from the Molecular Biology Program DNA Core Facility at the University of Missouri or from Genemed Biotechnologies (San Francisco, CA). Standard conditions of 10 µM primers, 4 ng template, and 2 mM MgSO4 in an (NH4)2SO4 buffer (46) were used for all PCR with Taq enzyme.

Site-Specific Mutagenesis
The site-specific mutation construct made in pLL{gamma}-187 (mutation I) was prepared by PCR using Pfu polymerase (Stratagene, La Jolla, CA) with the protocol from the manufacturer. DNA constructs with mutations B, J, and L in pLL{gamma}-232 were prepared by the megaprimer PCR method (47). DNA constructs with mutations B and J were made using Taq polymerase whereas that with mutation L was made with Pfu polymerase (each with the conditions specified above).

Constructs with mutations A, C, D, E, F, and G in pLL{gamma}-232 were produced by two-step PCR using Pfu polymerase with two flanking primers and a pair of primers complementary to each other. One of the complementary primers was used to make the mutation and upstream DNA, while in a separate reaction the other complementary primer was used to make the mutation and downstream DNA. The two products were used in the second PCR as overlapping templates that primed on each other.

For every construct the 3'-end of the {gamma}-sequence is at the +41 position. Constructs were sequenced to ensure that both the 3'- and 5'-junctions were correct and that PCR products contained only the desired mutations. Standard transformation protocols were used to amplify the constructs in DH5{alpha} Escherichia coli (Bethesda Research Laboratories, Rockville, MD) (48). Plasmid DNA was isolated by alkaline lysis (49) followed by purification over an anion exchange resin column (Tip 2500, QIAGEN, Chatsworth, CA). In addition, all DNA used for transfection into liver cells underwent a single cesium chloride gradient purification (48) to achieve optimal results in the transfection assays.

Liver Cell Culture and Transfection and Assays for Reporter Gene Activity
Adult female X. laevis (90–170 g) were treated with estradiol (Sigma Chemical Co., St. Louis, MO) (14) to obtain proliferating parenchymal cells (50). For each cell preparation, primary cells were purified from the livers of two or three frogs by an in situ perfusion method (14). Experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Transfection of freshly isolated hepatocytes was performed as previously described with the modification that 50 µg luciferase reporter DNA and 25 µg control plasmid pCMVßgal (51) were mixed with cells for electroporation (14). Cells were incubated as before with or without our standard hormone treatment consisting of 10-7 M dexamethasone and 10-9 M T3 (14). T3 is necessary for optimal fibrinogen gene induction by glucocorticoids, but has no effect by itself (12). After 48 h, cell extract was prepared using nonionic detergent lysis (14).

Luciferase activity in the cell extracts was assayed as described (14) except the assay buffer included Coenzyme A at a final concentration of 700 µM (52). For the assay, 150 µl of cell extract were added to 540 µl of assay buffer and mixed briefly. Then 100 µl of 1 mM luciferin were automatically injected by the luminometer (Analytical Luminescence Laboratory, Westlake Village, CA; model 2010) and light production measured for 20 sec.

The activity of the ß-galactosidase produced from the control plasmid was measured with the MUG assay (14) for some of the data in Fig. 2Go. This assay was replaced by the Galactolight method (Tropix, Bedford, MA) for the remainder of the data. Before either assay the endogenous hepatocyte galactosidase was inactivated by heating the extracts at 48 C for 1 h (Tropix and Ref.53). For the Galactolight assay, 6 µl cell extract diluted to 50% strength with lysis buffer were incubated with 67 µl diluted Galacton substrate for 1 h, 100 µl accelerator were injected, and light output was measured for 5 sec after a 3-sec delay.

Nuclear Extract Preparation
X. laevis primary hepatocytes were isolated as described (14) and incubated in the presence of 10-7 M dexamethasone and 10-9 M T3 (Sigma) for 4–12 days at 2.5–4 x 105 cells/cm2 on Primaria substrate (Falcon, Franklin Lakes, NJ), with one half of the medium replaced with fresh medium every 2 days. Nuclear extract was prepared by modification (54, 55) of Dignam et al. (56). The cells (2–3 x 107) from each flask were scraped from the substrate and washed three times with ice-cold Barth (88 mM NaCl, 1 mM K2SO4, 10 mM HEPES, pH 7.4), with pelleting each time in a microcentrifuge at 13,800 x g. All subsequent procedures were performed on ice in a 4 C room. The cells were resuspended in buffer A+ (10 mM HEPES-KOH, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonylfluoride (PMSF), 1 µg/ml pepstatin, 5 µg/ml leupeptin, 3 µg/ml aprotinin, 0.05 mM benzamidine), pelleted at 13,800 x g, and resuspended in 100 µl buffer A+ per 2.5 x 106 cells. NP40 was added to 0.3% (vol/vol), and the cells were incubated for 10 min and centrifuged at 735 x g in a microcentrifuge for 10 min to pellet the nuclei. The nuclei were resuspended in buffer A+, pelleted at 13,800 x g as before, resuspended in buffer C+ (10 mM HEPES-KOH, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 20% glycerol, 1 mM DTT, 1 mM PMSF plus protease inhibitors as in A+ above) at 15 µl per 5 x 106 cells, and incubated for 20 min. After centrifugation at 13,800 x g for 10 min, the supernatant was collected and buffer D (20 mM HEPES-KOH pH 7.9, 50 mM KCl, 0.2 mM EDTA, 0.2 mM EGTA, 20% glycerol, 1 mM DTT, 1 mM PMSF) was added (3.75 volumes buffer D per 1 volume supernatant). The nuclear extract was stored over liquid nitrogen.

Gel Shift Assays
Gel shift assays with purified rat GR DNA-binding domain (GR-DBD) (20) were performed at room temperature as previously described (14). Gel shift assays with hepatocyte nuclear extract followed a protocol (57) modified to match the buffer and salt conditions of the nuclear extract described above. For Figs. 4Go and 5Go the reactions contained in 14 µl final volume: nuclear extract (3 µg total protein in 5 µl), 2 µg poly(dI.dC), 3 µl of 5x binding buffer (100 mM HEPES-KOH, pH 7.9, 250 mM KCl, 5 mM EDTA, pH 8, 50% glycerol, 0.5% NP40, 5 mM DTT), and either no specific competitor or 20- or 100-fold molar excess of specific competitor DNA (adding 15 mM NaCl). The reaction conditions in Fig. 10BGo were the same except with 2 µl 5x binding buffer in a final volume of 9 µl. Reactions were incubated 15 min on ice. Radioactively labeled probe in 1 µl was added, and the incubation was continued for 30 min on ice. One-tenth volume of loading buffer (0.2% bromophenol blue and xylene cyanol in 40% glycerol, 250 mM Tris-Cl, pH 7.5) was added, and the free DNA was separated from the protein-bound DNA on a 5% polyacrylamide (75:1 acrylamide-bisacrylamide), 0.1% NP40 gel using 0.25x TBE running buffer (22.25 mM Tris, 22.25 mM borate, 0.625 mM EDTA). The gel was run for 1.5–2 h and dried at 80 C for 2 h, and the DNA was detected by exposure to XAR film (Kodak) at -80 C with one Lightning Plus intensifying screen (DuPont, Wilmington, DE).

The -200 to -115 probe used for nuclear extract gel shifts was produced by PCR with Taq enzyme. One of the primers used in the reaction was 5'-end-labeled (49). The radioactively labeled PCR product was purified on a native 6% polyacrylamide gel (48). Oligonucleotides used for double-stranded probes or competitors were purchased from the same sources as described above for PCR primers, purified through a denaturing 16% polyacrylamide gel (48), ethanol precipitated, and annealed (14). Those used as probes in Figs. 5BGo and 10CGo were end-labeled as before (49).

Methylation Protection Footprinting
Methylation protection probes extending from -232 to -6 or from -232 to -42 were produced by PCR with Taq enzyme. For selectively labeling either the top or bottom strand, the sense or antisense PCR primer was 5' end-labeled (49). The PCR product was purified through a native 6% polyacrylamide gel as above. The methylation protection assays were performed according to a modified procedure (58, 59) by incubating the labeled probe with 250 or 500 ng GR-DBD in a 20-µl volume for 20 min under the conditions described above for gel shifts with GR-DBD. The reactions were treated with 1 µl 10% dimethyl sulfate for 3 or 4 min at 22 C. Reactions were stopped by addition of 180 µl of 10 mM Tris-Cl, pH 8, 0.1 mM EDTA, 50 µl of 1.5 M sodium acetate, pH 7, 1 M ß-mercaptoethanol, and 3 µl of 200 µg/ml yeast RNA. After ethanol precipitation, the DNA was dissolved in 100 µl of 1 M piperidine and incubated at 90 C for 30 min, followed by lyophilization to remove the piperidine. The DNA was twice dissolved in 100 µl water and lyophilized before the products were analyzed on a denaturing 6% polyacrylamide gel (49). The G + A sequencing ladders were prepared by chemical sequencing methods (60).


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Figure 10A. 9, DNA only (9.2 fmol, 3.2–5 x 104 cpm per reaction). Lanes 2, 4, 6, 8, and 10, DNA incubated with GR-DBD. DNA probes: lanes 1 and 2, {gamma} (-189 to -157), the 33-bp double-stranded oligonucleotide of the {gamma}-sequence that includes the glucocorticoid-responsive region; lanes 3 and 4, {gamma} (-189 to -157) with mutation E; lanes 5 and 6, {gamma} (-189 to -157) with mutation F; lanes 7 and 8, {gamma} (-189 to -157) with mutation G; lanes 9 and 10, {gamma} (-189 to -157) with mutation C. F, Free DNA; I, monomer of GR-DBD bound to DNA; II, dimer of GR-DBD bound to DNA.

 

    ACKNOWLEDGMENTS
 
We thank Drs. Donald Brown for the Xenopus genomic library, Richard Maurer for pLuc-Link 2.0 vector, and Keith Yamamoto for the recombinant DNA-binding domain of the rat GR. We are grateful to Suzanne Simmons for the genomic sequencing experiments, Cindy Zhu for making one of the constructs, and Brian Morin and Dr. Mark Hannink for helpful suggestions on the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Lené J. Holland, Department of Physiology, MA415 Medical Sciences Building, University of Missouri School of Medicine, Columbia, Missouri 65212

This work was supported by the following grants from the National Heart Lung and Blood Institute: RO1-HL-39095 (to L.J.H.), postdoctoral and predoctoral training grant HL-07094 (to M.L. and R.N.W.), and Research Career Development Award HL-02934 (to L.J.H.).

1 Present address: Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, San Francisco, California 94141. Back

2 The genomic DNA sequence of the {gamma}-fibrinogen subunit gene from Xenopus laevis has been entered in Genbank, accession number U66896. Back

Received for publication December 11, 1996. Revision received February 11, 1997. Accepted for publication February 14, 1997.


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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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