[38]
PROTEIN-DNA
INTERACTIONS UPSTREAM OF
CYPIAI
403
[38] U s e of Gel R e t a r d a t i o n to A n a l y z e P r o t e i n - D N A I n t e r a c t i o n s U p s t r e a m of C Y P I A 1 G e n e By EMILY S. SHEN, CORNELIS J. ELFEmNK, and JAMES P. WHITLOCK, JR.
Introduction The gel retardation or gel mobility shift technique has become a standard tool for analyzing the interaction in vitro between DNA-binding proteins and their cognate genomic recognition sequences. The method relies on the fact that, during electrophoresis under nondenaturing conditions, protein-DNA complexes have a reduced mobility compared with protein-free DNA. Recent reviews provide a comprehensive description of the technique. 1,2 Here, we discuss our experience with the gel retardation method in analyzing the protein-DNA interactions at regulatory domains upstream of the CYPIAI gene. One interesting aspect of CYPIA1 gene regulation is that the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) activates gene transcription. The transcriptional response involves the binding of TCDD to an intracellular protein (the Ah receptor), followed by the binding of the liganded receptor to a dioxin-responsive enhancer located upstream of the CYPIA1 gene. 3 We have used gel retardation to analyze receptor-enhancer interactions in vitro. In this chapter, we discuss the method itself, with emphasis on the variables that influence gel retardation, and several uses of the method, as applied to the CYPIA1 gene. Gel Retardation Method
Several groups have used gel retardation to analyze TCDD-inducible protein-DNA interactions at regulatory domains upstream of the C YPIA1 gene. 4-7 The conditions used by each group vary somewhat, revealing 1 D. M. Crothers, Nature (London) 325, 464 (1987). 2 A. Revzin, BioTechniques 7, 346 (1989). 3 j. p. Whitlock, Jr., Annu. Rev. Pharmacol. Toxicol. 30, 251 (1990). 4 M. S. Denison, J. M. Fisher, and J. P. Whitlock, Jr., Proc. Natl. Acad. Sci. U.S.A. 85, 2528 (1988). 5 A. Fujisawa-Sehara, M. Yamane, and Y. Fujii-Kuriyama, Proc. Natl. Acad. Sci. U.S.A. 85, 5859 (1988). 6 j. Hapgood, S. Cuthill, M. Denis, L. Poellinger, and J.-A. Gustafsson, Proc. Natl. Acad. Sci. U.S.A. 86, 60 (1989).
METHODS IN ENZYMOLOGY, VOL. 206
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
404
TRANSCRIPTIONALREGULATION
[38]
some flexibility in the assay conditions. The conditions described below, which are modified from those published previously, 4 have yielded the best results for us. Nuclear extracts are prepared from mouse hepatoma cells as previously described. 4 The extracts are stored at - 7 0 ° in 25- to 50-/.,1 singleuse aliquots. Under these conditions, the extracts are relatively stable and yield reproducible results for 3-4 months. In a typical gel retardation assay, nuclear extract (3-5/~g of protein) is mixed with HEDG buffer [25 mM HEPES, 1 mM EDTA, 1 mM dithiothreitol, 10% (v/v) glycerol, pH 7.5], 0.25-1.0/zg poly[d(I-C)] (dissolved in HEDG buffer; see below), and an appropriate volume of HEDG buffer containing 1.0 M KCI to bring the final KC1 concentration in the reaction to 0.2 M. The reaction mixture (21/~1) is preincubated for 15 min at room temperature, after which 4/xl of 32p-labeled DNA [I-5 × 104 counts/min (cpm), 0.1-0.5 ng in 4/zl HEDG buffer] is added, and the incubation is continued for 15 min at room temperature. Sample buffer [2.8/xl of 25% (w/v) Ficol1400, 0.25% (w/v) bromphenol blue, 0.25% (w/v) xylene cyanol] is added, and 15 gl of the reaction is loaded onto a 4% polyacrylamide gel [acrylamide-bisacrylamide ratio of 30 : 1; acrylamide stocks are filtered through disposable Millipore (Bedford, MA) or Nalgene (Rochester, NY) membrane filters] containing TAE buffer (40 mM Tris base, 20 mM sodium acetate, 1 mM EDTA, pH 8.08). The gel is preelectrophoresed at 11 V/cm for 30-120 min. Electrophoresis is carried out at the same voltage until the protein-free DNA reaches the bottom of the gel (about 1.5-3 hr, depending on DNA length). Buffer is recirculated during electrophoresis. We observe that with relatively small DNA fragments [e.g., 30-40 base pairs (bp)], a 6% polyacrylamide gel may provide better resolution than a 4% gel. This can be determined empirically. We have also used gel retardation to analyze cytosolic proteins from both mouse hepatoma cells and rodent liver. In these studies, cytosol is prepared and the Ah receptor is transformed to a DNA-binding form by incubation with TCDD in vitro, as previously described. 9 The cytosolic preparations are stored under the same conditions as the nuclear extracts, and exhibit similar stabilities. Detection of an Ah receptor-DNA complex requires 50-100/zg of cytosol and reduction of the final KCI concentration
7 L. A. Neuhold, Y. Shirayoshi, K. Ozato, J. E. Jones, and D. W. Nebert, Mol. Cell. Biol. 9, 2378 (1989). 8 T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual," p. 454. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982. 9 E. C. Henry, G. Rucci, and T. A. Gasiewicz, Biochemistry 28, 6430 (1989).
[38]
PROTEIN-DNA INTERACTIONS UPSTREAMOF CYPIA1
405
to 100 mM. NaCI can substitute for KCI without detectably affecting the results. The salt conditions optimal for gel retardation are determined empirically. We find that, for the cytosolic Ah receptor transformed by TCDD in vitro, the range of useful concentrations is relatively restricted (50-150 mM KCI or NaCI). In contrast, for the liganded receptor extracted from nuclei, the range is somewhat broader (100-400 mM). The reason(s) for this differences is unknown. We find that the poly[d(I-C)], which is used as nonspecific DNA competitor, can be a source of substantial experimental variation. Poly[d(I-C)] is supplied as the sodium salt, and the average DNA length varies from batch to batch. Both salt and DNA size can influence the gel retardation assay. Therefore, we routinely precipitate the poly[d(I-C)] with ethanol prior to use, and we empirically determine the optimal concentration of each batch. Applications of Gel Retardation Method to CYPIA1 System Receptor-Enhancer Interactions
We used gel retardation to identify the DNA recognition sequence for the liganded Ah receptor. The approach involves analyzing different DNA fragments (previously shown to function in a TCDD-inducible, Ah receptor-dependent fashion) for their ability to participate in a TCDD-inducible, Ah receptor-dependent protein-DNA interaction. Comparison of the nucleotide sequences of such DNA fragments reveals that each contains a common motif 5' A-GCGTG 3' 3' A-CGCAC 5'
which represents the " c o r e " recognition sequence for the liganded receptor.~° Knowledge of this motif allows us to identify additional DNA domains (e.g., associated with other genes) that may respond to the liganded Ah receptor. In addition, the recognition motif may be useful as an affinity reagent for receptor purification. We used a modified gel retardation assay to determine the direct involvement of the liganded Ah receptor in the protein-DNA interaction. 10 Instead of labeling the DNA, we labeled the protein, using 2-[125I]iodo-7,8dibromodibenzo-p-dioxin, a high affinity agonist for the Ah receptor.l~ J0 M. S. Denison, J. M. Fisher, and J. P. Whitlock, Jr., J. Biol. Chem. 264, 16478 (1989). it C. A. Bradfield, A. S. Kende, and A. Poland, Mol. Pharmacol. 34, 229 (1988).
406
TRANSCRIPTIONALREGULATION
[38]
Migration of the 125I-labeled receptor-DNA complex to the same position in the gel as the TCDD-inducible protein-[32p]DNA complex implies that the protein-DNA complex contains the A h receptor.l° In principle, this approach can also be applied to other DNA-binding proteins that can be specifically labeled. Gel retardation is also useful for analyzing the effect of DNA mutations on receptor-enhancer interactions. For example, synthetic doublestranded oligodeoxyribonucleotides containing single base-pair substitutions within the receptor recognition motif can be used to study the details of the protein-DNA interaction. When radioactively labeled, these DNA fragments are employed in retardation assays, and, when unlabeled, they serve as specific competitors in the binding reactions. Differences among the mutants in their gel retardation properties reveal the relative contribution of specific base pairs to the protein-DNA interaction. Methylation Protection and Interference
Additional details of the protein-DNA interaction are revealed by methylation protection and interference studies. ~2 In these experiments the DNA is labeled with 32p on either the sense or antisense strand. For the protein-DNA binding reaction, the amounts of nuclear extract and poly[d(I-C)] are increased 2.5-fold from the standard reaction, and the amount of radiolabeled DNA is increased to 1 x l06 cpm. The methylation interference assays were performed essentially as described by Gilman et al. 13Briefly, the radiolabeled DNA (in 5/zl of glassdistilled water) is added to 200/zl of methylation buffer (50 mM sodium cacodylate, 1 mM EDTA, pH 8.0) and chilled on ice. Dimethyl sulfate (I /zl) is added to a final concentration of 50 mM and allowed to incubate at room temperature for 7 min to methylate purine residues. The incubation time is determined empirically. The reaction is stopped by the addition of 50/~1 ice-chilled stop buffer (1.5 M sodium acetate, 1 M 2-mercaptoethanol, 100/.~g/ml glycogen). The DNA is precipitated by the addition of 2 volumes of ethanol, dissolved in 250/zl of 0.3 M sodium acetate, and reprecipitated with ethanol. The DNA pellet is washed with 70% ethanol and resuspended in HEDG buffer for use in the scaled-up binding reaction. In the methylation protection experiments, dimethyl sulfate is added (to a final concentration of 40 mM) at the completion of the binding reaction. The methylation reaction is allowed to continue at room temperature for 5 min before the samples are loaded directly onto the gel used for the gel retardation assay. 12E. S. Shen and J. P. Whitlock, Jr., J. Biol. Chem. 264, 17754(1989). 13M. Z. Gilman, R. N. Wilson, and R. A. Weinberg, Mol. Cell. Biol. 6, 4305 (1986).
[38]
PROTEIN-DNA INTERACTIONS UPSTREAM OF C Y P I A I
407
For both the methylation protectionand methylation interference experiments, the protein-bound DNA is separated from protein-free DNA by gel retardation. The positions of the protein-bound and protein-free DNAs are determined by autoradiography of the wet gel. The DNAs are transferred onto NA45 membrane and eluted with a buffer containing 20 mM Tris-HC1, I mM EDTA, and 1 M NaC1 at pH 8.0. Carrier tRNA (10 /xg) is added to the eluates before each sample is purified by phenol-chloroform extraction and ethanol precipitation. The DNAs are chemically cleaved at methylated bases by incubation in 1 M piperidine for 30 min at 90°. The cleavage products are analyzed on a standard sequencing gel. The pattern of cleavage reveals both the location of the protein-DNA interaction (i.e., the major or minor groove) and the purine residues that contribute to the interaction.
Protein-DNA Stoichiometry Gel retardation can be used to determine the molar ratio between a specific DNA-binding protein and its cognate recognition motif. Both components are labeled (e.g., the protein with 125I and the DNA with 32p), and the nucleoprotein complex is isolated from the separate DNA and protein components by gel retardation. The retarded band is excised and counted, and the protein-DNA ratio is calculated using the known specific activities of the two components. For example, using this approach, we found that the liganded Ah receptor binds to its recognition motif in a 1 : 1 ratio. ~0
DNA Bending The binding of a protein to DNA can induce a bend in the nucleic acid) 4 Bent DNA fragments migrate more slowly during electrophoresis than straight DNA of the same length and base composition. The mobility of the DNA is influenced by the extent and location of bending. Gel retardation can be used to determine whether a protein-DNA interaction bends the DNA and, if so, the location of the bend. For example, we have used a series of five circularly permuted DNA fragments and the gel retardation technique to show that the binding of the liganded Ah receptor to its recognition motif produces a bend in the DNA at (or very near) the protein-binding site. 15 This change in DNA configuration may contribute to the activation of gene transcription by TCDD. 14 H. M. Wu and D. M. Crothers, Nature (London) 308, 509 (1984). 15 C. J. Elferink and J. P. Whitlock, Jr., J. Biol. Chem. 265, 5718 (1990).
408
TRANSCRIPTIONAL REGULATION
[38l
Analysis of Proteins Cross-Linked to DNA
DNA that contains bromodeoxyuridine (BrdU) in place of thymidine can be covalently cross-linked to bound proteins by exposure of the nucleoprotein complex to ultraviolet irradiation.16 To take advantage of this phenomenon, gel retardation can be performed using 32p-labeled, BrdUsubstituted DNA. To cross-link the transformed Ah receptor to DNA, a double-stranded BrdU-substituted oligonucleotide is made) 7 A synthetic oligonucleotide is annealed to a complementary 8-nucleotide primer. The primed template is end-filled using the DNA polymerase Klenow fragment in a reaction mixture containing 50/~M each of dCTP, dGTP, 5-bromo-2'deoxyuridine triphosphate, 5/zM [a-32p]dATP, 50 mM Tris-HCl (pH 7.5), 10 mM MgSO4, and 0.1 mM dithiothreitol. The standard binding assay containing the labeled, BrdU-substituted DNA is irradiated with a UV lamp (maximum wavelength, 302 nm; maximum intensity, 7000 ~W/cm 2) for 30-45 min at a distance of 4-5 cm from the source. The nucleoprotein complex of interest is separated from free DNA by gel retardation and is identified by autoradiography. Elution of the cross-linked protein-DNA complexes and analysis by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and autoradiography reveal the number and size of the proteins that contact the DNA in the vicinity of the BrdU residues. Evidence obtained using this approach suggests that the DNA-binding form of the Ah receptor is a heterodimer.17 Monitoring Protein Purification
Gel retardation is a simple, convenient assay for monitoring a DNAbinding protein during its purification. In principle, this approach should be useful during the purification of both the liganded Ah receptor and other DNA-binding proteins involved in the regulation of cytochrome P450 gene expression.
16 F. Hutchinson, Q. Reo. Biophys. 6, 201 (1973). 17 C. J. Elferink, T. A. Gasiewicz, and J. P. Whitlock, Jr., J. Biol. Chem. 265, 20708 (1990).
PROTEIN-DNA
INTERACTIONS UPSTREAM OF
CYPIAI
403
[38] U s e of Gel R e t a r d a t i o n to A n a l y z e P r o t e i n - D N A I n t e r a c t i o n s U p s t r e a m of C Y P I A 1 G e n e By EMILY S. SHEN, CORNELIS J. ELFEmNK, and JAMES P. WHITLOCK, JR.
Introduction The gel retardation or gel mobility shift technique has become a standard tool for analyzing the interaction in vitro between DNA-binding proteins and their cognate genomic recognition sequences. The method relies on the fact that, during electrophoresis under nondenaturing conditions, protein-DNA complexes have a reduced mobility compared with protein-free DNA. Recent reviews provide a comprehensive description of the technique. 1,2 Here, we discuss our experience with the gel retardation method in analyzing the protein-DNA interactions at regulatory domains upstream of the CYPIAI gene. One interesting aspect of CYPIA1 gene regulation is that the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) activates gene transcription. The transcriptional response involves the binding of TCDD to an intracellular protein (the Ah receptor), followed by the binding of the liganded receptor to a dioxin-responsive enhancer located upstream of the CYPIA1 gene. 3 We have used gel retardation to analyze receptor-enhancer interactions in vitro. In this chapter, we discuss the method itself, with emphasis on the variables that influence gel retardation, and several uses of the method, as applied to the CYPIA1 gene. Gel Retardation Method
Several groups have used gel retardation to analyze TCDD-inducible protein-DNA interactions at regulatory domains upstream of the C YPIA1 gene. 4-7 The conditions used by each group vary somewhat, revealing 1 D. M. Crothers, Nature (London) 325, 464 (1987). 2 A. Revzin, BioTechniques 7, 346 (1989). 3 j. p. Whitlock, Jr., Annu. Rev. Pharmacol. Toxicol. 30, 251 (1990). 4 M. S. Denison, J. M. Fisher, and J. P. Whitlock, Jr., Proc. Natl. Acad. Sci. U.S.A. 85, 2528 (1988). 5 A. Fujisawa-Sehara, M. Yamane, and Y. Fujii-Kuriyama, Proc. Natl. Acad. Sci. U.S.A. 85, 5859 (1988). 6 j. Hapgood, S. Cuthill, M. Denis, L. Poellinger, and J.-A. Gustafsson, Proc. Natl. Acad. Sci. U.S.A. 86, 60 (1989).
METHODS IN ENZYMOLOGY, VOL. 206
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
404
TRANSCRIPTIONALREGULATION
[38]
some flexibility in the assay conditions. The conditions described below, which are modified from those published previously, 4 have yielded the best results for us. Nuclear extracts are prepared from mouse hepatoma cells as previously described. 4 The extracts are stored at - 7 0 ° in 25- to 50-/.,1 singleuse aliquots. Under these conditions, the extracts are relatively stable and yield reproducible results for 3-4 months. In a typical gel retardation assay, nuclear extract (3-5/~g of protein) is mixed with HEDG buffer [25 mM HEPES, 1 mM EDTA, 1 mM dithiothreitol, 10% (v/v) glycerol, pH 7.5], 0.25-1.0/zg poly[d(I-C)] (dissolved in HEDG buffer; see below), and an appropriate volume of HEDG buffer containing 1.0 M KCI to bring the final KC1 concentration in the reaction to 0.2 M. The reaction mixture (21/~1) is preincubated for 15 min at room temperature, after which 4/xl of 32p-labeled DNA [I-5 × 104 counts/min (cpm), 0.1-0.5 ng in 4/zl HEDG buffer] is added, and the incubation is continued for 15 min at room temperature. Sample buffer [2.8/xl of 25% (w/v) Ficol1400, 0.25% (w/v) bromphenol blue, 0.25% (w/v) xylene cyanol] is added, and 15 gl of the reaction is loaded onto a 4% polyacrylamide gel [acrylamide-bisacrylamide ratio of 30 : 1; acrylamide stocks are filtered through disposable Millipore (Bedford, MA) or Nalgene (Rochester, NY) membrane filters] containing TAE buffer (40 mM Tris base, 20 mM sodium acetate, 1 mM EDTA, pH 8.08). The gel is preelectrophoresed at 11 V/cm for 30-120 min. Electrophoresis is carried out at the same voltage until the protein-free DNA reaches the bottom of the gel (about 1.5-3 hr, depending on DNA length). Buffer is recirculated during electrophoresis. We observe that with relatively small DNA fragments [e.g., 30-40 base pairs (bp)], a 6% polyacrylamide gel may provide better resolution than a 4% gel. This can be determined empirically. We have also used gel retardation to analyze cytosolic proteins from both mouse hepatoma cells and rodent liver. In these studies, cytosol is prepared and the Ah receptor is transformed to a DNA-binding form by incubation with TCDD in vitro, as previously described. 9 The cytosolic preparations are stored under the same conditions as the nuclear extracts, and exhibit similar stabilities. Detection of an Ah receptor-DNA complex requires 50-100/zg of cytosol and reduction of the final KCI concentration
7 L. A. Neuhold, Y. Shirayoshi, K. Ozato, J. E. Jones, and D. W. Nebert, Mol. Cell. Biol. 9, 2378 (1989). 8 T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual," p. 454. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982. 9 E. C. Henry, G. Rucci, and T. A. Gasiewicz, Biochemistry 28, 6430 (1989).
[38]
PROTEIN-DNA INTERACTIONS UPSTREAMOF CYPIA1
405
to 100 mM. NaCI can substitute for KCI without detectably affecting the results. The salt conditions optimal for gel retardation are determined empirically. We find that, for the cytosolic Ah receptor transformed by TCDD in vitro, the range of useful concentrations is relatively restricted (50-150 mM KCI or NaCI). In contrast, for the liganded receptor extracted from nuclei, the range is somewhat broader (100-400 mM). The reason(s) for this differences is unknown. We find that the poly[d(I-C)], which is used as nonspecific DNA competitor, can be a source of substantial experimental variation. Poly[d(I-C)] is supplied as the sodium salt, and the average DNA length varies from batch to batch. Both salt and DNA size can influence the gel retardation assay. Therefore, we routinely precipitate the poly[d(I-C)] with ethanol prior to use, and we empirically determine the optimal concentration of each batch. Applications of Gel Retardation Method to CYPIA1 System Receptor-Enhancer Interactions
We used gel retardation to identify the DNA recognition sequence for the liganded Ah receptor. The approach involves analyzing different DNA fragments (previously shown to function in a TCDD-inducible, Ah receptor-dependent fashion) for their ability to participate in a TCDD-inducible, Ah receptor-dependent protein-DNA interaction. Comparison of the nucleotide sequences of such DNA fragments reveals that each contains a common motif 5' A-GCGTG 3' 3' A-CGCAC 5'
which represents the " c o r e " recognition sequence for the liganded receptor.~° Knowledge of this motif allows us to identify additional DNA domains (e.g., associated with other genes) that may respond to the liganded Ah receptor. In addition, the recognition motif may be useful as an affinity reagent for receptor purification. We used a modified gel retardation assay to determine the direct involvement of the liganded Ah receptor in the protein-DNA interaction. 10 Instead of labeling the DNA, we labeled the protein, using 2-[125I]iodo-7,8dibromodibenzo-p-dioxin, a high affinity agonist for the Ah receptor.l~ J0 M. S. Denison, J. M. Fisher, and J. P. Whitlock, Jr., J. Biol. Chem. 264, 16478 (1989). it C. A. Bradfield, A. S. Kende, and A. Poland, Mol. Pharmacol. 34, 229 (1988).
406
TRANSCRIPTIONALREGULATION
[38]
Migration of the 125I-labeled receptor-DNA complex to the same position in the gel as the TCDD-inducible protein-[32p]DNA complex implies that the protein-DNA complex contains the A h receptor.l° In principle, this approach can also be applied to other DNA-binding proteins that can be specifically labeled. Gel retardation is also useful for analyzing the effect of DNA mutations on receptor-enhancer interactions. For example, synthetic doublestranded oligodeoxyribonucleotides containing single base-pair substitutions within the receptor recognition motif can be used to study the details of the protein-DNA interaction. When radioactively labeled, these DNA fragments are employed in retardation assays, and, when unlabeled, they serve as specific competitors in the binding reactions. Differences among the mutants in their gel retardation properties reveal the relative contribution of specific base pairs to the protein-DNA interaction. Methylation Protection and Interference
Additional details of the protein-DNA interaction are revealed by methylation protection and interference studies. ~2 In these experiments the DNA is labeled with 32p on either the sense or antisense strand. For the protein-DNA binding reaction, the amounts of nuclear extract and poly[d(I-C)] are increased 2.5-fold from the standard reaction, and the amount of radiolabeled DNA is increased to 1 x l06 cpm. The methylation interference assays were performed essentially as described by Gilman et al. 13Briefly, the radiolabeled DNA (in 5/zl of glassdistilled water) is added to 200/zl of methylation buffer (50 mM sodium cacodylate, 1 mM EDTA, pH 8.0) and chilled on ice. Dimethyl sulfate (I /zl) is added to a final concentration of 50 mM and allowed to incubate at room temperature for 7 min to methylate purine residues. The incubation time is determined empirically. The reaction is stopped by the addition of 50/~1 ice-chilled stop buffer (1.5 M sodium acetate, 1 M 2-mercaptoethanol, 100/.~g/ml glycogen). The DNA is precipitated by the addition of 2 volumes of ethanol, dissolved in 250/zl of 0.3 M sodium acetate, and reprecipitated with ethanol. The DNA pellet is washed with 70% ethanol and resuspended in HEDG buffer for use in the scaled-up binding reaction. In the methylation protection experiments, dimethyl sulfate is added (to a final concentration of 40 mM) at the completion of the binding reaction. The methylation reaction is allowed to continue at room temperature for 5 min before the samples are loaded directly onto the gel used for the gel retardation assay. 12E. S. Shen and J. P. Whitlock, Jr., J. Biol. Chem. 264, 17754(1989). 13M. Z. Gilman, R. N. Wilson, and R. A. Weinberg, Mol. Cell. Biol. 6, 4305 (1986).
[38]
PROTEIN-DNA INTERACTIONS UPSTREAM OF C Y P I A I
407
For both the methylation protectionand methylation interference experiments, the protein-bound DNA is separated from protein-free DNA by gel retardation. The positions of the protein-bound and protein-free DNAs are determined by autoradiography of the wet gel. The DNAs are transferred onto NA45 membrane and eluted with a buffer containing 20 mM Tris-HC1, I mM EDTA, and 1 M NaC1 at pH 8.0. Carrier tRNA (10 /xg) is added to the eluates before each sample is purified by phenol-chloroform extraction and ethanol precipitation. The DNAs are chemically cleaved at methylated bases by incubation in 1 M piperidine for 30 min at 90°. The cleavage products are analyzed on a standard sequencing gel. The pattern of cleavage reveals both the location of the protein-DNA interaction (i.e., the major or minor groove) and the purine residues that contribute to the interaction.
Protein-DNA Stoichiometry Gel retardation can be used to determine the molar ratio between a specific DNA-binding protein and its cognate recognition motif. Both components are labeled (e.g., the protein with 125I and the DNA with 32p), and the nucleoprotein complex is isolated from the separate DNA and protein components by gel retardation. The retarded band is excised and counted, and the protein-DNA ratio is calculated using the known specific activities of the two components. For example, using this approach, we found that the liganded Ah receptor binds to its recognition motif in a 1 : 1 ratio. ~0
DNA Bending The binding of a protein to DNA can induce a bend in the nucleic acid) 4 Bent DNA fragments migrate more slowly during electrophoresis than straight DNA of the same length and base composition. The mobility of the DNA is influenced by the extent and location of bending. Gel retardation can be used to determine whether a protein-DNA interaction bends the DNA and, if so, the location of the bend. For example, we have used a series of five circularly permuted DNA fragments and the gel retardation technique to show that the binding of the liganded Ah receptor to its recognition motif produces a bend in the DNA at (or very near) the protein-binding site. 15 This change in DNA configuration may contribute to the activation of gene transcription by TCDD. 14 H. M. Wu and D. M. Crothers, Nature (London) 308, 509 (1984). 15 C. J. Elferink and J. P. Whitlock, Jr., J. Biol. Chem. 265, 5718 (1990).
408
TRANSCRIPTIONAL REGULATION
[38l
Analysis of Proteins Cross-Linked to DNA
DNA that contains bromodeoxyuridine (BrdU) in place of thymidine can be covalently cross-linked to bound proteins by exposure of the nucleoprotein complex to ultraviolet irradiation.16 To take advantage of this phenomenon, gel retardation can be performed using 32p-labeled, BrdUsubstituted DNA. To cross-link the transformed Ah receptor to DNA, a double-stranded BrdU-substituted oligonucleotide is made) 7 A synthetic oligonucleotide is annealed to a complementary 8-nucleotide primer. The primed template is end-filled using the DNA polymerase Klenow fragment in a reaction mixture containing 50/~M each of dCTP, dGTP, 5-bromo-2'deoxyuridine triphosphate, 5/zM [a-32p]dATP, 50 mM Tris-HCl (pH 7.5), 10 mM MgSO4, and 0.1 mM dithiothreitol. The standard binding assay containing the labeled, BrdU-substituted DNA is irradiated with a UV lamp (maximum wavelength, 302 nm; maximum intensity, 7000 ~W/cm 2) for 30-45 min at a distance of 4-5 cm from the source. The nucleoprotein complex of interest is separated from free DNA by gel retardation and is identified by autoradiography. Elution of the cross-linked protein-DNA complexes and analysis by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and autoradiography reveal the number and size of the proteins that contact the DNA in the vicinity of the BrdU residues. Evidence obtained using this approach suggests that the DNA-binding form of the Ah receptor is a heterodimer.17 Monitoring Protein Purification
Gel retardation is a simple, convenient assay for monitoring a DNAbinding protein during its purification. In principle, this approach should be useful during the purification of both the liganded Ah receptor and other DNA-binding proteins involved in the regulation of cytochrome P450 gene expression.
16 F. Hutchinson, Q. Reo. Biophys. 6, 201 (1973). 17 C. J. Elferink, T. A. Gasiewicz, and J. P. Whitlock, Jr., J. Biol. Chem. 265, 20708 (1990).
