Species-specific binding of transformed Ah receptor to a dioxin responsive transcriptional enhancer

European Journal ()f Pharmacology - Encironmental Toxicology and Pharmacology Section, 228 (1992) 85-94

85

~:~ 1992 Elsevier Science Publishers B.V. All rights reserved 0926-6917/92/$1)5.00

EJPTOX 40012

Species-specific binding of transformed Ah receptor to a dioxin responsive transcriptional enhancer P a u l a A. B a n k ~, E v e l i n e F. Y a o ~', C y n t h i a L. P h e l p s ,l, P a t r i c i a A. H a r p e r b a n d M i c h a e l S. D e n i s o n 'l " Department of Biochemistry, Michigan State Unicersity, East Lansing, M148824, USA, and /' Dicision of Clinical Pharmacology and Toxicolog3', The ttospital for Sick Children, Toronto, Ontario M5G IX8, Canada

Received 23 December 1991, revised MS received 18 March 1992, accepted 31 March 1992

The Ah receptor (AhR) mediates many, if not all, of the toxic and biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related halogenated aromatic hydrocarbons. Although wide variations in species sensitivity to these compounds have been observed, numerous biochemical and physiochemical characteristics of the AhR appear similar among species. We have examined the ability of cytosolic AhR, from a variety of species (rat, rabbit, guinea pig, hamster, mouse, cow, sheep, fish, chicken and human), to transform and bind to its cognate DNA recognition sequence, the dioxin responsive enhancer (DRE), to evaluate the importance of these events in species variations in TCDD responsiveness. Gel retardation analysis using a murine DRE oligonucleotide has revealed that cytosolic AhR from a wide variety of species can transform in vitro and bind to the DRE and demonstrates that all of the factors necessary for AhR transformation and DNA binding are present in cytosol. In addition, DNA-binding analysis using a series of mutant DRE oliognucleotides has indicated no apparent species- or ligand-dependent, nucleotide-spccific difference in AhR binding to the DRE. These studies support a highly conserved nature of the DRE and AhR (at least in DNA binding) and imply that a sequence closely related to the murine consensus DRE sequence is responsible for conferring AhR-dcpendent. TCDD responsiveness in each of these species. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin); Ah receptor

1. Introduction

Halogcnated aromatic hydrocarbons represent a large group of widely distributed, persistent and highly toxic environmental contaminants. In experimental animals, exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), the prototypical and most potent halogenated aromatic hydrocarbon, results in a wide variety of species- and tissue-specific toxic and biological effects, including: teratogenesis, immuno- and hepatotoxicity, tumor promotion and induction of numerous enzymes, including m i c r o s o m a l cytochrome P450IA1 (Poland and Knutson, 1982; Safe, 1986). Numerous biochemical and genetic studies (Poland eta[., 1976; Legraverend et al., 1982; Whitlock, 1986) have indicated that the induction of cytochrome P450IA1 is mediated by a soluble, intracellular protein, the Ah receptor (AhR), which binds T C D D saturably and with high affinity. Additional studies (Safe, 1986;

Correspondence to: M.S. Denison, Department of Biochemistry, Biochemistry Building, Michigan State University, East Lansing, MI 48824, USA. Tel. (517) 353-8786; Fax (517) 353-9334.

Whitlock, 1986) have suggested that many, if not all, of the observed TCDD-inducible responses are AhR-dependent. Mechanistically, induction of the cytochrome P450IA1 involves the binding of T C D D to the A h R protein, 'transformation' of the T C D D : A h R complex to its DNA-binding form, and subsequent accumulation of transformed T C D D : A h R complexes within the cell nucleus (Whitlock and Galeazzi, 1984; Henry et al., 1989; Denison and Yao, 1991). Recent studies (Elferink et al., 1990; Gasiewicz et al., 1991) have indicated that the DRE-binding form of the A h R is a heterodimer, containing only one ligand binding subunit and possibly a distinct DNA-binding subunit. In fact, a protein factor required for A h R nuclear l o c a l i z a t i o n / D N A binding has been recently cloned (Hoffmann et al., 1991) and may represent the DNA-binding A h R subunit, however, this remains to be determined. The binding of these t r a n s f o r m e d T C D D : A h R heterodimeric complexes to specific cis-acting dioxin responsive enhancers (DREs) adjacent to the CYPIA1 gene results in enhanced transcription of the CYPIAI gene (Whitlock, 1986, 1990; Denison et al., 1988a; Fisher et al., 1990). Significant differences in the ability of T C D D to elicit toxic and biological responses in various species

86 have been reported (Poland and Knutson, 1982; Safe, 1986). Species variation in T C D D responsiveness could be due to a variety of species- and tissue-specific biochemical and physiological characteristics, including, but not limited to, differences in T C D D pharmacokinetics, pharmacodynamics and metabolism, AhR functionality, cellular AhR subunit concentrations, the presence or absence of intracellular/extracellular T C D D binding sites (such as P450IA2 (Poland et al., 1988)) and species- and tissue-specific regulatory effectors. Additionally, since in depth studies have demonstrated that the AhR in these differentially responsive species appears to be very similar (with only minor differences in physiochemical properties, TCDD-binding affinity and the relative cellular concentration (Gasiewicz and Rucci, 1984; Denison and Wilkinson, 1985; Denison et al., 1986b,c), a detailed examination of AhR transformation and DNA binding may provide some insight into the observed species variations. Recently, we demonstrated that rat hepatic cytosolic AhR could be transformed in vitro to a form which bound to a DRE-containing oligonucleotide, specifically and with high affinity (Denison and Yao, 1991). Although some species differences in AhR transformation and binding to DNA have been reported (Henry et al., 1989; Gasiewicz et al., 1991), a comparative assessment of the ability of AhR, from a wide variety of species, to transform and bind to its cognate DNA recognition site, the DRE, has not been carried out. Since modulation of AhR functionality could significantly alter T C D D responsiveness in a given species we have examined the ability of T C D D : A h R complexes from a variety of species to transform and bind, in a nucleotide-specific manner, to a D R E oligonucleotide.

2. Experimental procedures 2. I. Materials [3H]TCDD (37 Ci/mmol), unlabeled T C D D and 2,3,7,8-tetrachlorodibenzofuran (TCDBF) were obtained from Dr. S. Safe (Texas A & M University). These compounds are extremely toxic substances and were handled with special precautions, including the use of disposable benchtop paper, gloves, plasticware and glassware.

2.2. Animals Male Sprague-Dawley rats (250-500 g), Golden Syrian hamsters (125 g) and C57BL/6N, B A L B / c , DBA and C3HeN mice (20 g) were obtained from Charles River Breeding Laboratories (Wilmington, DE); male SWR, AKR and B 6 / D 2 mice (20 g) from the Jackson Laboratories (Bar Harbor, ME); male Hartley guinea

pigs (250-400 g) were from the Michigan State Department of Health (Lansing, MI); and male New Zealand White rabbits (2 kg) were from Baileys (Alto, MI). Ovine and bovine hepatic tissue samples were obtained from the Michigan State University Meat Science Laboratory (East Lansing, MI); rainbow trout (200 g) from Dr. J. Giesy (Department Fisheries and Wildlife, Michigan State University) and white leghorn chickens (100 g) from Michigan State University Laboratory Animal Care Services (East Lansing, MI). Laboratory animals were exposed to 12 h of light and 12 h of dark daily and were allowed free access to food and water.

2.3. Cell culture The human intestinal cell line, LS180, was obtained from the American Type Tissue Culture Collection (Rockville, MD) and maintained as described by the supplier.

2.4. Preparation of cytosol Cytosol was prepared in H E D G buffer (25 mM HEPES, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol and 10% (v/v) glycerol) from the liver as described by Denison et al. (1986b) and from LS180 cells as described by Harper et al. (1991). Sample aliquots were stored at - 80°C until use. Protein concentrations were determined by the method of Bradford (1976) using bovine serum albumin as the standard.

2.5. AhR ligand-binding assay Specific binding of [3H]TCDD to cytosol was measured using the hydroxylapatite adsorption assay as described previously (Denison et al., 1986a). Aliquots (0.5 ml) of cytosol (2 m g / m l ) were incubated with 2 nM [3H]TCDD in the absence or presence of the competitor T C D B F (200 riM). Following incubation for 2 h at 20°C, 0.2 ml aliquots of the incubation mixture was added to tubes containing hydroxylapatite (125 mg hydroxylapatite in 250/~1 of H E D G buffer). After a 3(t rain incubation, with gentle vortexing every 10 rain, hydroxylapatite pellets were washed three times with 1 ml of H E D G buffer containing 0.05% (v/v) Tween 80 to remove unbound ligand. After the last wash, 1 ml of scintillation fluid was added to each tube, the pellets resuspended and the mixture transferred to a 7 ml scintillation vial containing 4 ml of scintillation fluid. The incubation tube was rinsed with 1 ml of 95% ethanol and this rinse was also added to the scintillation vial. Radioactivity was quantitated in a Packard Tricarb liquid scintillation counter. Specific binding of [3H]TCDD to the AhR was computed by subtracting the amount of [3H]TCDD bound in the presence of

87

TCDBF from the amount of [3H]TCDD bound in the absence of competitor.

TCDD

+

2.6. Oligonucleotides A complementary pair of synthetic oligonucleotides containing the sequence 5'-GATCTGGCTCTTCTCACGCAACTCCG-3' and 5'-GATCCGGAGTTGCGTGAGAAGAGCCA-3' (corresponding to the wild type AhR binding site of DRE3 (Denison et al., 1988a) and designated as the DRE oligonucleotide) or complimentary pairs of DRE oligonucleotides containing single nucleotide substitutions were synthesized, purified, annealed and radiolabeled with 7-32p-ATP as previously described (Denison et al., 1988b). Annealing generates a double-stranded DNA fragment with a BamHI cohesive end at its 3' terminus and a BglII cohesive end at its 5' terminus, relative to its normal orientation with respect to the CYPIA1 gene promoter (Jones et al., 1986).

2. 7. Gel retardation analysis Cytosol (16 mg protein/ml for tissue and 2-4 mg protein/ml for cell extracts) was incubated with DMSO (20/xl/ml), 20 nM TCDD or in the ligand-dependent experiments with 100 nM 3-methylcholanthrene, benzanthracene or/~-naphthoflavone, in DMSO, for 2 h at 20°C (Denison and Yao, 1991). An aliquot of the incubation mixture (5 /xl) was mixed with 16 #I of HEDGK (HEDG buffer containing 0.15 M KC1) containing poly dI -dC (225 ng for guinea pig cytosol and 113 ng for all other species examined) and incubated for 15 rain at room temperature. The 32p-labeled oligonucleotide (100,000 cpm, 0.8-1.4 ng, in 4 #1 of HEDG buffer) was added and the incubation continued for an additional 15 rain at room temperature. The final buffer conditions in the binding reaction were 25 mM HEPES, pH 7.5, 1.0 mM EDTA, 1 mM DTT, 10% (v/v) glycerol and 96 mM KC1. Protein-DNA complexes were analyzed by polyacrylamide gel electrophoresis and autoradiography as previously described (Denison et al., 1988b; Denison and Yao, 1991).

3. Results and discussion

3.1. Species differences in TCDD-inducible protein-DNA complex formation Incubation of rat hepatic cytosol with 32p-labeled DRE oligonucleotide resulted in the formation of two distinct protein-DNA complexes (fig. 1), one of which was TCDD-inducible and the other constitutive. We have previously shown (Denison and Yao, 1991) that the TCDD-inducible protein-DNA complex represents

Fig. 1. Interaction between the liganded cytosolic AhR and DNA. Rat hepatic cytosol was incubated with DMSO (lane 1) or 20 nM TCDD (lane 2) for 2 h at 20°C, followed by addition of 32p-labeled D R E oligonucleotide as described under materials and methods section. Specific protein-DNA complex formation was determined by gel retardation analysis. The arrow indicates the position of the TCDD-inducible protein-DNA complex.

the binding of transformed TCDD-AhR complexes to double-stranded 32P-labeled DRE, whereas the constitutive protein-DNA complex appears to represent the binding of (an) unknown protein(s) to radiolabeled single-stranded DRE oligomer present in the reannealed oligonucleotide preparation. Additionally, the binding of transformed rat hepatic cytosolic TCDD:AhR complex to the DRE-containing oligonucleotide occurs specifically and with high affinity (Denison and Yao, i991). To assess the ability of TCDD:AhR complexes from different species to bind to the DRE oligonucleotide we carried out gel retardation analysis, as described above, using cytosol from various species. Incubation of TCDD-treated cytosol from rat, rabbit, guinea pig, hamster, mouse, sheep, cow and chicken liver, as well as cytosol from the human cell line LS180, with 32p_ labeled DRE oligonucleotide results in formation of two protein-DRE complexes comparable similar to those observed above (fig. 2). Similarly, a recent study by Gasiewicz and coworkers (1991) has also demonstrated a TCDD-inducible protein-DNA complex using hepatic cytosol from rat, guinea pig and hamster. In contrast, no TCDD-inducible complex was observed using hepatic cytosol from rainbow trout (fig. 2) or from blue gill sunfish, chinook salmon or yellow perch (data not shown). Comparison of the relative amount

88 TABLE 1

to contain significantly more AhR (Lorenson and Okey, 1991), can bind to the D R E oliognucleotide. The small amount of inducible protein-DNA complex formed using C57 mouse hepatic cytosol, which contains AhR levels comparable to most other species (table 1), is most likely due to the fact that C 5 7 B L / 6 mouse AhR appears to transform/dissociate much more slowly in vitro than AhR from other species (Denison et ah, 1988b; Henry et al., 1989; Denison and Vella, 1991). Ligand binding experiments (table 1) indicate that although relatively high levels of [~H]TCDD specific binding is observed in hamster hepatic cytosol, relatively low levels of inducible complex formation arc observed (fig. 2). These results may be due to differences in the hamster AhR itself (when compared to other species) a n d / o r the presence or absence of tt t r a n s f o r m a t i o n / D N A binding modulatory factor in hamster cytosoh We have also not observed any significant difference in DRE-binding of transformed T C D D : A h R complex from cytosol prepared male and female rats and rabbits (data not shown). In addition, the lack of any apparent age-dependent alteration in TCDD-induciblc protein-DRE complex formation (data not shown), combined with the fact that the animals utilized in these studies were all relatively young, would indicate that the apparent species differences we have observed are not simply due to differences in age between the animals. Species variation in the relative migration of the TCDD-inducible protein-DNA complex were also

Specific binding of 3H-TCDD to hepatic cytosol from various species. Species

[3H]TCDD specific binding ( f m o l / m g protein ;')

Relative DRE binding i~

Sprague-Dawley rat New Zealand White rabbit Hartley guinea pig Hamster Sheep Cow C57mouse RainbowTrout Human LS18(1 cells Chicken

51.0 + 2.[/ 75.1 +_ 3.5 43.2 + 4.5 50.4+ 5.4 42.1+_ 10.0 35.9+ 4.4 41.2_+ 1.7 5.1 + 2.6 260.0_+ 3.0 41.3+ 2.3

+ + + + + + +

+ + + + + + + + + + ++

+ + + +

'~ Values are expressed as the mean + S.D. of duplicates of at least four determinations, b The symbols represent the relative amount of TCDD-inducible complex formed (see fig. 21, ranging from no inducible complex ( ) to relatively high amounts of inducible complex

(+++).

of TCDD-inducible protein-DNA complex formed to the concentration of AhR ([3H]TCDD specific binding) present in the same cytosolic preparation, as determined by hydroxylapatite-binding, is presented in table 1. The absence of a TCDD-inducible protein-DNA complex using rainbow trout may be due to the low levels of AhR in this cytosolic sample a n d / o r instability of fish AhR. We are currently examining whether fish cytosolic/nuclear T C D D : A h R complexes, isolated from a rainbow trout hepatoma cell line recently shown

/ TCDD

+

-

+

+

+

-

+

-

+

+

-

+

+

-

+

Fig. 2. TCDD-inducible protein-DNA complex formation using TCDD-treated cytosol from various species. Cytosol prepared from liver of the indicated species or from human LS180 cells was incubated in the absence ( - TCDD) or presence ( + TCDD) of 20 nM TCDD and protein-DNA complexes were resolved by gel retardation.

89

readily apparent (fig. 2) and may be due to previously reported differences in the molecular weight of the A h R (Poland and Glover, 1988) a n d / o r the presence of additional proteins in the inducible protein-DNA complex (Elferink et al., 1990; Gasiewicz et al., 1991). In addition, preliminary studies have indicated that the relatively broad TCDD-inducible band observed with some species (i.e., guinea pig and rabbit) appears to result from the presence of multiple inducible proteinD N A complexes contained within this band (data not shown). We are currently attempting to characterize these multiple TCDD-inducible p r o t e i n - D R E complexes in greater detail and to determine whether multiple D R E binding proteins are present in other species as well. Two classes of inbred mouse strains have been described which differ in their response to T C D D and related aromatic hydrocarbons (Poland et al., 1976). 'Responsive' inbred mice (typified by the C 5 7 B L / 6 strain) respond to T C D D and polycyclic aromatic hydrocarbons, such as 3-methylcholanthrene, with the induction of cytochrome P450IA1, while in 'nonresponsive' mice (typified by the D B A / 2 strain) P450IA1 is not induced by 3-methylcholanthrene but i s i n d u c e d by T C D D (although a ten-fold greater concentration of T C D D is required for a comparable response in the latter strains). Hepatic cytosol from C 5 7 B L / 6 mice contains A h R which exhibits high affinity T C D D binding, while A h R from D B A / 2 mice binds T C D D with a ten-fold lower affinity (Poland et al., 1976; Okey et al., 1989). Gel retardation analysis of the ability of hepatic cytosol from various responsive and nonresponsive inbred mouse strains to form TCDD-inducible protein-

mDRE2

A GIC TIAIG e

mDRE3 mDRE4 mDRE5 rXREI rXRE2

G A A G T

DRE CONSENSUS Fig.

4.

AIG CIG GIC AIG CIC

T[T[G TIGIG TICIG TITIG TIAIG

C C C C C

T G G G G G G

T T T T T

G O G G G

o A T A A A

A T A A G

G G G G C

G N N N C T N G C G T G N G A N N N C C G C T G

Nucleotide

sequence

alignment

of

DREs

identified

in

the

5'-flanking region of the mouse (Denison et al., 1988) and rat (Fujisawa-Sehara et al., 1987) CYPIA1 gene and the derived DRE consensus sequence. D N A complexes is shown in fig. 3. Inducible complex formation was observed with all strains examined, although the amount of complex in nonresponsive D B A and SWR strains was barely detectable. Interestingly, the amount of complex formation using 'nonresponsive' A K R hepatic cytosol was comparable to that obtained with the responsive C57 and B 6 / D 2 strains. The above results demonstrate that the degree of A h R transformation and D N A binding can vary widely between inbred mouse strains and this apparent difference in complex formation is not simply due to differences in A h R concentration, since significant differences in complex formation was observed in strains which contain relatively similar receptor levels (compare C57 and C3H mice (table 2)). The above results demonstrate that cytosolic A h R from a variety of different species can be transformed in vitro, in a ligand-dependent manner, to a form(s) which recognizes and binds to a DRE-containing oligonucleotide indicating that all of the 'factors' nec-

/ +

TCDD

•

+

;

-

+ -

+

+

-

+

-

+

-

b

Fig. 3. TCDD-inducible protein-DNA complex formation using TCDD-treated cytosol from various inbred strains of mice. Cytosol prepared from liver of the indicated mouse species was incubated in the absence ( - TCDD) or presence (+ TCDD) of 20 nM TCDD and protein-DNA complexes were resolved by gel retardation.

91)

3.2. Effect of DRE mutagenesis on TCDD-inducible protein-DNA complex formation

TABLE 2 Specific binding of [3H]TCDD to hepatic cytosol from various species. Species

[3H]TCDD specific binding ( f m o l / m g protein ")

Relative DRE binding t,

Responsive C57BE/6N C3HeN BALB/c B6D2 F1 backcross

41.3 + 1.5 77.2 + 1.8 50.8+_3.7 47.6+9.2

+ + + + + + + +

Nonresponsive DBA AKR SWR

5.6 + 1.9 12.6_+ 1.8 21/.3 + 4.6

+/ + +/ -

Previous studies (Denison et al., 1989; Elferink et al., 1990; Denison and Yao, 1991) in addition to those described above have demonstrated the specificity of the D N A binding of transformed mouse and rat T C D D : A h R complexes, however, a detailed examination of species differences in D N A binding has not been performed. Alignment of known rat and mouse D R E s (fig. 4) has allowed us to derive a putative D R E consensus sequence ( G / C N N N G / C T N G C G T G N G / C T / A N N N G / C ) from which we can carry out experiments designed to examine nucleotide-specificity of T C D D : A h R : D R E complex formation. The D R E consensus sequence contains an invariant core sequence of T N G C G T G and several variable nucleotides flanking the 'core'. Several of the nucleotides contained within the consensus D R E core motif ( T N G C G T G ) have been shown by methylation interference experiments (Shen and Whitlock, 1989; Saatcioglu et al., 1990) to be in close contact with the A h R complex and are thus critical for complex formation (specific critical bases are indicated in bold type). Initially, we utilized a mutant D R E oligonucleotide which contains a single C to A substitution in one of these critical bases in the core motif ( G C G T G to G A G T G ) in order to examine the importance of the 'core' motif in species specificity for binding of A h R to the DRE. We have previously observed that this single nucleotide substitution can

~' Values are expressed as the m e a n +S.D. of duplicates of at least four determinations, b The symbols represent the relative amount of TCDD-inducible complex formed (see fig. 3), ranging from barely detectable complex ( + / - ) to relatively high amounts of inducible complex ( + + + ).

essary for transformation and D N A binding must be present in the cytosol of these species. In addition, our data indicates a lack of correlation between measurable A h R concentrations ([3H]TCDD specific binding) and the degree of A h R t r a n s f o r m a t i o n / D N A binding and demonstrate the lack of utility of gel retardation analysis as a predictor of T C D D (or H A H ) responsiveness. These results do indicate, however, that transformed A h R from a variety of species can recognize and bind to the same D N A regulatory element.

°+ TCDD

4.

-

+

4-

4-

+°+°° I 4-

-

4-

4-

-

4-

Fig. 5. Binding of cytosolic T C D D : A h R complex from various species to a m u t a n t D R E oligonucleotide. Cytosol from the indicated species was incubated in the absence ( - T C D D ) or presence ( + T C D D ) of 20 nM T C D D followed by the addition of 32P-labeled mutant D R E oligonucleotide. Specific protein-DNA complex formation was resolved by gel retardation analysis.

91 TABLE 3 DRE substitution mutant oligonucleotides used in direct binding and competitive binding experiments. DRE nucleotide position Mutant oligo W T

1

1

2

3

4

5

6

7

8

9

l0

11

12

13

14

15

16

17

18

19

C

G

G

A

G

T

T

G

C

G

T

G

A

G

A

A

G

A

G

a

Tb

2 3 4 5 6 7 8 9 l0 11

A G A

T G T C

Wild-type (WT) DRE oligonucleotide containmg no nucleotide substitution, b Indicates the specific nucleotide substitution of the wild-type DRE.

d e c r e a s e the D R E b i n d i n g affinity of t r a n s f o r m e d g u i n e a pig T C D D : A h R c o m p l e x by a b o u t 2000-fold ( Y a o a n d D e n i s o n , u n p u b l i s h e d observations). Similarly, P o e l l i n g e r a n d c o w o r k e r s (Cuthill et al., 1991) d e m o n s t r a t e d that s u b s t i t u t i o n of this n u c l e o t i d e elimin a t e d D R E b i n d i n g of t r a n s f o r m e d rat T C D D : A h R complex. W h e n gel r e t a r d a t i o n analysis was c o n d u c t e d using the 32p-labeled m u t a n t D R E o l i g o m e r as a p r o b e , no T C D D - i n d u c i b l e p r o t e i n - D N A c o m p l e x was ob-

Oligo

#

1

DRE O l i g o m e r

C

Oligo

TCDD

G

WT -

+

G

1 +

A

2 +

2

3

G

T

3 +

served using h e p a t i c cytosol from several species (fig. 5), i n d i c a t i n g the i m p o r t a n c e of this c o n s e r v e d base in TCDD:AhR:DRE complex formation. Although DNA b i n d i n g of t r a n s f o r m e d T C D D : A h R c o m p l e x from a variety of species was similarly e l i m i n a t e d by the above single n u c l e o t i d e s u b s t i t u t i o n within the D R E ' c o r e ' motif, it is not c l e a r w h e t h e r s u b s t i t u t i o n s of o t h e r c o n s e r v e d D R E n u c l e o t i d e s will also affect D N A binding o f A h R from t h e s e species similarly.

T

4 +

4

5

6

7

8

-

9

10

-

-

G

C

G

T

G

A

G

A

A

G

5 +

6 +

7 +

8 +

9 +

i0 +

11 A

G

11 +

Fig. 6. Nucleotide-specific binding of transformed guinea pig hepatic cytosolic TCDD:AhR complex. Guinea pig cytosol was incubated in the absence ( - TCDD) or presence (+ TCDD) of 20 nM TCDD and 32pqabeled wild-type or mutant DRE oligonucleotide as described in Materials and methods and specific protein-DNA complexes resolved by gel retardation. The specific mutant oligomers are numbered as indicated in table 3.

92 OLIGO

To address whether species-specific differences in D R E binding of AhR exist, we examined the ability of transformed AhR from various species to bind to a series of mutant D R E oligonucleotides which contain single nucleotide substitutions in the D R E consensus. Initially, to test the ability and extent to which the T C D D : A h R complex recognizes and binds to these mutant DREs, double-stranded wild type and mutant D R E oligonucleotides (table 3) were radiolabeled with 32p and the ability of transformed guinea pig T C D D : A h R complex to bind to these oligomers directly analyzed by gel retardation (fig. 6) No TCDD-inducible complex was formed when certain of the 'core' consensus bases were substituted (specifically the bases C G T G at position 9, 10, 11, 12 (table 3)). Substitutions of several of the variably conserved flanking nucleotides (positions 8 and 15) resulted in a modest decrease in complex formation, while others (positions 1, 5, 6 and 19) had no apparent effect on complex formation (fig. 6). Thus, based on our mutagenesis experiments, we have deduced an optimal T C D D : A h R DNA binding consensus sequence of G C G T G N N T / A N N N G / C for guinea pig hepatic AhR. To examine whether DRE-binding of transformed T C D D : A h R complex from several other species exhibits the same nucleotide specificity, we examined the ability of TCDD-treated cytosol from rat, C3HeN mouse and hamster liver and cytosol from human LSI80 cells to bind to radiolabeled wild-type and mutant D R E oliogonucleotides (fig. 7). No species-specific differences in nucleotide specificity of D R E binding of transformed T C D D : A h R complex were readily apparent. These results indicate the critical nature of the core consensus sequence for T C D D : A h R : D R E complex formation and the apparent lack of importance (at least in DNA binding) of the conserved nucleotides 5'-ward of the core. This is supported by the observation that the binding of transformed T C D D : A h R complex to a D R E oligonucleotide in which all three of the 5' conserved nucleotides (positions 1, 5 and 6) had been substituted was not significantly different from that of the wild-type D R E oligomer (Yao and Denison, unpublished observation). Although our results indicate that the primary interaction of transformed T C D D : A h R complex with the D R E occurs specifically with the C G T G sequence of the 'core' motif and are consistent with previous methylation interference studies (Shen and Whitlock, 1989; Saatcioglu et al., 1990), in that methylation of the core nucleotides blocked TCDD-inducible complex formation. We have previously observed, however, that nucleotides outside of the core motif are also required for D R E enhancer activity (Denison et al., 1988b). We are currently examining the effect of these mutations on transcriptional enhancer activity and expect that decreased AhR DNA binding will coincide with decreased enhancer activity,

TCDD

wt

wt

1

2

3

4

5

6

7

8

9

10

11

+

+

+

+

+

+

+

+

+

+

+

+

RAT

MOUSE

HAMSTER

RABBIT

H ~

Fig. 7. Nucleotide-specific binding of transformed cytosolic T C D D : A h R complex from a variety of species, ttepatic cytosol from the indicated species or cytosol from human LS180 cells was incubated in the absence ( - T C D D ) o r presence ( + T C D D ) o f 2{) nM T C D D and 3zP-labeled wild-type or mutant D R E oligonucleotide as described in Materials and methods and specific protein-DNA complexus resolved by gel retardation. The specific mutant oligomcrs are numbered as indicated in table 3.

as has been observed with other transcriptional factors (Glass et al., 1988; Schule et al., 1990). Thus, the n u c l e o t i d e - s p e c i f i c i n t e r a c t i o n of t r a n s f o r m e d T C D D : A h R complex with the D R E is highly conserved between species and is suggestive of a functional role for this sequence in T C D D responsiveness of these species.

3.3. Effect of t~arious AhR ligands on nucleotide-specific DRE-binding of guinea pig hepatic AhR Differences in the biological potency of various AhR ligands has been attributed to differences in their relative AhR binding affinity a n d / o r ability to be metabolically degraded (Poland et al., 1976). It is also possible that different AhR ligands could induce an alteration(s) in the AhR which results in ligand-dependent differences in nucleotide-specific DRE-binding and an alteration in D R E transcriptional enhancer activity. To

93 Oligo

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TCDD

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3.4. General conclusions

. +

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Fig. 8. Effect of various AhR ligands on nucleotide-specific binding of transformed guinea pig hepatic cytosol. Guinea pig hepatic cytosol was incubated in the absence ( - ) or presence ( + ) of 20 nM TCDD or 100 nM 3-methylcholanthrene (MC), benzanthracene (BA), /3naphthoflavone (BNF) or benzo(a)pyrene for 2 h at 20°C. After the incubation period -~2p-labeled wild-type or mutant DRE oligonucleotide was added to the mixture and specific protein-DNA complexes resolved by gel retardation analysis. The specific mutant oligomers are numbered as indicated in table 3.

e x a m i n e w h e t h e r different A h R ligands alter the nucleotide-specific i n t e r a c t i o n of t r a n s f o r m e d A h R with the D R E we e x a m i n e d the effect of various A h R ligands (TCDD, 3-methylcholanthrene, benzant h r a c e n e , b e n z o ( a ) p y r e n e a n d /3-naphthoflavone) on i n d u c i b l e p r o t e i n - D N A complex formation. I n c u b a t i o n of these ligands with g u i n e a pig hepatic cytosol a n d s u b s e q u e n t gel r e t a r d a t i o n analysis using r a d i o l a b e l e d wild-type a n d m u t a n t D R E o l i g o n u c l e o t i d e s is shown in fig. 8. N o l i g a n d - d e p e n d e n t differences in nucleotide-specific D R E b i n d i n g were observed in these e x p e r i m e n t s implying that the D N A - b i n d i n g d o m a i n of the t r a n s f o r m e d A h R was not significantly affected or altered by any of the A h R ligands tested. O u r data also suggest that the biological p o t e n c y of a given A h R ligand does not the result from differences in the specific i n t e r a c t i o n of the [iganded A h R with D N A but m o r e likely that of o t h e r factors, such as differences in individual ligand p h a r m a c o k i n e t i c a n d p h a r m a c o d y n a m i c p r o p e r t i e s a n d distribution, affinity for the A h R a n d / o r differences in their rate of m e t a b o l i c d e g r a d a tion.

We have d e m o n s t r a t e d that cytosolic A h R from a variety of species can be t r a n s f o r m e d in vitro, in a l i g a n d - d e p e n d e n t m a n n e r , to a form which can b i n d to a D R E - c o n t a i n i n g oligonucleotide. T h e s e results indicate that all of the factors necessary for A h R transform a t i o n a n d D N A b i n d i n g are c o n t a i n e d within the cytosol of these species. Gel r e t a r d a t i o n analysis using a series of m u t a n t D R E oligonucleotides has shown that the primary i n t e r a c t i o n of the T C D D : A h R complex with a m u r i n e D R E occurs primarily with the n u c l e o t i d e s of the core c o n s e n s u s motif, since single s u b s t i t u t i o n of any of these bases resulted in a dramatic decrease in inducible p r o t e i n - D N A complex formation. No a p p a r e n t species- or l i g a n d - d e p e n d e n t , nucleotide-specific differences in T C D D - i n d u c i b l e A h R b i n d i n g to the D R E were observed, s u p p o r t i n g a highly conserved n a t u r e of the A h R (at least in D N A b i n d i n g ) a m o n g various species. O u r results suggest that D N A b i n d i n g of t r a n s f o r m e d A h R requires a highly conserved D R E s e q u e n c e a m o n g species and f u r t h e r support the role of the D R E as a u b i q u i t o u s s e q u e n c e i m p o r t a n t in c o n f e r r i n g T C D D responsiveness in multiple species.

Acknowledgements We thank Dr. S. Safe for [3H]TCDD, unlabeled TCDD and TCDBF and Drs. D. Dewitt, W. Helferich and M. EIFouly for helpful comments and critical review of this manuscript. Acknowledgment is made to the American Cancer Society (grant #CN-4), the International Life Sciences Institute and the Michigan Agriculture Experiment Station for its support of this research. Some of these data were presented in preliminary form at the Banbury Conference on The Biological Basis for Risk Assessment of Dioxins and Related Compounds (Cold Spring Harbor, NY, October 199(/).

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