Identification and Characterization of Single Strand DNA-Binding Protein That Represses Growth Hormone Receptor Gene Expression

Ram K. Menon, Hui Cheng and Manbir Singh

Department of Pediatrics University of Pittsburgh School of Medicine Children’s Hospital of Pittsburgh Pittsburgh, Pennsylvania 15213


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The GH receptor is essential for the actions of GH on growth and metabolism. Electromobility shift assay established that a 42-bp enhancer element in the promoter of the L1 transcript of the murine GH receptor bound nuclear proteins specific for the coding strand or the DNA duplex. Using methylation interference footprinting and electromobility shift assay with mutant oligonucleotides, the DNA-binding sites for the single-strand DNA-binding protein (SSBP) and the double-strand DNA-binding protein (DSBP) were mapped and shown to be contiguous with partial overlap. Shift-Western analysis indicated that the SSBP was a component of the DSBP complex. A functional interaction between SSBP and DSBP was suggested by the effect of the exclusion of SSBP on equilibrium binding and dissociation rate ("off rate") of the DSBP-DNA complex. Experiments using the anionic detergent deoxycholate provided evidence for a direct protein-protein interaction between SSBP and DSBP. Using lectin-affinity chromatography, discordance between the pattern of O-glycosylation of SSBP and DSBP was demonstrated. Transient transfection experiments support the role of SSBP as a repressor of DSBP’s activation of transcription of the GH receptor gene. Southwestern analysis indicated that a protein of molecular mass 23-kDa exhibited binding activity specific to the coding strand of the enhancer element. We conclude that single- and double-strand DNA-binding proteins conjointly regulate the expression of the murine GH receptor gene.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Pituitary GH is essential for postnatal growth in animals. In addition to growth, GH affects the metabolism of fat, protein, and carbohydrate. GH exerts these actions both by its direct effect on target organs and by stimulating the production of insulin-like growth factor I (IGF-I). At the tissue level, these pleiotropic actions of GH result from the interaction of GH with a specific cell surface receptor, i.e. GH receptor. The association of GH with the GH receptor triggers the postreceptor signaling systems, which culminate in the biological actions of GH. GH receptors are present in all the tissues toward which GH actions are directed. Thus the ability of GH to exert biological effects is intimately linked to the number and function of GH receptors in these tissues.

The GH receptor belongs to a gene family that includes the receptors for PRL, a number of cytokines such as granulocyte colony-stimulating factor, erythropoietin, granulocyte macrophage colony-stimulating factor, and a wide variety of interleukins (1). The identification and partial characterization of the promoter-regulatory regions of the murine (2) and ovine (3) GH receptor gene were recently reported. The organization of the 5'-region of the GH receptor gene is complex. In the mouse, two 5'-untranslated regions (UTR), termed L1 and L2, have been identified (2, 4). Southard et al. (4) determined that GH receptor transcripts containing the L1 UTR are expressed preferentially in liver compared with the placenta. In contrast, L2 UTR- containing transcripts are preferentially expressed in the placenta. A similar scheme of multiple 5'-UTRs for the GH receptor gene exists in the human (5).

The expression of the GHR is tissue- and development-specific. Thus expression is highest in the liver, with lesser amounts being expressed in heart and kidney. In the liver, expression of GH receptor transcripts is very low in the fetus and increases postnatally to maximal during pregnancy (1, 4). Nutritional status (1), thyroidal status (6), and diabetes mellitus (7) are some of the states in which expression of GH receptor is altered. Whereas detailed information about the molecular mechanisms involved in regulating the expression of the GH gene is available (8), much less is known about the factors modulating the expression of the GH receptor (1). Previous reports from this laboratory have described the identification and partial characterization of two enhancer elements for the L1 transcript of the murine GH receptor gene. Our analysis had indicated that the cognate trans-acting factor for one these elements belongs to the CTF/NF-1 family of transcription factors (9). In another report we presented evidence that the second enhancer element, termed FP1, defined a novel protein-DNA binding motif and is involved in the developmental expression of the murine GH receptor gene (2).

Based on experiments detailed in the current report, we have expanded by 12 nucleotides the enhancer element FP1 described in the previous report (2). We now present results to support the conclusion that the expanded 42-bp enhancer element, which we term FP42, binds both a single strand (SSBP) and a double-strand DNA-binding protein (DSBP). We demonstrate that the binding sites for the SSBPs and DSBPs are distinct, although they are contiguous with partial overlap. We provide proof that there is physical and functional interaction between SSBP and DSBP and that these two proteins conjointly regulate the expression of the murine GH receptor gene. Our studies indicate that the SSBP serves as a repressor of DSBP’s role in the activation of the transcription of the GH receptor gene.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
SSBP Specifically Recognizes a Site on the Coding Strand of the GH Receptor Promoter
We had previously identified and partially characterized an enhancer element located about 3.4 kb upstream of the major transcription start site for the L1 transcript of the mouse GH receptor gene (2). A double-strand oligonucleotide probe (FP42-DS; Table 1Go) containing the enhancer element can form a single sequence-specific protein-DNA complex with liver nuclear extracts (2). Whereas the noncoding strand (FP42-LS) failed to form a DNA-protein complex (data not shown), the coding (FP42-US) of this oligonucleotide formed a protein-DNA complex (Fig. 1Go). To determine whether this SSBP-DNA complex was sequence specific, we performed competition experiments in which the binding reaction was carried out in the presence of increasing concentrations of unlabeled FP42-US, FP42-DS, and a single-stranded oligonucleotide with random sequence. Whereas 100-fold molar excess of FP42-US eliminated the formation of the SSBP-DNA complex, neither FP42-DS nor the random sequence oligonucleotide at even a 200-fold molar excess altered this binding (Figs. 1Go and 2BGo). These results demonstrate that the SSBP-DNA complex is sequence specific. This SSBP-DNA complex has an electrophoretic mobility distinct from that observed for the double-strand DNA-binding protein (DSBP) complex formed by the double-strand oligonucleotide FP42-DS (Fig. 3Go). SSBP was expressed in a variety of murine tissues (Fig. 4AGo); the paucity of SSBP-DNA complex formed with fetal liver extracts suggests that the expression of this protein is developmentally regulated (Fig. 4AGo). Whereas crude nuclear extracts from HeLa, COS-7, and HepG2 cells formed DNA-protein complexes with FP42-US, the mobility of these complexes was distinct from those formed with murine tissues (Fig. 4Go). The binding of HepG2 nuclear proteins to FP42-US was sequence-dependent and exhibited nucleotide specificity for DNA binding similar to that observed with mouse liver nuclear proteins (Fig. 4BGo). Proof for the presence of a true primate homolog of the rodent FP42-US SSBP will have to await the identification of the protein(s) that represent SSBP activity on electrophoretic mobility shift assay (EMSA).


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Table 1. Sequence of Oligonucleotides Used in These Experiments

 


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Figure 1. Nuclear Proteins from Mouse Liver Bind to FP42-US

32P-labeled FP42-US was incubated with nuclear extracts prepared from liver of adult female mice, electrophoresed, and subjected to autoradiography as described in Materials and Methods. Competition between labeled and unlabeled specific (FP42-US, lanes 2–4) or double-strand oligonucleotide (FP42-DS, lanes 5–7) at molar ratios of 25 (lane 2), 50 (lanes 3 and 5), 100 (lanes 4 and 6), and 200 (lane 7) is shown. The band representing the specific single-strand DNA-protein complex is indicated as SSBP.

 


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Figure 2. Effects of Mutations in the SSBP-Binding Site on Protein-DNA Binding

32P-labeled FP42-US was incubated with nuclear extracts prepared from liver of adult female mice, electrophoresed, and subjected to autoradiography as described in Materials and Methods. Panel A, Competition between labeled and unlabeled wild type sequence (FP42-US, lanes 2–4) or mutant oligonucleotide (M2FP42-US, lanes 5–7; M4FP42-US, lanes 8–10) at molar ratios of 25 (lanes 2 and 5), 50 (lanes 3, 6, and 8), 100 (lanes 4, 7, and 9) and 200 (lane 10) is shown. The band representing the specific single-strand DNA-protein complex is indicated as SSBP. Panel B, Competition between labeled and unlabeled wild type sequence (FP42-US, lanes 2–4), mutant oligonucleotide (M3FP42-US, lanes 5–7), or oligonucleotide with random sequence (lanes 8–10) at molar ratios of 25 (lanes 2 and 5), 50 (lanes 3, 6, and 8), 100 (lanes 4, 7, and 9) and 200 (lane 10) is shown. The band representing the specific single-strand DNA-protein complex is indicated as SSBP. Panel C, Gels represented in panels A and B and for mutant oligonucelotide M1FP42-US (data not shown) were subjected to analysis using a PhosphorImager. The data from three independent EMSA analyses are plotted as the percent change (mean ± SEM) in the intensities of the protein-DNA complexes formed in the presence of molar excess (open bar = 25-fold; solid bar = 50-fold; hatched bar = 100-fold) of unlabeled competitor compared with that formed in the absence of unlabeled competitor.\.

 


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Figure 3. Effect of Mutations in the SSBP-Binding Site on DSBP Binding

With (lanes 2, 4, 6, 8, and 10) or without (lanes 1, 3, 5, 7, and 9) the addition of the respective unlabeled complimentary oligonucleotide, the indicated 32P-labeled oligonucleotides were incubated with nuclear extracts prepared from liver of adult female mice. These oligonucleotides were either with wild type sequence (FP42, lanes 1–2) or with specific mutations (M1FP42, lanes 3 and 4; M2FP42, lanes 5 and 6; M4FP42, lanes 7 and 8; M3FP42, lanes 9 and 10). The sequences of the oligonucleotides are defined in Table 1Go. The reactions were electrophoresed and subjected to autoradiography as described in Materials and Methods. The bands representing the specific single (SSBP) and double-strand (DSBP) binding proteins are indicated.

 


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Figure 4. Tissue and Species Distribution of SSBP

Panel A, 32P-labeled FP42-US was incubated with nuclear extracts prepared from the indicated tissues, electrophoresed, and subjected to autoradiography as described in Materials and Methods. Panel B, 32P-labeled FP42-US (lane 1) was incubated with nuclear extracts prepared from HepG2 cells (lanes 2–7) or liver of adult female mice (lane 8), electrophoresed, and subjected to autoradiography as described in Materials and Methods. Competition between labeled and unlabeled oligonucleotides (FP42-US, lane 3; FP42-DS, lane 4; M1FP42-US, lane 5; M2FP42-US, lane 6; M3FP42-US, lane 7) at molar ratios of 25 is shown. The band representing the specific single-strand DNA-protein complex is indicated as SSBP.

 
Partial Overlap of Contiguous DNA-Binding Sites for SSBP and DSBP
To define the binding sites of the SSBP and the DSBP, the contact points with purines for both SSBP and DSBP were determined by methylation interference footprinting. Chemical modification of the purines present in FP42-US revealed that methylation of four G residues (designated GI, GII, GIII, and GIV) interfered with the binding of SSBP; these four G residues were located at the 5'- (GI), middle (GII), and 3'- (GIII, GIV) regions of FP42-US (Fig. 5AGo). In contrast, methylation of the purines of FP42-DS revealed that the contact points (GIII, GIV, and GV) of the DSBP on the coding strand were restricted to the 3'-end and overlapped with the contact points GIII and GIV of the SSBP (Fig. 5BGo). On the noncoding strand of FP42-DS the contact points (designated GVI, GVII, and GVIII) flanked the contact points of the coding strand (Fig. 5CGo). These results reveal that whereas the DNA-binding sites of SSBP and DSBP are distinct, there is overlap of these contiguous binding sites.



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Figure 5. Methylation Interference Footprint of Single- and Double-Strand DNA-Protein Complex

Panel A, Oligonucleotide FP42-US was end-labeled with [{gamma}32P]ATP and T4 polynucleotide kinase. This probe was used in the methylation interference assay in conjunction with adult mouse liver nuclear proteins. Either unbound (Free) DNA, or DNA recovered from the DNA-protein complex (Bound) was isolated and the DNA cleaved at the methylated guanine residues by piperidine. Asterisks indicate the reduction in the intensity of bands (numbered I-IV) that is facilitated by binding of protein(s) to a specific sequence of DNA; ***, for >80% interference; **, for 50–80% interference; and * for 20–50% interference. Maxim-Gilbert sequencing reactions (C, C/T, and G) of FP42-US (the sequence of which is indicated) were electrophoresed concurrently to identify the location of the footprints. Panel B, A PCR-generated fragment containing the FP42 enhancer site was labeled on the sense strand of the DNA duplex and methylation interference assay was carried out as described above in panel A. Asterisks indicate the reduction in the intensity of bands (underexposed gel depicted in inset) numbered (III-V) that is facilitated by binding of protein(s) to a specific sequence of DNA; ***, >80% interference; **, 50–80% interference. The sequence of the FP42 enhancer site is indicated. Panel C, Sequence of both strands of the enhancer element FP42. *, Bases whose methylation reduces the proportion of FP42-US DNA in the bound fraction; the bases whose methylation decreases the proportion of FP42-DS in the bound fraction are underlined. \.

 
The functional significance of the contact points identified by the methylation interference assays was investigated by electromobility shift assay (EMSA). Compared with the wild type sequence, oligonucleotides (Table 1Go) with mutation at the GI (M2FP42-US), GI and GII (M3FP42-US), or GIII and GIV (M1FP42-US) had decreased affinity for SSBP. This was demonstrated by the decreased ability of the mutant oligonucleotides to compete with the wild type sequence for binding to SSBP (Fig. 2Go). In contrast, an oligonucleotide (M4FP42-US; Table 1Go) with mutations of all the four Gs identified to make contact with the SSBP completely failed, even at a 200-fold molar ratio, to compete with the wild type oligonucleotide (Fig. 2Go, A and C). These results confirm that the Gs identified as contact points by the methylation interference assay are essential for binding of the SSBP to FP42-US, and that the entire DNA-binding site is needed for binding of SSBP to DNA with full affinity.

Physical and Functional Interaction between SSBP and DSBP
The interrelationship between the binding sites for the SSBP and the DSBP was investigated by using the wild type and mutant oligonucleotides in EMSA. Addition of unlabeled noncoding strand (FP42-LS) to a binding reaction containing labeled coding strand (FP42-US) resulted in the abolishment of binding of SSBP and the appearance of binding of DSBP (Fig. 3Go, lane 2). This result is consistent with the above discussed findings indicating specificity of the SSBP for the coding strand and suggest that formation of the DNA duplex excludes the binding of the SSBP. In a similar series of EMSA using M1FP42-US, M2FP42-US, or M4FP42-US as the labeled probes, the addition of the respective unlabeled complimentary oligonucleotide resulted in differential effects on the formation of the DSBP-DNA complex. Whereas the oligonucleotide mixtures containing 32P-labeled M1FP42-US or M4FP42-US oligonucleotide could not bind DSBP, addition of the unlabeled oligonucleotide complimentary to labeled M2FP42-US resulted in the formation of the DSBP (Fig. 3Go). These results indicate that the G residues (GIII and GIV) at the 3'-end of the enhancer element are essential for binding of the DSBP and that the binding of SSBP to the 5'-end of the element alone will not result in the binding of the DSBP.

Analysis of the steady state binding of the DSBP to the DNA duplex indicated that the binding of the DSBP to oligonucleotides with mutant binding site that abrogated the binding of the SSBP (M2FP42-DS and M3FP42-DS) was increased 4- to 5-fold compared with the DNA duplex with the wild type sequence (Fig. 6Go). To quantitatively analyze the effect of the discrete mutations in the SSBP DNA-binding site on the DSBP DNA binding, we next estimated the dissociation rates of the mutated binding sites relative to the wild type FP42 site. We approached this analysis by measuring the dissociation rates in terms of the half-life, determined by estimating the amount of bound DNA measured at time points subsequent to the addition of a vast excess of specific competitor DNA to a binding reaction that has reached equilibrium (10). Figure 7AGo represents an example of such an experiment performed with FP42-DS and M2FP42-DS. The data are plotted on the accompanying graph (Fig. 7BGo) as the log of the percent change in binding from that at time zero vs. minutes after the addition of the competitor DNA. The results of these analyses reveal that the half-life (t1/2) of the protein-DNA complex formed by the DNA (M2FP42-DS) with the mutant binding site that abrogated the binding of the SSBP was significantly decreased from that of the protein-DNA complex formed with the wild type sequence FP42-DS (Fig. 7BGo and Table 2Go). Interestingly, the decrease in the dissociation rate of the DSBP-DNA complex was roughly proportional to the extent of the alteration of the SSBP DNA-binding site. Hence, the half-life of the DSBP-DNA complex formed with M3FP42-DS was even shorter than that formed with M2FP42-DS (Fig. 7BGo and Table 2Go).



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Figure 6. Effects of Mutations in the DNA-Binding Site or DOC on the Relative Affinities of the DSBP-DNA Complex

To quantitate the effect of mutations in the DNA-binding site by either alterations in the nucleotide sequence or presence of anionic detergent (DOC), EMSA was carried out with equal amounts of liver nuclear extracts and equimolar amounts of the indicated 32P-labeled double-strand DNA probes in the absence of unlabeled specific competitor. After electrophoresis, the dried gels were subjected to analysis using a PhosphorImager, and the amount of bound DNA-protein complex was calculated as a percent of the total amount of probe added per reaction. The data represent mean ± SEM of three independent experiments. *, P < 0.05 as compared with FP42-DS by one-way ANOVA with Duncan’s Correction for Multiple Comparisons.

 


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Figure 7. Determination of Dissociation Rates (Half-Life) of the Cognate DNA-Binding Protein for FP42-DS, M2FP42-DS, and M3FP42-DS

Panel A, 32P-labeled FP42-DS (lanes 1–5) or M2FP42-DS (lanes 6–10) was incubated with nuclear extracts prepared from liver of adult female mice. After the reactions had reached equilibrium (20 min), a 400-fold excess of homologous unlabeled double-stranded oligonucleotide was added, and aliquots of the mixture were loaded onto a running gel at the indicated time points. After electrophoresis, the dried gels were subjected to analysis using a PhosphorImager. Panel B, Data (mean ± SEM; n = 3) from the PhosphorImager analysis of gels similar to that shown in panel A are plotted as the log of the percent bound probe relative to probe bound at the time of addition of the unlabeled competitor (time 0), as a function of time. The half-life of the complexes were derived from the slope of the curves (10 ); FP42-DS(t1/2) = 25 min, M2FP42-DS(t1/2) = 14 min, and M3FP42-DS(t1/2) = 10 min.

 

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Table 2. Dissociation Rate of DSBP-DNA Complex: Effects of Mutations in the DNA-Binding Site and Deoxycholate

 
The experiments analyzing the effect of the mutated DNA-binding site on the dissociation rate of the DSBP-DNA complex suggested an interaction between the SSBP and DSBP. To determine whether this interaction involved direct physical interaction between SSBP and DSBP, we exploited the property of the mild anionic detergent deoxycholate (DOC) to inhibit protein-protein interactions without ordinarily altering protein-DNA interaction (11, 12, 13). We reasoned that if the increase in steady state binding and the shortening of the half-life of the DSBP-DNA complex observed with M2FP42-DS and M3FP42-DS resulted from the lack of interaction between SSBP and DSBP, then perturbation of the putative protein-protein interaction between SSBP and DSBP by DOC should also result in similar alterations in the binding characteristics of the DSBP complex formed by the wild type DNA-binding site, i.e. FP42-DS. Therefore, 32P-labeled FP42-DS was incubated with liver nuclear extracts in the presence or absence of varying (0.09% and 0.15%) DOC and the steady state binding and dissociation rate in terms of half-life of the DNA-protein complex estimated as described above. Whereas DOC did not significantly alter the DNA binding of SSBP (data not shown), there was a dose-dependent increase in the steady-state binding of the DSBP to the wild type FP42 (Fig. 6Go). Furthermore, Fig. 8Go illustrates that in the presence of DOC there was a decrease in the half-life of the protein-DNA complex formed by FP42-DS. This decrease in the half-life of the DSBP-DNA complex was roughly proportional to the concentration of DOC (Table 2Go). To exclude the possibility that this effect of DOC was due to the dissociation of an oligomeric DSBP-DNA complex that did not involve SSBP, we studied the effect of DOC on the half-life of the DSBP-DNA complex formed with M3FP42-DS (Table 2Go). Since M3FP42-DS contains a mutant binding site that abrogates binding of SSBP but not of DSBP, the lack of effect of DOC on the half-life of the DSBP-complex formed by M3FP42-DS indicates that the effect of DOC on FP42-DS binding was due to perturbation of protein-protein interaction(s) mediated by the SSBP-binding site. We conclude from these experiments that there is a functional association between the SSBP and the DSBP and that SSBP serves to stabilize the DSBP complex as evidenced by the decrease in the half-life of the DSBP complex when the SSBP is excluded from this complex.



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Figure 8. Effect of DOC on Dissociation Rate of the Cognate DNA-Binding Protein for FP42-DS

In the presence of the indicated concentration of DOC, 32P-labeled FP42-DS was incubated with nuclear extracts prepared from liver of adult female mice. After the reactions had reached equilibrium (20 min), a 400-fold excess of unlabeled FP42-DS was added, and aliquots of the mixture were loaded onto a running gel at the indicated time points. After electrophoresis, the dried gels were subjected to analysis using a PhosphorImager, and the data (mean ± SEM; n = 3) plotted as the log of the percent bound probe relative to probe bound at the time of addition of the unlabeled competitor (time 0), as a function of time. The half-life of the complexes was derived from the slope of the curves (10 ); t1/2 (min) = 25 (0% DOC), 21 (0.09% DOC) and 14 (0.15% DOC).

 
A limitation of the experiments described above is that they relied on an in vitro assay, i.e. EMSA, to analyze the putative interaction between the SSBP and DSBP. We next studied the in vivo functional significance of the SSBP-DSBP interaction by testing the ability of the enhancer element with the respective mutations to exhibit activity in the context of an heterologous thymidine kinase promoter contained in the luciferase vector pTK81. In transient transfection experiments using COS-7 cells, consistent with our previous findings, the wild type enhancer sequence pTKFP42 exhibited 2- to 3-fold activity over the activity of the vector alone (Fig. 9Go and Ref. 2). Whereas the plasmid pTK-M4FP42, containing the enhancer sequence with mutations that abolished both SSBP and DSBP DNA-protein binding in EMSA, did not exhibit significant activity compared with the vector alone, the plasmid pTK-M2FP42 containing mutations that selectively abrogated the binding of the SSBP, but retained DSBP binding, exhibited a 6- to 7-fold increased luciferase activity compared with the vector alone and 2- to 3-fold increased activity compared with pTK-FP42. These results support the conclusions that the SSBP-DSBP interaction is not essential for the transcriptional activity of DSBP and that SSBP acts as a repressor of activity of the DSBP in vivo. Since it was not possible to selectively abolish SSBP while retaining DSBP binding, we cannot discern whether SSBP has transcriptional regulatory activity independent of DSBP.



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Figure 9. Transient Expression Analysis of Enhancer Element FP42 in an Heterologous Promoter

Expression plasmids were generated by inserting the wild sequence GH receptor enhancer FP42 or oligonucleotide with mutations in the binding sites of the SSBP and DSBP (open bars) into the plasmid pTK81 containing the luciferase gene (shaded bars) driven by the thymidine kinase (TK) promoter (solid bars). Equimolar amounts of the vector pTK81 and luciferase fusion plasmids were transfected into COS-7 cells and luciferase activity measured as described in Materials and Methods. Luciferase specific activity in cell homogenates is expressed as relative light units equalized for transfection efficiency monitored by cotransfection of a plasmid expressing ß-galactosidase. Results represent the mean ± SEM of four independent transfections performed in quadruplicate. Solid triangles indicate the location of the mutated nucleotides; the sequence of the oligonucleotides are defined in Table 1Go. Using Student t test, * = P < 0.0001 compared with pTK81; ** = P < 0.0001 compared with FP42.

 
SSBP is a Component of the DSBP-DNA Complex
The results of the EMSA and methylation interference assays suggested an interrelationship between the SSBP and the DSBP. To investigate the possibility that the SSBP is a component of the DSBP-DNA complex, we used the technique of Shift-Western analysis. These experiments established that DSBP complex, transferred to nitrocellulose, exhibited binding to FP42-US (Fig. 10Go, panel 1) and FP42-DS (Fig. 10Go, panel 5) but not to FP42-LS (Fig. 10Go, panel 4). In competition experiments a 50-fold molar excess of unlabeled FP42-US (Fig. 10Go, panel 2) or FP42-DS (Fig. 10Go, panel 6) significantly decreased the binding of the respective labeled probe to the DSBP on the nitrocellulose membrane. In contrast, a similar 50-fold molar excess of an oligonucleotide (M4FP42-US; Table 1Go), with mutations of all the four Gs identified to make contact with the SSBP, did not significantly effect binding of labeled FP42-US (Fig. 10Go, panel 3). These results demonstrate that the binding of FP42-US and FP42-DS to the DSBP protein complex on the nitrocellulose membrane is sequence specific and provide evidence that the SSBP is a component of the DSBP complex.



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Figure 10. Detection of SSBP in DSBP-DNA Complex by Shift-Western Blot Analysis

Unlabeled FP42-DS was incubated with liver nuclear extracts, and the resultant protein-DNA complexes were separated by electrophoresis through a 6% nondenaturing gel. After localization of the protein-DNA complex by alignment with a concurrently electrophoresis reaction with 32P-labeled FP42-DS, the proteins were electro-transferred onto nitrocellulose membrane. The nitrocellulose membrane was then probed with 32P-labeled FP42-US (lanes 1–3), FP42-LS (lane 4) or FP42-DS (lanes 5–6). Competition between labeled and unlabeled specific (FP42-US, lane 2; FP42DS, lane 6) or mutant (M4FP42-US, lane 3) oligonucleotides at molar ratio of 50 is shown; the sequences of these oligonucleotides are specified in Table 1Go.

 
Posttranslational Modifications of SSBP and DSBP
Many transcription factors have their activity regulated by posttranslational modifications, such as phosphorylation (14, 15) and O-glycosylation (16). In an attempt to define the relationship between SSBP and DSBP, we investigated the status of these common posttranslational modifications for these two proteins. We first examined whether phosphorylation of SSBP and DSBP was important for their DNA-binding activity. The inability of either calf intestinal alkaline phosphatase or antiphosphotyrosine antibodies to alter the binding of SSBP or DSBP indicates that the DNA-binding activities of SSBP and DSBP are not significantly modulated by their phosphorylation status (data not shown).

Glycosylation of transcription factors is a well established posttranslational modification. In all characterized examples, this involves N-acetlyglucosamine (GlcNAc) residues attached to serine and/or threonine residues of the protein (16). Since proteins bearing GlcNAc residues are often bound by the lectin wheat-germ agglutinin (WGA) (17, 18), we used WGA affinity chromatography and EMSA to test for O-glycosylation of the SSBP and the DSBP. DSBP binding activity could be recovered in the eluate ensuing after the application of the competitor sugar N-acetylglucosamine to the WGA column equilibrated with an aliquot of liver nuclear extract (Fig. 11Go). In contrast, no SSBP-binding activity could be demonstrated in the same eluate (Fig. 11Go). These results suggest that the DSBP is significantly glycosylated, while the SSBP does not undergo a similar posttranslational modification. Since O-glycosylation of DNA-binding proteins does not affect the DNA-binding properties of proteins (19), this discordance in the glycosylation status cannot be implicated for the differential DNA-binding properties of the SSBP and the DSBP and would support the possibility that the SSBP and DSBP activities represent two distinct proteins and not modifications of the same protein.



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Figure 11. WGA Affinity Chromatography of DSBP and SSBP Binding Activities

Autoradiography of EMSA with 32P-labeled FP42-DS (lanes 1–5) or FP42-US (lanes 6–10) of either crude liver nuclear extracts (lanes 1 and 6) or proteins eluted by the application of n-acetylglucosamine to a WGA column that had been allowed to equilibrate with crude liver nuclear extracts (lanes 3–5 and 8–10). The binding activities of the proteins eluted in the wash solution immediately before the application of the n-acetlyglucosamine is also shown (lanes 2 and 7). The bands representing specific double (A) or single (B) strand DNA-binding proteins are indicated.

 
Single-Strand DNA Binds to a 23-kDa Protein
To identify the molecular size of the protein interacting with the coding strand of the GH receptor enhancer (FP42-US), Southwestern analysis was performed. These experiments established that, after SDS-PAGE and transfer to nitrocellulose membrane, a 23-kDa liver nuclear protein exhibited binding to FP42-US (Fig. 12Go). To determine whether the binding of FP42-US to the 23- kDa protein was sequence specific, we performed competition experiments in which the binding reaction was carried out in the presence of unlabeled FP42-US, M4FP42-US, or an oligonucleotide with random sequence (Table 1Go). Whereas a 50-fold molar excess of unlabeled FP42-US significantly abrogated the binding of the labeled FP42-US to the 23-kDa protein, a similar 50-fold molar excess of M4FP42-US or the random sequence oligonucleotide did not significantly alter this binding (Fig. 12Go). These results demonstrate that the binding of the 23-kDa protein in liver nuclear extracts to FP42-US is sequence specific.



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Figure 12. Determination of Molecular Weight of SSBP by Southwestern Blot Analysis

Fifteen-microgram aliquots of nuclear extracts from mouse liver were electrophoresed through a 12% SDS-polyacrylamide gel and transferred onto nitrocellulose. The nitrocellulose membrane was then probed with 32P-labeled FP42-US in the absence (lane 4) or presence of 50-fold molar excess of the wild type sequence FP42-US (lane 3), M4FP42-US (lane 2), or an oligonucleotide with random sequence (lane 1); the sequences of these oligonucleotides are specified in Table 1Go. Position of concurrently electrophoresed protein size markers are indicated.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have demonstrated that the coding strand of an enhancer region regulating the expression of the murine GH receptor gene binds to a sequence-specific DNA-binding protein. Although this is the first example of a SSBP modulating the transcription of the GH receptor gene, there is increasing evidence that sequence-specific, SSBPs are involved in the regulation of gene expression (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). Thus, recent reports have provided evidence for the role of SSBPs in the transcriptional regulation of genes such as the TSH receptor (23, 24), 3-hydroxy-3-methylglutaryl enzyme A reductase (25), tyrosine aminotransferase (26), adipsin (27), ß-casein (28, 29), actin (30, 31), platelet-derived growth factor A-chain (32), estrogen receptor (33), and the GH (34) genes. DNA is a structurally dynamic macromolecule that is able to adopt a number of alternative conformations. This conformational plasticity is believed to play an important role in the regulation of DNA replication, recombination, and transcription. Although the precise mechanisms by which the local topological features of these cis-acting elements may regulate gene transcription is unclear, one of the possibilities is that conformational changes of these DNA elements may alter the characteristics of the interaction between trans-acting factors and their cognate DNA elements. Whereas most eukaryotic sequence-specific DNA-binding proteins exclusively recognize native double-strand sequences, Larsen and Weintraub (35) originally proposed the existence of a class of regulatory proteins that do not recognize the classic B form DNA but specifically recognize altered structures of DNA. These altered forms of DNA include H-DNA form, an intramolecular triplex/single-strand structure, and an alternative intramolecular triplex structure that can form with a G-G-C composition (36, 37, 38). Some sequence-specific SSBPs recognize double-stranded DNA (21, 39), whereas others (32), such as the SSBP described in the current report, bind almost exclusively to single-strand DNA.

The identity of the SSBP characterized in this report remains to be defined. Computer analysis of the nucleotide sequence of the enhancer element (FP42) did not reveal any potential DNA-binding sites for proteins binding to either single-strand or double-strand DNA. Furthermore, the inability of an oligonucleotide incorporating the consensus binding motif for the single-strand DNA-binding protein, Y-box protein (24), to compete for binding of SSBP to FP42-US (data not shown) suggests that these two proteins are not similar. Many of the reported SSBPs have been shown to bind to cis-acting promoter elements that are hypersensitive to DNase I and/or single-strand-specific nucleases (32, 39). The intracellular milieu strongly favors the maintenance of a double-stranded DNA configuration, thereby restricting access to SSBPs. The current hypothesis is that the sensitivity of these regions to nucleases, resulting from alterations in the structure of active chromatin with differential dissociation of nucelosomes and/or local alteration in the conformation of the DNA, facilitates the interaction of the SSBP with DNA. In addition, some of these regions exhibit a strong purine/pyrimidine asymmetry and have been termed CT elements (39, 40). Inspection of the nucleotide composition of the FP42 enhancer element does not reveal a purine/pyrimidine asymmetry; whether this region exhibits nuclease hypersensitivity remains to be determined.

The DNA-binding site of the SSBP characterized in this report is contiguous with and partially overlaps the binding site of a DSBP. Previous reports have also described the proximity of binding sites for SSBPs and DSBPs on the cis-acting promoter elements of other genes (23, 34, 41), suggesting that the coexistence of the binding sites for SSBPs and DSBPs may not be a random association and may be necessary for the function of these trans-acting factors. At least two lines of evidence suggest that there is direct physical interaction between the SSBP and DSBP described in this report. First, the experiments using the Shift-Western technique demonstrated that the SSBP is present within the DSBP-DNA complex. Second, DOC, an agent that disrupts protein-protein interaction (13), alters the dissociation rates of the DSBP-DNA complex. The claim that the effect of DOC on the dissociation rate is because of inhibition of a putative interaction between SSBP and DSBP is substantiated by the finding that selective mutation of the SSBP DNA-binding site to exclude SSBP from the complex also results in a similar effect on the dissociation rate of the DSBP-DNA complex. Hence, the congruity of the results from these two approaches indicate that, at least in vitro, SSBP serves to stabilize the DSBP complex and that this function involves physical interaction between the SSBP and DSBP.

Some sequence-specific SSBPs can exhibit enhancer (26) and others can exhibit repressor (24, 25, 34) activities. The results of the transient transfection experiments in our study indicate that the SSBP regulating the activity of the GH receptor gene promoter represses the activity of the cis-element with which it interacts. Our results suggest that SSBP serves to repress this activity of the enhancer element by interfering with the function of the cognate DSBP. This model is supported by the following results. First, delineation of the DNA-binding sites established that there is partial overlap of the binding sites for the SSBP and DSBP. Whereas the binding site of the DSBP on the coding strand is narrowly restricted to three contiguous nucleotides, the binding site of the SSBP is spread over the entire 42-bp enhancer element. It is of interest to note that this broad distribution of the DNA-protein contact points is a feature common to many previously characterized SSBPs (22, 34, 38, 41). Second, the site-directed mutations in the enhancer element that selectively disrupted the binding sites unique to the SSBP, while retaining the binding sites common to the SSBP and DSBP, resulted in an increase in the binding of DSBP to the enhancer element. This finding suggests that SSBP restricts the access of the DSBP to the cognate DNA-binding site. This interaction could result from a variety of mechanisms including steric hinderance or alteration in the conformation of the DSBP molecule. Due to the fact that it was not possible to abolish binding of DSBP without concomitantly abrogating binding of the SSBP, we are at the present time unable to discern whether SSBP has transcriptional activity independent of the cognate DSBP.

The DSBP and SSBP present in adult female liver are differentially O-glycosylated. Alteration in the O-glycosylation status has been implicated in the modulation of trans-activation properties of transcription factors (19, 42). The current hypothesis for the role of O-glycosylation is based on the observation that in well characterized examples, the O-glycosylated amino acid residue on the transcription factor is adjacent to a phosphorylated amino acid residue. Thus the current model envisages a reciprocal relationship between O-glycosylation and phosphorylation, with O-glycosylation indirectly modulating DNA binding and/or transactivation by regulating the phosphorylation status of the trans-acting factor (19). Due to constraints on the choice of experiments because of lack of knowledge of their identity, the phosphorylation status of SSBP and DSBP could only be assayed by DNA binding. Our experiments indicate that phosphorylation does not significantly alter the DNA-binding properties of either SSBP or DSBP. However, the significant O-glycosylation of the DSBP predicts a role for phosphorylation in the function of DSBP. We speculate that the ability of DSBP to trans-activate the GH receptor gene, a property we could not assess directly, could be regulated by DSBP’s phosphorylation status. It is noteworthy that the demonstration of physical interaction between SSBP and DSBP is compatible with the observation that all O-glycosylated proteins, thus far identified, form reversible multimeric complexes (19).

The in vivo biological role of the SSBP identified in this report is not clear. Our previous work indicated that the cognate enhancer element FP42 and the DSBP play a role in the development-specific expression of the GH receptor gene (2). Because the levels of the SSBP as estimated by EMSA are actually significantly lower in the fetal liver, the demonstration of a repressor role for this protein in transient transfection experiments suggests that lack of the SSBP in the fetal tissue cannot be implicated in the decreased expression of the GH receptor in the fetal liver. The L1 transcript is selectively up-regulated in the liver during pregnancy (4). Since there was no significant change in the level of the SSBP in nuclear extracts from the liver of pregnant mice, we cannot ascribe a role for this protein in the increased expression of the GH receptor during pregnancy. However, the complex nature of the interaction between the enhancer element FP42 and its cognate DSBP and SSBP proteins leaves open the possibility that the modulation of the status of one of them could have a significant effect on the activity of the complex. Hence it is possible that a change in the topology of the enhancer element FP42 could restrict the interaction of the SSBP with the DSBP and FP42 and result in derepression of the activity of the enhancer element and increased expression of the GH receptor gene. Similarly, the posttranslational status (e.g. phosphorylation) of the DSBP could affect the interaction with SSBP and thereby modulate the transactivation potential of DSBP. The verification of these complex hypothetical models would necessarily involve the identification and further characterization of the proteins that represent the SSBP and DSBP activities on EMSA.

In summary, we have identified and partially characterized the DNA-protein interactions between a SSBP and its cognate cis-element in the promoter of the murine GH receptor gene. We have further demonstrated a physical and a functional interaction between this newly identified SSBP and a previously identified DSBP that also interacts with this cis-element. Our experiments suggest that, in vivo, SSBP decreases the activity of the GH receptor gene promoter by modulating the ability of the DSBP to trans-activate GH receptor gene expression.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
EMSA
Nuclear extracts from mouse liver were prepared as described by Gorski et al. (43). Protease inhibitors (leupeptin, 2 µg/ml; pepstatin, 1 µg/ml; and aprotinin 1%) were included in the buffers used to prepare the nuclear extracts. Custom synthesized oligonucleotides were used for single-stranded DNA probes and double-stranded DNA fragments used as probes were obtained by annealing complementary single-stranded oligonucleotides. The DNA was end-labeled with [{gamma}32P]ATP and T4 polynucleotide kinase. Approximately 6 fmol DNA were added to 1–4 µg nuclear extract in a final volume of 50 µl containing either 2 µg poly(deoxyadenylic-deoxythymidylic acid), 25 mM HEPES (pH 7.2), 75 mM KCl, 25 mM NaCl, 2.5 mM MgCl2, BSA (250 µg/ml), 10% glycerol, 0.025% NP-40, and 1 mM dithiothreitol (DTT) (for single-strand DNA probes) or 2 µg poly(deoxyadenylic-deoxythymidylic acid), 20 mM Tris-HCl (pH 8.0), 50 mM NaCl, BSA (50 µg/ml), 1% NP-40, 1 mM EDTA, 10% glycerol, and 1 mM DTT (for double-strand DNA probes). After incubation at room temperature for 30 min, DNA-protein complexes were resolved on a 6% nondenaturing polyacrylamide gel with 90 mM Tris-borate, 2 mM EDTA buffer. Where indicated, the gels were dried and sequentially subjected to autoradiography with intensifying screens (DuPont, Wilmington, DE) at -80 C and analysis via PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Competition experiments included the addition of excess unlabeled DNA fragments to the reaction mix. In some experiments nuclear extracts were incubated with the indicated amounts of antiphosphotyrosine antibodies for 120 min at 4 C before addition to the binding reactions. The antiphosphotyrosine mouse monoclonal antibodies tested were PY20 (ICN Biomedicals,, Inc., Costa Mesa, CA) and 4G10 (Upstate Biotechnology Inc., Lake Placid, NY).

Methylation Interference Assay
Gel-purified single-stranded oligonucleotide was end-labeled with [{gamma}32P]ATP and T4 polynucleotide kinase. Double-stranded 100-bp DNA probes that contained the FP42 enhancer element and labeled either on the coding or noncoding strand were generated using end-labeled oligonucleotides in PCR. The single- or double-stranded probes were modified with dimethyl sulfate for 40 min on ice (44). For the preparative EMSA, protein-DNA complexes were formed by incubating {gamma}32P-labeled or unlabeled double- stranded FP42 DNA duplex liver nuclear extract as described above for EMSA. After electrophoresis the undried gel was analyzed by PhosphorImager, and the regions corresponding to the protein-DNA complex and unbound probe were excised, eluted, and then precipitated. Base elimination and strand scission reactions at adenine and guanine (A < G) were performed as described (44). The samples were then lyophilized, resuspended in water, relyophilized (three times), and analyzed by electrophoresis through a 12% sequencing gel. The dried gel was sequentially subjected to autoradiography and analysis via PhosphorImager (Molecular Dynamics).

DNA Sequencing
Sequencing was carried out by the dideoxynucleotide chain termination method of Sanger et al. (45) using the Sequenase 2.0 kit (U.S. Biochemical Corp., Cleveland, OH). Sequencing primers were either complementary to the canonical T3, T7, or SP6 sites flanking the multiple cloning site of the vector or were complementary to experimentally established sequences. The sequence data were managed using the sequence analysis program MacVector5.0 (Oxford Molecular Group, Campbell, CA).

Reporter Gene Constructs
The activity of the enhancer element FP42 was tested via the ability to exhibit activity in the context of an heterologous thymidine kinase promoter contained in the luciferase vector pTK81 (ATCC, Rockville, MD). Double-stranded oligonucleotides with either the wild type enhancer element sequence (pTK-FP42) or enhancer element with mutations (pTK-M2FP42, pTK-M4FP42) were ligated into the vector pTK81 by exploiting convenient restriction sites in the vector. All constructs were sequenced through the vector-insert junctions to ensure nucleotide fidelity and verify directionality.

Transient Expression of Reporter Gene
The culture media used for tissue culture experiments were obtained from Life Technologies (Gaithersburg, MD) unless otherwise stated. COS-7 cells (ATCC) were maintained in Eagles MEM (with nonessential amino acids, sodium pyruvate, and Earle’s balanced salt solution), 10% FBS and penicillin G (100 U/ml), and streptomycin (100 µg/ml). Cells (1 x 106) were plated on 60- mm plates 24 h prior to transfection. Fifteen micrograms of plasmid DNA were transfected per plate using the calcium phosphate transfection method (Life Technologies, Gaithersburg, MD). After 6 h incubation the cells were washed with PBS and then supplemented with medium for 40 h before harvest for luciferase assay. For estimation of luciferase activity the plates were rinsed twice with PBS, and the cells were harvested by the addition of 200 µl lysis buffer (25 mM Tris-phosphate, pH 7.8, 2 mM DTT, 2 mM 1, 2 diaminocyclohexane-N,N,N',N-tetraacetic acid, 10% glycerol, 1% Triton X-100). After a brief freeze-thaw cycle, the insoluble debris was removed by centrifugation at 4 C for 2–3 min at 12000 x g. The supernatant was then immediately assayed for luciferase activity. All transfections were performed at least in quadruplicate. Transfection efficiency was monitored by cotransfection of 1 µg of the plasmid, pßgal-Control (CLONTECH Lab, Palo Alto CA). Protein concentration of the supernatant was determined using the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA).

Luciferase Activity Assay
Luciferase activity was measured in the cell lysates using reagents from Analytical Luminescence Laboratory (San Diego, CA). Briefly, using the automatic injectors of the Monolight 2010 Luminometer (Analytical Luminescence), 100 µl ({approx} 200 µg) of cell extract were mixed sequentially with 100 µl buffer (3 mM ATP, 15 mM MgSO4, 30 mM Tricine, 10 mM DTT, pH 7.8) and 100 µl 1 mM luciferin, and the light output was measured for 30 sec. For all the samples, determination of the ß-galactosidase activity (see below) was used to equalize the luciferase raw activity values for transfection efficiency. The results of the luciferase assay are expressed in relative light units equalized for transfection efficiency.

ß-Galactosidase Activity Assay
ß-Galactosidase activity was estimated by the Luminescent ß-Galactosidase Detection Kit II (CLONTECH Lab). Briefly, 50 µl of cell lysate were mixed with 100 µl of the reaction buffer, and, after an 60-min incubation period, the light output was measured for 5 sec in a Monolight 2010 Luminometer (Analytical Luminescence).

Shift-Western Analysis
To investigate whether the single-strand binding protein is a constituent of the double-strand DNA-protein complex, a modification of the Shift-Western blotting method was employed (46). Briefly, for the preparative EMSA, protein-DNA complexes were formed by incubating {gamma}32P-labeled or unlabeled double-stranded FP42 DNA duplex liver nuclear extract as described above for EMSA. After electrophoresis through a 6% nondenaturing gel, the localization of the double-strand DNA-protein complex was achieved by analyzing the undried gel on a PhosphorImager. The proteins were then transferred electrophorectically onto nitrocellulose membranes using a Mini-Protein Blotting system (Bio-Rad Laboratories). Blotting was done at 4 C for 1 h at 100 V in 25 mM Tris, 192 mM glycine, 20% (vol/vol) methanol, pH 8.3. After transfer, the portion of the nitrocellulose membrane corresponding to the different lanes was excised. The nitrocellulose filters were then soaked overnight at 4 C in 5% nonfat dry milk, 50 mM Tris (pH 7.5), 50 mM NaCl, 1 mM EDTA, and 1 mM DTT. The filters were then hybridized for 1 h at room temperature with binding buffer (20 mM HEPES, pH 7.9, 100 mM KCl, 5% glycerol, 1 mM EDTA, 1 mM DTT, and 15 µg/ml salmon sperm DNA) containing 106 cpm/ml of the 32P-labeled FP42-US probe, after which the filters were washed in the binding buffer at room temperature for 20 min. The filters were autoradiographed with an intensifying screen at - 80 C; in some instances, for purposes of quantification, the filters were analyzed using a PhosphorImager (Molecular Dynamics, CA). Competition experiments included the addition of molar excess of unlabeled DNA fragments to the hybridization mixture.

Effect of Alkaline Phosphatase on Single- and Double-Strand DNA-Binding Activities
Aliquots of nuclear extracts were incubated for 30 min at 25 C with the addition of buffer alone or the indicated amounts of calf intestinal phosphatase (New England Biolabs, Beverly, MA) and then subjected to EMSA with DNA labeled with [32P]dATP using terminal deoxynucleotide transferase (Promega, Madison, WI). Heat (80 C for 10 min)-inactivated alkaline phosphatase served as control for these experiments.

Lectin Affinity Chromatography
To test for O-glycosylation of the DNA-binding proteins, an aliquot of liver nuclear extracts (0.8–1 mg) was applied at a rate of 3 ml/h to a 1-cm Wheat Germ-Sepharose (Sigma, St. Louis, MO) column equilibrated in wash buffer (20 mM HEPES-KOH, 0.1 M KCl, 0.2 mM EDTA, 1 mM phenylmethylsulfonylfluoride, and 20% glycerol). After allowing the proteins to interact with the WGA matrix for 3 h, the column was washed with 100 ml of wash buffer. The proteins bound to the column were then eluted with buffer (25 mM HEPES-KOH, 0.1 M KCl, 12.5 mM MgCl2, 0.1% NP40, 1 mM DTT, 0.1 µM ZnSO4, 1 mM phenyl-methylsulfonylfluoride) containing 0.3 M N-acetylglucosamine. The eluate was assayed for the presence of the SSBPs and DSBPs by EMSA with the respective probe.

Southwestern Blot Analysis
Fifteen-microgram aliquots of nuclear protein from mouse liver were heated for 3 min at 90 C in 62.5 mM Tris-HCl, 10% (vol/vol) glycerol, 5% (vol/vol) 2-mercaptoethanol, 1.05% SDS, and 0.004% bromophenol blue. The protein samples were then electrophoresed through a 4% stacking, 12% resolving, discontinuous SDS-polyacrylamide gel in 25 mM Tris-HCl, 192 mM glycine, and 0.1% SDS buffer. High and low molecular weight markers (Bio-Rad Laboratories) were also concurrently electrophoresed. After electrophoresis, the proteins were transferred to nitrocellulose by electroblotting (SemiPhor, Hoefer Scientific, San Francisco, CA) for 2 h. The nitrocellulose filters were then soaked overnight at 4 C in 5% nonfat dry milk, 50 mM Tris (pH 7.5), 50 mM NaCl, 1 mM EDTA, and 1 mM DTT. The filters were then hybridized for 1 h at room temperature with binding buffer (20 mM HEPES, pH 7.9, 100 mM KCl, 5% glycerol, 1 mM EDTA, 1 mM DTT, and 15 µg/ml salmon sperm DNA) containing 106 cpm/ml of the radiolabeled probe, after which the filters were washed in the binding buffer at room temperature for 20 min. The filters were autoradiographed with an intensifying screen at -80 C; in some instances for purposes of quantification the filters were analyzed using a PhosphorImager (Molecular Dynamics). Competition experiments included the addition of molar excess of unlabeled DNA fragments to the hybridization mixture.


    ACKNOWLEDGMENTS
 
The generosity of Dr. Mark A. Sperling in providing support and encouragement is gratefully acknowledged. The technical suggestions given by Drs. Rudert, Anand, and Zou are greatly appreciated.


    FOOTNOTES
 
Address requests for reprints to: Ram K. Menon, M.D., Division of Endocrinology, Department of Pediatrics, Children’s Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, Pennsylvania 15213.

Supported by NIH Grants K08-HD-000986 and R29-DK-49845, Genentech Foundation for Growth and Development, Children’s Hospital of Pittsburgh, and the Vira I. Heinz Foundation.

Received for publication December 30, 1996. Revision received April 18, 1997. Accepted for publication April 26, 1997.


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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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