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0031-6997/87/i1902-0 t 47S02-00/0 PHARMACOLOGICAL REVIEWS Copyright,s 1987 by The American 8ociety for Pharmacologv and Experimental Therapeutics The Regulation of Gene Expression by 2, 3, p7,'8- Tetrachlorodibenzo-p-dioxin* JAMES P. WHITLOCK, JR. Department of Pharmacology, Stanford University School of Medicine, Stanford, California 94305 Vol. 39, tio. 2 Printed in (-.S.A, I. Background ......................................................................... 147 II. .IE6dence for a TCDD receptor ......................................................... 148 III. Bi,xhemical properties of the TCDD receptor ............................................ 150 IV. Function ofthe TCDD receptor ........................................................ 152 V. 7.'(:DD-responsive genomic elements .................................. . . . . . . . . : . . . , ..... 154 VI.)t'uture prospects .......................... . ........................................ 156 I. Background THE saIctop®a,McAL-climate in the U. S. during the 1960s helped draw attention to the chemical that we have come to know as "dioxin." In the first place, cognizance of the potential risks associated with environmental con- tamination was on the increase; for example, in 1962 the publication of Silent Spring by Rachel Carson (17) gen- erated particular concern about the increasing use of pesticides and herbicides. At the same time, there was growing r estiveness about the conduct of the Vietnam war; one particular tactic, chemical defoliation of the countryside (Operation Ranch Hand), again focused at- tention on the possible adverse effects of the herbicides used to kill crops and vegetation in Southeast Asia. One particular herbicide (Agent Orange) used in Vietnam was a 1:1 mixture of (the n-butyl esters 'of) 2,4-dichlorophen- oxyacetic acid (2,4-D)t and 2,4,5-trichlorophenosyacetic acid (2,4,5-'1:'); both, compounds were also widely used as weed killers in the U. S. In 1970, an article in the New Yorker by ']:`homas Whiteside. (172) publicized the sus- picion thait 2,4,5-T might,cause birth defects. Against this background, the report (24) that 2,4,5-T was teratogenic in rodents understandably aroused con- siderable concern among the public, environmental groups, the chemical industry, U. S. regulatory agencies, and Congre:ss and led to restrictions on the use of the ' The work in my laboratory has been supported by research grants from the American Cancer Society and the NIH. t Abbreviations used are: AHH, aryl hydrocarbon hydrozylase; b- ALAS, d-aminelevulinic'acid synthetase; HAH, halogenated aromatic hydrocarbon; PAH, polycyclic aromatic hydrocarbon; TCDD, 2,3,7,8- tetrachlorodibenzo-p-diozin; 2,4-D, 2,4-dichlorophenoxyacetic acid; 2.4,5-T, 2,4,5-r.richlorophenozyacetic acid; 3MC, 3-methylcholan- threne; l3NF, a-naphthoflavone; CAT, chloramphenicol acetyltransfer- ase; DRE, dioxLn-responsive element; GRE, glucocorticoid-responsive element; HMG, high-mobility group; LD,), median lethal dose; ED50, median effective dose; BP, benzo(a)pyrene; QSAR, quantitative struc- ture-activity relationship; DBBD, 2,2-dimethvl-5-t-butvl-1,3-benzo- dioxole. - herbicide. The results of subsequent studies (20, 23, 160) implied that the actual teratogen was probably 2,3,7,8- tetrachlorodibenzo p-diozin (TCDD), a contaminant that forms during the commercial synthesis of 2,4,5-T (fig. 1). Public and scientific attention then shifted from 2,4,5-T to TCDD (often described simply as "dioxin") and its potential risk to human health. The remarkable potency of TCDD (in its acute lethality for guinea pigs), combined with'the relative resistance of TCDD to chem- ical and biological degradation, contributed to the fear that soon was associated with the dioxin. Several indus- trial accidents, episodes of leakage or improper disposal of chemical waste, and lawsuits brought by veterans who might have been exposed to Agent Orange have tended to keep TCDD in the public eye ever since. Despite the scientific and lay scrutiny that dioxin has received, it has been difficult to document that TCDD poses a major health hazard for humans. Studies in animals reveal marked quantitative differences in their sensitivity to TCDD; for example, the acute oral median lethal dose (LDm) of TCDD is about 5000-fold higher for the ham- ster than for the guinea pig. In addition, the qualitative spectrum of effects produced by chronic exposure to TCDD varies substantially among animal species (140). These observations make it unusually difficult to extrap- olate the results of animal studies to man. Long-term follow-up of individuals exposed to TCDD in an indus- trial setting does not implicate the dioxin as a cause of excess mortality or serious morbidity for humans (110, 161, 180). However, the number of individuals followed has been relatively small. Its teratogenic effects in rodents stimulated scientific interest in TCDD and related chlorinated hydrocarbons. By the early 1960s, TCDD had been implicated in the etiology of chloracne in humans- (6, 90, 91) and chick edema disease (69), but its other effects and its mecha- nism of action were unknown. Today, we know that 147

148 a TCB a NaOH -------- to wHITLOCK a oNa ~la aCH2C00Na -~ /o~cr+2cQONa ~ i- 1a , TCBO i a 2,4,5 - T 0 a O ):~D a 0 2,3,7,8 - TCDD FiG. 1. :Farmation of 2,3,7,8-tetrachlorodibenz.o p-dioiin (2,3,7,8- TCDD) during the synthesis of 2,4,5-trichlorophenoryacetic acid (2,4,5-?'). The first step in the industrial production of 2,4,5-T involves the alkaline laydrolysin of 1,2,4,5-tetrachlorobenzene (TCB) to form sodium 2,4,li-trichlorob'enzeneozide (TCBO). In the second step, TCBO reacts with ctdoroethanoate to form 2,4,5-T. If the temperature of the fust step er.eceds about 160'C, two molecules of TCBO can react in a double nuclecphilic displacement to form 2,3,7,8-TCDD. Higher tem- perature and higher pH increase the formation of 2,3,7,8-TCDD. The side reaction is itself ezothermic, possibly leading to even higher temperatums and uncontrolled reaction conditions (1Q8, 143). TCDD eidcits a broad spectrum of biological effects, which vary according to the system in which the com- pound is tcsted. For example, in addition to its terato- genic effeci;s, TCDD also produces several species- and tissue-dependent changes in epithelial tissues, immuno- logical alterations, a wasting syndrome, tumor promo- tion, and the induction of several enzyme activities (139, 140). Therefore, models which seek to explain the mech- anism of TCDD action must account for the diversity of effects that the compound produces. One reasonable hypothesis is that TCDD, acting by means of an intra- cellular receptor protein(s), alters the expression of a different set of genes in each TCDD-responsive cell type (54,140). This particular model for TCDD action resem- bles that described for several steroid hormones, which also elicit diverse effects in receptor-dependent and tis- sue-specific fashion (146, 178). The evidence for and the molecular aspects of this model constitute the subject of this review. The development of TCDD-responsive cell culture sys- tems, combined with the use of recombinant DNA and gene transfer methods, has facilitated the analysis of TCDD action at the molecular level. We now know that TCDD can activate the rate of transcription of a gene that encodE;s a specific cytochrome P-450 isozyme (see below). In addition, exposure to TCDD produces phe- notypic changes suggestive of altered differentiation in epidermal cells in culture (53, 75, 93, 122, 145) and in cultured thirmic epithelium (25, 52). Furthermore, TCDD promotes the expression of a transformed phenotype in C3H10TI/s cells (1). Although the mechanism(s) by which TCDD produces these altered phenotypes is not yet known, it seems quite likely that changes in the expres- sion of specific genes are involved. We also know that othe,- halogenated aromatic hydrocarbons (HAHs) that are related structurally to TCDD (e.g., dibenzo p-dioains, dibenzofurans, biphenyls, biphenylenes, naphthalenes, and azoxybenzenes) produce similar patterns of toxicity, although the compounds differ greatly in potency. There- fore, we assume that these HAHs share a common mech- anism of action. Because it is the most potent, TCDD is the prototype, and it has been studied much more inten- sively than the other HAHs. Early studies (15, 55, 56) revealed that TCDD induces hepatic, drug-metabolizing enzyme activities that are catalyzed by cytochrome P-450 isozymes. This class of microsomal hemoproteins oxygenates lipophilic sub- strates and contributes to many different bialogical proc- esses, ranging from steroid biosynthesis to chemical car- cinogenesis (44, 99, ] 68). At the time when TCDD was beginning to undergo intensive study, certain chemicals were already known to induce one (or more) of the various cytochrome P-450 isozymes. The effect of TCDD was similar to that of 3-methylcholanthrene (3MC), a polycyclic aromatic hydrocarbon (PAH) that preferen- tially induces a specific form of cytochrome P-450 (des- ignated cytochrome P-450c in the rat and cytochrome Pi-450 in the mouse) (15, 55, 56). This particular cyto- chrome P-450 isozyme catalyzes aryl hydrocarbon hy- drozylase (AHH) activity, which is present in many tissues and which is assayed using a simple and sensitive fluorescence technique (112). Therefore, measurement of AHH induction became a convenient way to determine if a particular tissue or cell type can respond to TCDD. (Note: the failure of TCDD to induce AHH activity in a particular cell type does not necessarily mean that the cell cannot exhibit some other response to the dioxin.) Given the long-established relationship between TCDD action and AHH induction, together with more recent achievements in the purification of cytochrome P-450 isozymes and the cloning of cytochrome P-450 genes (3, 173), it is not surprising that the most detailed knowledge of the mechanism of TCDD action has come from the study of TCDD-responsive cytochrome P-450 genes. We assume that TCDD influences the activity of other genes (i.e., those responsible for other phenotypic changes in- duced by the dioxin) by similar mechanisms. In retro- spect, it is interesting that what began as a toxicological evaluation of a potent environmental contaminant has had unanticipated benefits. Analyses of TCDD action at the cellular and molecular levels have revealed a pathway by which an extracellular chemical signal can be trans- duced to the cell nucleus to activate the transcription of a specific gene. Further study of this TCDD-responsive signalling system in the future has the potential to reveal novel aspects of the mechanisms that control mamma- lian gene expression. H. Evidence for a TCDD Receptor The unusual potency of TCDD was the first clue that the dioxin might act through a specific receptor(s). For

R.EGULATION OF GENE EXPRESSION BY TCDD example, :Poland and Glover observed that TCDD was orders of magnitude more powerful than other com- pounds in inducing both AHH activity and 8-aminolev- ulinic acid synthetase (b-ALAS) activity in the chick embryo (131, 132). In addition, studies of TCDD conge- ners reve:aled a relationship between dioxin structure and potency as an inducer (132). On the basis of these data, Poland and'Glover postulated that TCDD acts by means of am "induction receptor" to elevate AHH and S- ALAS activities. They also suggested that the hypothet- ical receptor might mediate other effects of TCDD, be- cause th c l,xtencies of the halogenated dibenzo p-diozins as enzyme inducers paralleled their toxic potencies. Sub- sequent studies of AHH induction in rat liver confirmed the potency of TCDD; the dioxin was 30,000•times more potent tlhrm the prototypical AHH inducer, 3MC (133). The foregoing results naturally led to the study of TCDD-inducible AHH activity in inbred strains of mice that were known to respond differentially to 3MC. Two groups had shown that, in certain mouse strains (typified by C57BL/6), 3MC induced hepatic AHH activity; how- ever, in other stra.ins (typified by DBA/2), it did not. In crosses l~*tween these strains, AHH inducibility segre- gated as :an autosomal dominant trait (133, 164). Fur- thermore, other responses to PAHs exhibited a similar segregation pattern (152). Therefore, the genetic locus that cor,.ferred these phenotypes was thought to be reg- ulatory amd was designated Ah (for aromatic hydrocar- bon responsiveness). Mouse strains in which 3MC in- duced hel3atic AHH activity were considered "respon- sive," a dcminant, trait governed by the Ahb allele. Mouse strains in which 3MC did not induce hepatic AHH activ- ity were considered "nonresponsive," a recessive trait governed by the Ahd allele (50). Against this background, it was noitable when Poland et al. reported that TCDD induced AHH activity to equally high levels in both C57BL/6 and DBA/2 mice (137). This observation in- dicated that the so-called nonresponsive DBA/2 strain could, in fact, exhibit a responsive phenotype if TCDD was the inducer instead of 3MC. This finding raised the possibili.ty that the DBA/2 strain might contain an al- tered regiilat®ry protein (i.e., receptor) to which 3MC bound pOorly and, therefore, failed to elicit a response. However, the properties of TCDD might be such that it could still, bind tightly enough to the altered receptor to induce AHH activity. The finding that the median effec- tive dose i(ED5o) for AHH induction by TCDD was about 20-fold higher in DBA/2 mice than in C57BL/6 mice was consistent with the idea that the DBA/2 strain contained a recepto e with a lower binding affinity for the inducer (134). Subsequently, Poland et al., using [3H]TCDD and a charcoal/dextran binding assay, identified in C57BL/ 6 hepatic cytosol a protein which bound the dioxin sat- urably and with high affinity, thus providing biochemical evidence for the eaistence of a TCDD receptor. DBA/2 hepatic c;rtosol did not contain a detectable protein that 149 bound TCDD with a similar high affinity. Furthermore, competition studies with TCDD congeners revealed that their binding affinities paralleled their induction poten- cies, suggesting a functional role for the receptor in the mechanism of AHH induction (136). These biochemical observations complemented the genetic evidence for the existence of a TCDD receptor. Because it is (presumably) encoded by the Ah locus, the TCDD receptor is also known as the Ah receptor. The high affinity of TCDD for the TCDD receptor contributes to the high potency of the dioxin. In addition, TCDD's resistance to degradation means that the bio- logical half-life of the compound is relatively long (9, 111, 123, 130). Thus, TCDD may produce sustained effects upon the cell, in comparison to those of other ligands for the receptor [e.g., 3MC or fl-naphthoflavone (#NF)], whose biological half-lives are much shorter because the compounds are readily metabolized. It is not yet clear whether TCDD's ability to produce a prolonged biological response contributes substantially to the tox- icity of the dioxin (54, 140). In addition, it is unclear why we have a receptor for TCDD at all. It is possible that the dioxin is only mimicking the binding of a "physiolog- ical" ligand to the receptor. However, the existence and properties of this hypothetical ligand (i.e., is it exogenous or endogenous; are its effects transitory or prolonged?) remain completely speculative (54, 140). Studies of cells in culture have provided additional details about the involvement of a receptor in the re- sponse to TCDD. Hankinson exploited the observation of Gelba~n et al. (45) that the PAH benzo(a)pyrene (BP) is toxic to cells that oxygenate the compound by means of the AHH system. Thus, he was able to select for AHH- defective cell6 by growth in the presence of BP (61). Miller and Whitlock took advantage of the fluorescence properties of BP and utilized the fluorescence-activated cell sorter to isolate cells that exhibit low (or no) AHH activity (107). Both groups identified two classes of re- ceptor-defective mouse hepatoma cells. In one class, rel- atively few (i.e., 5 to 10% of wild-type) TCDD-receptor complexes form; however, those complexes that do form interact normally with a component(s) of the cell nu- cleus. These variants respond poorly to TCDD, as meas- ured by AHH induction. In the other class, the formation of TCDD-receptor complexes appears normal. However, the complexes fail to interact normally at the nuclear level, and the variants fail to respond to TCDD at all. These results imply that AHH induction requires not only the formation of the TCDD-receptor complex but also a particular interaction between the complex and a component of the cell nucleus (97, 106). Cell fusion studies indicate that both variant phenotypes are reces- sive with respect to wild-type and that the variants belong to different complementation groups (62, 106). Thus, receptor function requires the contribution of (at least) two genes. The complementation analyses are open

150 WHITLOCK to several interpretations. One possibility is that the TCDI) receptor has distinct subunits; perhaps, one gene encodes a TCDD-binding subunit, and a second gene encodes a chromatin-binding subunit. Another possibil- ity is that the'TCDD-receptor complex requires enzy- matic modification to convert it to a chromatin-binding species; in this scenario, one gene encodes the receptor, and a second gene encodes the modifying enzyme. The available data do not allow us to distinguish between these and other possibilities. Progress in receptor puri- fication and characterization should allow the testing of these hypotheses in the future. Okey et al. (119) have analyzed the TCDD receptor in a clone of C3H/10T1/z mouse fibroblasts in which some PAHs (and TCDD) induce AHH activity, but 3MC does not. [3H:]3MC can bind to the receptor in cell extracts; however, it apparently is unable to do so in the intact cell. The basis for this interesting and unusual phenotype is unknown. More detailed studies of these cells (e.g., the dominant/recessive nature of the trait, struqture-activity analysen of ligand binding) have the potential to reveal novel aspects of receptor structure and function in the future. Genetic evidence for the Ah locus exists only in mice; phenotyges analogous to the responsive and nonrespon- sive mouse strains have not been observed in other species. However, other species, including humans, do contain a TCDD-binding protein(s) whose biochemical properties are similar to those of the mouse receptor (43, 68, 10D). Therefore, the equivalent of the Ah locus pre- sumably also exists in other species. In addition, crosses other than the prototypical C57BL/6 X DBA/2 mating imply that the mouse Ah regulatory system may be quite complicated. In some crosses (e.g., C3H/He X DBA/2), the induction of AHH, activity by 3MC segregates as a codominant trait; this fmding may indicate the existence of a th&d Ah allele (163). Furthermore, there is a single report that, in' the C57BL/6N X AKR/N mating, the domin.ance is reversed, and the nonresponsive phenotype segregates as an autosomal dominant trait (148). This unusual observation, if confirmed, remains to be ex- plained. Therefore, additional studies of these inbred mouse strains and their progeny seem worthwhile, in order to determine if the genetic findings are associated with d',ifferences in TCDD receptor structure or function. The chromosomal location, organization, and struc- ture of i;he Ah locus are unknown. Studies of somatic cell hybrids suggest that mouse chromosome 17 contains a gene that regulates AHH inducibility; however, there is no d'ue,rt evidence that it encodes the receptor protein (98). Furthermore, the number of alleles at the Ah locus and the number of proteins encoded by the locus are unknown. Success in cloning the gene(s) for the TCDD receptor presumably will allow -these issues to be ad- dressed in the future. III. Biochemical Properties of the TCDD Receptor Assays of the TCDD receptor require measuring the specific binding of a radiolabelled ligand to a protein that is a minor component of a crude cell extract. The major problem is distinguishing between specific and nonspe- cific binding. The limited aqueous solubility of TCDD tends to increase nonspecific binding and compounds the difficulty of the assay. The potential usefulness of more hydrophilic ligands, such as 3MC or,6NF, is negated by their substantially lower affmity for the receptor. Several investigators have utilized different techqiques to im- prove upon the original assay, which employed dextran- coated charcoal to remove unbound [3H]TCDD (136). Either adsorption of ligand-receptor complexes to hydroxylapatite (41, 127) or precipitation of ligand-re- ceptor complexes with protamine sulfate (26) is a con- venient, simple, and rapid method for assaying large numbers of samples. However, compared to more com- plicated techniques, these procedures tend to lack spec- ificity, because they do not reveal any properties of the molecules to which TCDD is bound. In contrast, assays which involve centrifugation of TCDD-labelled material through sucrose gradients (116, 166) can verify that the TCDD-binding species has the appropriate sedimenta- tion coefficient; however, such techniques are time-con- suming, expensive, and relatively impractical for large numbers of samples. Other assays, such as isoelectric focusing in polyacrylamide gels (16) and gel permeation chromatography (42), have similar limitations. In prac- tice, a combined approach seems reasonable. Impurities in the radiolabelled TCDD (28), contamination of the cell or tissue extract with serum proteins (129), and the presence of other PAH-binding proteins in the cell ex- tract (18, 65, 165, 181) can introduce substantial artifacts into studies of the TCDD receptor. These factors need to be considered when interpreting the experimental data. Several investigators (28, 43, 65, 101, 129) have com- pared the properties of the TCDD receptor from various animal species and/or tissues to find differences that might account for the diversity of TCDD's effects. In general, the results reveal that the hydrodynamic prop- erties and the ligand-binding properties of the TCDD receptor are similar, but not identical, in various systems. In solution, the receptor behaves as a larger species (apparent Mr~250,000) in 0.1 ht KCl and as a smaller species (apparent M,-120,000) at 0.4 M KC1. This be- havior may reflect the dissociation of an oligomeric spe- cies as the ionic strength is raised. If so, we do not yet know whether the receptor is homomeric or heteromeric. Both the faster and slower sedimenting species behave as asymmetric molecules, with axial ratios in the rangr of 11 to 12. Dissociation constants for TCDD fall in tht range of 0.1 to 2 nht, and, in liver tissue, the number of TCDD binding sites is in the range of 30 to 60 fmol/mg

REGULATION OF GENE EXPRESSION BY TCDD 151 protein. Hydrodynamic differences among various TCDD receptors appear to be relatively small. For ex- ample, Denison et al. (28) found about a 10% difference between Sprague-Dawley rats and C57BL/6N mice in the relative molecular mass of the hepatic TCDD recep- tor. In addition, the rat receptor readily changes to the smaller, more slowly sedimenting form in 0.4 M KCl whereas the mouse receptor is relatively resistant to this salt effect. Furthermore, the rat and mouse receptors differ sornewhat in their ligand-binding preferences. We do not know whether these biochemical variations are associated with meaningful differences in receptor func- tion. Therefore, based on our present knowledge, differ- ences in the properties of the TCDD receptor among animal s}?x,cies and/or tissues do not easily account for the qualit.-Aive and quantitative differences in TCDD's effects in various experimental systems. Several investigators (18, 73, 165, 181) have character- ized in rats and mi.ce another intracellular protein(s) that binds PAHfs (e.g., 3MC) with higher affinity than TCDD. The hycb-odynamic and ligand-binding properties of this protein distinguish it from the TCDD receptor (18, 74). In addition, the production of the protein does not seg- regate with the Ah locus in inbred mouse strains (118). The func:ti•on of this protein remains unknown; it might be involved in the regulation of the rat cytochrome P- 450c gene (74). However, this remains to be demon- strated rigorously. The recent purification of the mouse protein (19) should allow the preparation of antibodies, which will be helpful in studying its structure and regu- lation in greater detail. If this PAH-binding protein does, in fact, i.niluence gene expression, it will be interesting to compare its mechanism of action with that of the TCDD receptor. Both the hydrodynamic properties of the TCDD re- ceptor and its apparent mechanism of signal transduc- tion are analogous to those of'Several steroid receptors (146, 178). These similarities have led several investiga- tors to compare the properties of steroid and TCDD receptors in detail (178). The ligand-binding properties of the receptors are quite different; steroids do not exhibit high affinity f`or the TCDD receptor, and vice-versa. On the other hand, the TCDD receptor and the glucocorti- coid receptor are similar with respect to their chromato- graphic behavior on DNA-cellulose and heparin-Sephar- ose (175), Studies involving limited proteolysis reveal that, like steroid receptors, the TCDD receptor has a ligand-biriding domain that is distinct from a DNA- binding domain'~~! (64, 175). Molybdate stabilizes the higher molecular weight, ligand-binding form of steroid receptors; the compound has less effect on the TCDD receptor (29). The significance of this observation is not clear. Overall, the results of biochemical studies reveal some relatively crude structural similarities between the TCDD :receptor and steroid receptors. In addition, both the TCI)D receptor and steroid receptors transduce their respective chemical signals by mechanisms that appear similar at our relatively superficial level of knowledge (see below). These structural and functional similarities suggest that both types of receptor might belong to a family of proteins which evolved from a common ances- tor. On the other hand, variant cells that contain defec- tive TCDD receptors fall into several complementation groups, whereas, in the glucocorticoid-responsive system, the analogous receptor variants are all in the same com- plementation group (179). These genetic findings might reflect important structural or functional differences be- tween the TCDD receptor and steroid receptors and could mean that the biochemical similarities between them do not reflect their evolution from a common ancestor. Purification and characterization of the TCDD receptor in the future will permit a more meaningful comparison with steroid receptors and a more rigorous evaluation of their possible evolutionary relatedness. Several groups have characterized the ligand-binding site of the TCDD receptor using structure-activity anal- yses. Initial studies, involving several series of HAHs, revealed that the ligands with the highest binding affinity were essentially planar and would fit into a rectangle approximately 3 x 10 A, with halogen atoms at each corner (138, 140). However, this particular view of the binding site cannot easily account for the efficacy of ligands like 3MC or ONF, which are substantially differ- ent in structure from the HAHs. More recently, studies of a series of indoles (including ONF) suggested that viewing the binding site as a rectangle of 6.8 X 13.7 A could more easily account for all of the data (47). Quantitative structure-activity relationship (QSAR) methods (10) have also been used to study the interac- tions betweer; various ligands and the TCDD receptor. In this approach, one studies a series of structurally related ligands, whose physicochemical properties (e.g., hydrophobicity, electronegativity, hydrogen-bonding ca- pacity, van der Waals volume) can be estimated in quan- titative terms. Multiple linear regression analysis is used to determine which physicochemical property(s) corre- lates with the ligand's ability to produce the effect being studied (e.g., binding to the receptor). Safe and coworkers (150) found that the binding affinity of 33 chlorinated dibenzo-p-dioxins and dibenzofurans correlated with the hydrophobicity of the compounds (within limits imposed by the volume of the ligand). These observations imply that the ligand-binding site of the TCDD receptor is very hydrophobic (31, 32, 150). QSAR analysis of a series of halogenated biphenyls suggested that, for these ligands, hydrophobicity, electronegativity, and hydrogen bond- accepting ability all enhance ligand-receptor binding (5). One potential limitation of the QSAR approach is that the data may not be amenable to unambiguous interpre- tation. For example, McKinney and coworkers have in- terpreted the halogenated biphenyl binding data to mean that dispersive interactions are the primary forces that

152 WHITLOCK stabilize the ligand-receptor complex (102, 103). A sec- ond, and perhaps more serious, limitation of the QSAR approach ;involves the potential artifacts associated with the study of very insoluble ligands. For example, in some cases, tb e dissociation constant calculated for receptor binding substantially exceeds the aqueous solubility of the ligancL This ; raises questions as to the biological significa:nce of the binding data. In general, the QSAR analyses ,,uggest that the interactions which stabilize ligand-remptor binding are primarily hydrophobic, but can vary to some extent, depending upon the properties of the ligEmd. The ligand-binding site appears to be a hydrophot ic pocket of somewhat undefined volume. It is conceivable that the binding site is somewhat flexible; the receptor might undergo small changes in conforma- tiorn so as to optimize the binding interactions for any given ligand. To put the QSAR data in some perspective, it is wortla noting that a thermodynamic analysis of glucocorticoid-receptor interactions implies that the forces which stabilize the hormone-receptor complex in that system are also primarily hydrophobic (177). In the fu ture, the QSAR approach might provide clues about the Eunction of the TCDD-receptor complex. For example, Denomme et al. observed that, for £wo series of chlorinated dibenzo p-diozins and dibenzofurans, the re- ceptor binding affinity correlated with lipophilicity alone, whereas the ability to induce AHH activity cor- related with lipophilicity plus a steric factor (31, 32). These fmdLings suggest that the formation of a ligand- receptor complez ;does not by itself suffice to evoke a biological iresponse. Denomme et al. (31, 32) infer that the ligand-receptor complex must undergo a subsequent biochemicFa change(s) (perhaps conformational) in order to become functional. This interpretation is consistent with other studies, of the TCDD receptor, involving dif- ferent eiqaerimental techniques (see below). Also, in stud- ies of the estrogen receptor, Hanson and Gorski, using a thermodynamic analysis, have reached a similar conclu- sion (66). Overall, despite its limitations, the QSAR approach appears useful for studying TCDD receptor structure and function in the future, particularly if used in conjunction with other experimental approaches. Despite its biochemical similarities to steroid recep- tors, the TCDD receptor has been refractory to substan- tial purification by techniques used successfully for ste- roid recepl:ors. Several factors have contributed to the difficulty. 17irst, the relatively low receptor concentration (of the order of 105 molecules/cell, assuming one TCDD- binding sits per receptor) necessitates extensive purifi- cation. Second, like many proteins, the receptor tends to interact nonspecifically with other macromolecules dur- ing attempts at purification. Third, the extreme hydro- phobicity of the ligand, combined with low receptor concentrations, aggravates the problem of nonspecific binding. Fourth, the noncovalent nature of ligand bind- ing does not permit the use of denaturing procedures. To address the last factor, Poland et al. (135) have synthe- sized an "I-labeled, 2-azido-3-iodo-7,8-dibromodibenzo- p-diogin as a photoaffmity reagent. They have used this compound to specifically covalently label in C57BL/6J mouse liver a protein that is likely to be the TCDD receptor (135). The protein migrates in denaturing poly- acrylaniide gels with an apparent molecular weight of about 95,000. The development of this reagent will per- mit a substantially greater degree of receptor purification (albeit in denatured form) than has previously been possible. In principle, this will lead to the generation of antibodies, which would be very powerful reagents for studying the structure and function of the TCDD recep- tor and could permit the cloning of its gene(s). Thus, studies during the next few years may proct`iice substan- tial advances in our knowledge of the biochemical prop- erties of the TCDD receptor. IV. Function of the TCDD Receptor Our understanding of the mechanism by which the TCDD receptor transduces a chemical signal into a cel- lular response is sketchy. Much of the current thinking is based on the apparent functional analogies between the TCDD receptor and steroid receptors, which have been studied more extensively. The hydrophobic ligand apparently enters the cell by passive diffusion; there is no evidence that active transport is required. The binding of TCDD to its receptor occurs inside the cell and ap- parently requires both ATP (58) and reduced sulfhydryl groups (30, 88). These findings may mean that the TCDD receptor undergoes cyclic phosphorylation/dephosphor- ylation during signal transduction and that the cell con- tains an enzyme system that can maintain the receptor in a reduced state. However, these hypotheses remain to be tested. The location of the unoccupied receptor in the intact cell is open to question. In homogenates of untreated cells, the unoccupied receptor distributes primarily to the cytosolic fraction; conversely, in homogenates of TCDD-treated cells, the ligand-receptor complex distrib- utes largely to the nuclear fraction (116, 117). One inter- pretation of these data is that, in the intact cell, the unoccupied receptor is in the cytoplasm and that ligand binding produces a "translocation" of the TCDD-recep- tor complex to the nucleus (116, 117). However, the TCDD receptor can redistribute between cytoplasm and nucleus during cell homogenization and fractionation (27, 174). Therefore, data from broken-cell experiments are difficult to interpret unambiguously. An alternative interpretation is that the unoccupied receptor is primar- ily nuclear and that the binding of TCDD increases the affinity of the ligand-receptor complex for a nuclear component (e.g., chromatin), thus reducing the tendency of the complex to redistribute into the cytosol during cell fractionation (174). Studies of the distribution of the TCDD receptor in cells enucleated by cytochalasin B are also difficult to interpret unambiguously because expo-

REGULATION OF GENE EXPRESSION BY TCDD sure of cells to the antibiotic results in the loss of most TCDD-binding activity (59). Analogous studies of steroid receptors in cell homogenates are not particularly helpful either; for example, under aerobic conditions, the unoc- cupied estrogen receptor appears to reside in the nucleus (92, 171)vwhereas the unoccupied glucocorticoid receptor is apparently cytoplasmic (4). Interestingly, in ATP- depleted cells, the unoccupied glucocorticoid receptor appears to be nuclear (104). This may mean that the release of receptors from the nucleus is an energy-re- quiring event. Perhaps the simplest interpretation of the available data is that, in the intact cell, the unoccupied TCDD receptor is neither entirely cytoplasmic nor en- tirely nuclear but is in equilibrium between the two compartments. Despite the uncertainty about the intracellular loca- tion of the unoccupied TCDD receptor, it seems clear that the biological response to TCDD requires an action of the inducer-receptor complex at the nuclear level. The most compelling evidence on this point stems from stud- ies of receptor-defective cells. Two groups have isolated variant mouse hepatoma cells in which the TCDD-recep- tor complex apparently forms normally, but the complex binds weakly to a component of the nucleus (97, 106). This class of variant cells fails to transcribe the cyto- chrome :P„-450 gene in response to TCDD (63, 77, 78). These findings imply that, in order to evoke a response, the TCDD-receptor complex must interact with an ele- ment in the cell nucleus. The p:henotype', of these variant cells implies that the binding of TCDD to its receptor is not sufficient to generate a functional inducer-receptor complex. This conclusion is consistent with the observation that, if the TCDD-receptor complex forms at 4°C (as opposed to 37°C), it fails to bind strongly to the nucleus (117, 174). Thus, the generation of a functional TCDD-receptor complex a,pparently requires a temperatzire-dependent "activation" event(s). The temperature-dependent step has the effect of increasing the affinity of the TCDD- receptor compleg _ for nuclear binding sites, presumably on chromatin (116, 174). In addition, ligand binding enhances the affinity of the TCDD receptor for DNA- cellulose cr QNA-Sepharose in vitro (40, 64). However, we know virtually nothing about the mechanism of ac- tivation. For example, the temperature dependence could reflect a conformational change in the TCDD-receptor complex or a dissociation of subunits (which could expose a chromatin-binding domain), an enzymatic modification of the complex (which could alter its affinity for a nuclear binding si!te), or a combination of such events. In fact, studies of estrogen and glucocorticoid receptors suggest that several steps occur during the activation of the steroid-receptor complex to its functional form (67, 153, 154, 158). More detailed biochemical analysis of the activation phenomenon (e.g., after antibodies for the TCDD rereptor become available) seems to be a poten- 153 tially fruitful area for future research. In addition, the isolation of variants in the activation pathway would permit genetic analyses of the event(s) involved. The interaction of the activated TCDD-receptor com- plex with the nucleus can lead rapidly to a biological response. For example, the increase in cytochrome Pl- 450 gene transcription is half-maximal about 15 min after eaposure of mouse hepatoma cells to TCDD (78). Furthermore, the response occurs in the absence of on- going protein synthesis (76). These findings imply that the TCDD-receptor complex can activate gene transcrip- tion directly, without a requirement for intervening bio- chemical events, such as the generation of "second mes- sengers" or the induction of other proteins. Studies in XB mouse teratoma cells support this conclusion, in that no evidence for the participation of several second mes- sengers in the response to TCDD could be demonstrated (95). We know very little about the factors that regulate the concentration of the TCDD receptor within the cell. There is disagreement in the literature as to whether exposure to TCDD-like ligands alters the intracellular receptor concentration (33, 157). However, the experi- ments are inherently difficult to interpret, because the only way to measure the receptor is with a ligand-binding assay, and the hydrophobicity of the ligand makes the studies technically difficult. In the future, it will be interesting to determine if TCDD regulates the expres- sion of the TCDD receptor gene by a feedback mecha- nism, as may occur in the glucocorticoid-responsive sig- nalling system (121). Such studies await the development of antibody probes for the TCDD receptor and the clon- ing of the TCDD receptor gene. A priori, there is no obvious reason to think that other inducers of cyto- chrome P-450 enzyme activities should influence the level of the TCDD receptor within the cell. (See ref. 173 for a discussion of the cytochrome P-450 isozymes and the different types of cytochrome P-450 inducers.) Yet, several investigators have reported that compounds of the "phenobarbital type" produce a 2- to 3-fold increase in the concentration of the hepatic TCDD receptor in rats and mice (33, 120). We know neither the mechanism by which this effect occurs nor its functional significance. Other workers have reported that 2,2-dimethyl-5-t-butyl- 1,3-benzodioxole (DBBD), which is an "isosafrole type" of cytochrome P-450 inducer, apparently produces about a 2-fold decrease in the hepatic TCDD receptor in Dub:ICR and C57BL/6 mice (22). Again, the mechanism by which this reduction occurs is unknown. Furthermore, DBBD-treated mice also exhibit decreased enzyme in- duction in response to 3MC (a "TCDD-type" ligand), suggesting that the decrease in the TCDD receptor is functionally significant. However, this result seems to conflict with findings in C57BL/6 X DBA/2 mice, which indicate that a 2-fold reduction in receptor concentration has no apparent effect on maximal AHH induction by

154 W HITLOCK TCDD (101). Overall, it seems premature to draw firm conclusions about the regulation of the intracellular TCDD ;receptor concentration and the quantitative re- lationship between receptor concentration and a partic- ular bioloj;ical response. Development of antibody probes for the receptor will greatly facilitate the experimental analysis of these issues in the future. V. 7:'CDD-u°esponsive Genomic Elements Studies in variant cells imply that the induction of cytochrome P-450 gene transcription requires an inter- action(s) 'between the TCDD-receptor complex and an element in the cell nucleus (78). Furthermore, the TCDD-re+septor complex is a DNA-binding protein (40, 64). These observations suggested that the inducer-re- ceptor complex might act at a "genomic switch" that is located near the start site of transcription for the cyto- chrome Pi-450 gene. To test this idea, several groups have utilizsd a strategy (87) that involves (a) ligating the putative genomic switch to a heterologous "indicator" gene and (b) testing the hybrid gene for function by transfection (fig. 2). For example, in studies of mouse hepatoma cells, Jones et al. (83) isolated DNA from the region upeitream of the cytochrome P1-450 gene, ligated it to the bacterial chloramphenicol acetyltransferase (CAT) gene, and transfected the recombinant molecules into ce1Hs that contained a normal TCDD receptor. TCDD induced CAT activity in the transfected cells, implying that the hybrid gene contained a TCDD-re- sponsive DNA element. Furthermore, CAT induction had the expected sensitivity (ED50) to TCDD, and other ligands such as 3MC and flNF also induced CAT expres- sion. Tr&lsfection of the hybrid gene into receptor- defective variant cells resulted in loss of TCDD respon- siveness, indicating that the induction of CAT activity 5' 3' DFlE P 0 3' DRE P Indicator Gene FIG. 2. Identification of diozin-responsive elements. The top dia- gram depict~~ a TCDD-inducible gene (e.g., cytochrome Pt-450), con- txii~na a dio,sin-responaive element (DRE) and a promoter (P), which are located upstream of the transcription start site (arrow ). The middle diagram depicts the control region, which has been isolated from its homologovs structural gene after cleaving the DNA with a restriction endonucleauas(s). The bottom diagram depicts a hybrid gene, constructed by ligating tlle control region to a heterologous indicator gene, whose product (mRNA or protein) is convenient to assay. In the hybrid, the indicator gene (in principle) becomes responsive to TCDD. This hy- pothesis is tested by transfecting the hybrid gene into suitable (i.e., receptor-positive) cells, and determining if TCDD induces the product of the indicator gene.'A positive result implies that the control region contains a DRE. required a functional TCDD receptor. Together, these observations imply that the DNA that flanks the 5'-end of the cytochrome P1-450 gene contains a domain(s) that functions as a dioxin-responsive •element (DRE). Other workers have used a similar approach to find TCDD- responsive domains upstream of the cytochrome P1-450 gene in C57BL/6 mice (49) and in the corresponding cytochrome P-450 genes in rats (39, 159) and humans (85). Thus, the current evidence suggests that TCDD acts by similar mechanisms in these different species. Furthermore, the functions of the TCDD receptor and its cognate DRE apparently have been conserved during evolution. For example, the DRE of the mouse responds to TCDD even when transfected into human cells (82), and the genomic elements of the rat (39, 159) and human (85) function when transfected into mouse cells. Thus, the TCDD receptor from one species apparently can recognize and act at a DRE from a heterologous species. These findings imply that the TCDD-responsive signall- ing system evolved prior to the divergence between mouse and man. Future studies in other species may substantiate this point more firmly. From an evolution- ary standpoint, it is interesting that even some bacteria activate gene transcription by means of a receptor-de- pendent mechanism that responds to certain flavones as chemical signals (35, 125, 144). This (or a similar) system might represent the forerunner of the TCDD-responsive pathway present in eukaryotic cells. The DNA that flanks the 5'-end of the cytochrome P1-450 gene in mouse hepatoma cells contains other regulatory components in addition to the DRE. Jones et al. (83) used an exonuclease to produce progressively smaller DNA fragments, which were tested for function by transfection, after insertion into a CAT expression vector. These deletion analyses revealed an element that appears to function as a transcriptional promoter and confers constitutive expression upon the CAT gene. Still another functional domain is located at least 600 base pairs upstream of the promoter and acts to inhibit pro- moter function. Presumably, this inhibitory element in- teracts with a regulatory p:„~_in (i.e., a repressor), al- though this hypothesis remains to be tested. Further- more, the mechanism by which inhibition occurs from such a distance is unknown; the situation is reminiscent of "silencer" elements in other systems (12, 96). The TCDD-responsive genomic domain is located upstream of the inhibitory element, at least 1500 base pairs away from the transcription start site. The ability to activate transcription from a distance is typical of "enhancer" control systems. This observation provided a clue that the TCDD-responsive element might function as a tran- scriptional enhancer (see below). Together, the deletion analyses indicate that the DNA which flanks the 5'-end of the cytochrome PI-450 gene in mouse hepatoma cells contains a combination of (at least) three different gen- omic control elements, each of which presumably inter-

REGULATION OF GENE EXPRESSION BY TCDD acts with specific regulatory proteins. Gonzalez and Ne- bert (49) have 'made similar observations in a C57BL/6 mouse liver system. Thus, in the case of the cytochrome PI-450 gene, the TCDD-responsive system functions in a context that'also includes inhibitory and constitutive regulatory components. This sort of combinatorial con- trol of transcription may prove to be typical of many eukaryotic genes (14). Transcriptioi~al enhancers are DNA elements that bind specific proteins and thereby augment gene expres- sion. In contrast to other types of regulatory components (e.g., promoters), enhancers function relatively inde- penden ly of their distance and orientation with respect to the regulated gene (89,156). The ability of the TCDD- responsive domain to function at a distance from the transcri.Ation start site suggested that the DRE might be an enhancer (83). To test this possibility, Jones et al. isolated the TCDD-responsive domain and inserted it into a CAT expression vector, which was designed to evaluate the enhancer properties of the insert. Analyses of the recombinants by transfection revealed that (a) the DRE can function independently of the inhibitory and constitutive regulatory components to which it is linked in vivo; (b) the DRE can activate transcription from a heterologous promoter, (c) the DRE functions relatively indepen dently of its distance from the promoter; and (d) the DRE functions relatively independently of its ori- entation with respect to the promoter. These findings indicate that the DRE has properties characteristic of enhancers. Transfections into receptor-defective variant cells revealed that the DRE requires a functional TCDD receptar (82). Therefore, the DRE, together with the TCDD receptor, constitutes a dioxin-responsive enhan- cer system. Others have made similar observations using an analogous experimental approach (39, 115). The mechanism(s) by which enhancers activate transcription from a distance is unknown. For example, enhancers might (a) produce a change in chromatin structure that can be propagated and that converts the nucleoprotein to a"trFmscriptionally active" form, (b) provide a binding site for a factor(s) that then "slides" along the genome to the promoter and initiates transcription, or (c) pro- duce "looping"; of the genome and the formation of a stable nucleoprotein complex that is required for the activation of transcription (37, 142). The TCDD-respon- sive enhancer constitutes a system appropriate for test- ing these hypotheses in the future. The fact that the dioxin-responsive signalling pathway can function relatively independently of the other control compon.ents (i.e., constitutive and inhibitory) to which it is linked would appear to increase the versatility of the system as a mechanism for regulating gene expression. In principle, the system could function in diverse regu- latory contexts that generate different patterns of gene expression. To begin to test this concept, Jones et al. (82) inserted into a CAT expression vector both a DRE 155 and a glucocorticoid-responsive element (GRE) in two different linear arrangements. When the DRE was po- sitioned upstream of the GRE (i.e., the arrangement was 5'-DRE-GRE-promoter-CAT-3'), both TCDD and dex- amethasone induced CAT activity independently, and CAT expression was additive in the presence of both inducers. Thus, in this context, both the dioxin-respon- sive system and the glucocorticoid-responsive system appear to function relatively independently of each other. In contrast, when the DRE was positioned downstream of the GRE (i.e., the arrangement was 5'-GRE-DRE- promoter-CAT-3'), TCDD by itself could induce CAT expression, but dexamethasone produced a response only if TCDD also was present. Thus, in this context, the dioxin-responsive system appears to exert a "permissive" effect on the glucocorticoid-responsive system. These findings suggest that two different inducible enhancer systems can become interdependent when linked and can exhibit altered responsiveness, depending upon the reg- ulatory context in which they are placed. It is relatively easy to envision that, in other contexts, the response of a particular gene to TCDD may be a function not only of the TCDD-responsive system itself but also of the other control components with which it is linked. This might be a mechanism which could account for (at least some of) the species and tissue specificity that is char- acteristic of the biological responses to TCDD. The in- teraction of the Ah and hr loci (94, 141) is a possible example of how regulatory systems might act in combi- nation to control gene expression. In receptor-positive (Ah+) hairless (HRS/J) mice, TCDD produces epidermal hyperplasia and promotes skin papillomas only in homo- zygous animals (hr /hr ) bearing a recessive mutation at the hr locus (94, 141). These observations may indicate the egistenZ;e of a regulatory system that can block the response to TCDD. For example, suppose the hr locus encodes a regulatory•protein that blocks gene expression by binding to a cis-acting genomic control element. Fur- thermore, suppose that the inhibitory (hr) system dom- inates thp stimulatory (Ah) system when the two are linked. Then, a (hypothetical) keratinocyte gene that is under the control of both systems will not respond to TCDD unless the two hr alleles have been inactivated. This type of model might account for the responsiveness of hr /hr mouse skin to TCDD. The model makes predictions that are testable, in principle. However, the mechanisms by which control systems act in combination to regulate gene expression remain to be determined. Knowledge of the principles and mechanisms that govern combinatorial control of gene transcription appears fun- damental to an understanding of major biological phe- nomena, such as differentiation or carcinogenesis. [See, for example, studies of the mouse alpha-fetoprotein gene (60)]. The TCDD-responsive system appears potentially useful for analyzing the mechanisms of combinatorial control in the future.

156 WHITLOCK The number of DREs in a regulatory hierarchy may also influence the response of the linked gene to TCDD. Deietion analyses suggested the presence of multiple TCDD-rrnsponsive elements upstream of the cytochrome P1-450 gene (83). To examine this possibility, Jones et al. perfo;rnned a more detailed study of the TCDD-re- sponsive region in mouse hepatoma cells. Their findings revealed the existence of (at least) two distinct, non- overlapp; ng DNA fragments, each of which functions as a TCDD-responsive element when inserted into a CAT expression vector and transfected into wild-type cells. Transfections into receptor-defective cells imply that each element requires the TCDD receptor for its func- tion. Each element has the properties of a transcriptional enhancer, and each can function independently of the other. The combined effects of the two elements are (at least) adctitive (81). Sogawa et al. (159) have also reported findings that are consistent with the existence of multiple TCDD-responsive domains upstream of the cytochrome P-450c gene of the rat. The significance of these obser- vations is unknown at present. It is possible that the association of multiple DREs with the cytochrome P-450 gene is atypical and that other TCDD-responsive genes are linked to a single DRE. A second possibility is that multiple DREs are typical of TCDD-responsive genes and are advantageous in some way. For example, linking DREs in tIndem may allow the formation of additional protein-poutein interactions (e.g., between adjacent TCDD-roceptor complexes) that stabilize a productive transcriptional complex, thereby permitting more effec- tive gene eapression. If so, then increasing the number of linked DREs might have a synergistic effect on gene ezpresakis, In addition, the spacing between DREs (which aretld affect protein-protein interactions) might also infiwauce the!response of the linked gene to TCDD (see, for a¢ample, refs. 11 and 162). Experiments de- signed to loest these ideas are feasible in principle and may rewdaadditional details of the mechanism of TCDD action i.Ilae future. The aeMfration of cytochrome P1-450 gene expression requires irpth the TCDD receptor and the DRE. How- ever, tbe, Ibct that both components are required does not n.peNwily demand that they physically interact during OhlM1 process of signal transduction. To address this iewrb,-Vurrin and Whitlock (36) utilized an assay which neaures the accessibility of the DRE in situ (determia* by its susceptibility to digestion by an eg- onucl+rrufts a function of exposure of the cell to TCDD. Their s11d in mouse hepatoma cells revealed that (a) a speA ~ A reo on upstream of the cytochrome Pl- 450 g~ protected from exonuclease digestion in TCDD-il0 ed cells, but not in uninduced cells; (b) protedir1Ms not occur in receptor-defective cells; (c) protecliAqcurs within 1 h of exposure of the cell to TCDIY.44 rotection occurs in the absence of ongoing proteioill esis; (e) the protected region is in a domain that functions as a DRE. These observations imply that both the DRE and the TCDD-receptor complex contrib- ute to the formation of a stable nucleoprotein structure that is relatively resistant to exonuclease attack. These findings strongly imply that the TCDD-receptor complex and the DRE interact in vivo to activate the transcription of the cytochrome Pt-450 gene. Others have made similar observations in studies of the glucocorticoid-responsive system (8). The details of the protein-DNA interactions and the possible participation of other proteins in the activation of gene transcription are interesting issues requiring additional research. ,. The properties of the chromatin recognition site(s) for the TCDD-receptor complex remain to be determined in more detail. Sogawa et al. (159) have proposed that the inducer-receptor complex recognizes a specific "consen- sus" decanucleotide sequence that is present in multiple copies in the DNA that flanks the 5'-end of the rat cytochrome P-450c gene. Interestingly, they observed that a synthetic concatemer of one such decanucleotide augmented the response of a linked CAT gene to 3MC. On the other hand, it is not yet clear that this effect is dependent upon the TCDD receptor, because the con- struct was not tested in receptor-defective cells. Also, two copies of the putative recognition sequence are lo- cated in a DNA region that does not exhibit responsive- ness to 3MC (159). Thus, the specific chromatin struc- ture that the TCDD-receptor complex recognizes re- mains uncertain. While a specific DNA sequence may be a necessary constituent of the recognition site, it may not be sufficient. In other systems, the binding of a regulatory protein to a specific DNA sequence does not generate a response unless additional specific protein- protein interactions can also occur (13, 57, 71, 86). An analogous situation may also exist for the TCDD-respon- sive system. According to this view, the TCDD-receptor complex could bind to a specific DNA sequence; however, the binding will not produce a response unless the com- plex can also form additional interactions with other proteins that bind to adjacent regions of the genome. Thus, both DNA and protein would contribute to a functional recognition site for the TCDD-receptor com- plex. VI. Future Prospects The purification and characterization of TCDD recep- tors remain important areas of research for the future. Major advances in this area may occur during the next several years. For example, the ability to covalently label the receptor with an affinity reagent will allow the use of denaturing conditions during the isolation of the TCDD-binding protein; this will lead to a much greater degree of purification than has been possible previously. Antibodies raised against the purified protein (either in its denatured form or, possibly, after renaturation) should be useful reagents for studying the structural and functional domains of the receptor and for its isolation

REGULATION OF GENE EXPRESSION BY TCDD 157 using imnnuinoaffinity techniques. Anti-receptor anti- bodies sheuld permit more detailed analyses of receptor heterogeneity, receptor modification, receptor synthesis and degradFition, and the temperature-dependent acti- vation event that occurs during transduction of the TCDD signal. In addition, sequence analysis of the de- natured prol;ein should permit the synthesis of an oligon- ucleotide(s) that might be used to isolate the correspond- ing gene. Other approaches to receptor purification may also be useful. Foi. - example, the functional similarities between TCDD receptors and steroid receptors suggest that struc- tural simiLarities may also exist. Therefore, it may be possible to find antibodies, raised against purified steroid receptors, that cross-react with TCDD receptors. Such antibodies could be used in receptor purification. In addition, we, may find that the TCDD-receptor complex recognizes a specific DNA sequence. If so, oligonucleo- tides that contain this sequence may be useful affinity reagents for the purification of the TCDD-receptor com- plex (see, for example, refs. 84 and 149). Anti-receptor antibodies presumably could be used to clone the gene(s) for the TCDD receptor. An alternative approach ndght be to insert either genomic DNA or cDNA into an appropriate expression vector and to use the recombinant to complement the lesion in receptor- defective variant cells, with the selection procedure de- veloped by van Gurp and Hankinson (167). Cloning and characterizEition of,the TCDD receptor gene(s) will per- mit studies of its expression and lead to a better under- standing of the factors which regulate the intracellular concentra tion of the receptor. The TCDD receptor presumably consists of multiple functional domains, including a ligand-binding domain, a DNA (chromatin)-binding domain, and possibly, a domain(s) tlhat interacts with other transcription factors. Cloning and expression of cDNA for the TCDD receptor, when coml ined with mutagenesis and gene transfer methodologies, should permit a detailed analysis of its functional domains (see, for example, refs. 46, 48, and 105). Furthermore, given the similarities between the TCDD receptor and steroid receptors described above, it will be intriguing to learn whether the TCDD receptor is a member of the hormone receptor family that is related to the viral erb A oncogene (51). Variant cells have been very useful in characterizing the TCDD-responsive system to date; the study of addi- tional variants would seem to be worthwhile in the future. For example, Hankinson and coworkers (62) have al- ready identified by complementation analysis cells which presumab'';ly contain defects at other steps in the signal transducti.on pathway. In the future, the isolation of temperature-sensitive variants would allow us to analyze the reversibility of particular steps in signal transduction and to study the requirements for the maintenance of TCDD-induced changes in gene expression. Selection of cells that overproduce TCDD receptors might be useful for purifying the receptor and for cloning its gene, as well as for studying quantitative aspects of signal trans- duction. A great deal remains to be learned about the mecha- nism by which the dioxin-responsive element, together with the TCDD-receptor complex, functions as a tran- scriptional enhancer. Mutagenesis and gene transfer techniques can be used to define the functional bounda- ries of various DREs. DNA sequence analyses should reveal whether each DRE contains a specific sequence that forms part of the recognition site for the TCDD- receptor complex. The development of an enhancer- dependent in vitro transcription system (see, for exam- ple, ref. 151) would facilitate the functional analysis of the dioxin-responsive pathway. In view of what is known about other enhancer systems (126, 155), it seems likely that the DRE will be found to interact with several other proteins, in addition to the TCDD-receptor complex. If so, the task of understanding the mechanism by which the inducer-receptor complex activates transcription will become substantially more complicated. The chromatin structure (124, 169, 170) of TCDD- responsive genes is an interesting area for future study. For example, we know very little about the nucleoprotein organization of the DRE and other linked regulatory components (38). Are these elements associated with histones or other chromosomal [e.g., high-mobility group (HMG)] proteins? Are they organized into nucleosomes in vivo? If so, how do these structural features influence the function of the regulatory elements? If (as seems more likely) the DRE does not assume a nucleosomal structure in,vivo, why not? What determines the chro- matin structure of the DRE? Does the nucleoprotein structure of the element change upon its interaction with the TCDD-receptor complex? If so, is the structural alteration local or does it propagate along the chromatin fiber? What is the mechanism by which a change in structure leads to activation of gene transcription? Fu- ture studies that address these issues may generate in- teresting information that is relevant to transcriptional enhancement in general. In addition, studies in other systems suggest that transcriptionally active regions of chromatin may be preferentially associated with the nu- clear matrix (80, 114). The role that the nuclear matrix plays in the cellular response to TCDD may also be a productive area for future,research. We know that TCDD induces the activity of UDP- glucuronyltransferase and NADPH:quinone reductase, apparently by activating the transcription of the corre- sponding gene (79, 147, 176). However, we do not yet know whether the activation of these other genes occurs in the absence of ongoing protein synthesis (i.e., if in- duction reflects a primary response to the TCDD-recep- tor complex). For example, others have proposed that TCDD induces a protein that secondarily activates a

158 W HI'1'LOCK battery o f other genes (54, 140). In fact, some glucocor- ticoid-responsive genes appear to display this type of regulation (2, 7). The study of additional (i.e., non- cytochronse P-450) TCDD-responsive genes might pro- vide evidence for a protein(s) that mediates a TCDD- induced cascade of biological responses. The isolation and chamcterization of such a factor would be funda- mental to our understanding of the mechanism by which TCDD elicits its diverse effects. The TCDD-responsive signalling system could also diminish the rate of tran- scriptiornn of some genes, either directly via the TCDD- receptor complea, or indirectly, via the synthesis of an inhibitory factor. This idea is testable, in principle. Also, the study,of additional TCDD-responsive genes can in- crease our knowledge of how the dioxin-responsive en- hancer system functions in other regulatory contexts, in combination with different promoters, silencers, and en- hancers. Such information could make a valuable contri- bution to our understanding of the principles that govern the combinatorial control of gene transcription. Appro- priate T(' MD-responsive cell systems are available to begin the study of these problems (1, 25, 93, 122, 145). The results of on-going epidemiological inyestigations suggest that exposure to TCDD poses less of a human health risk than was once feared, although the issue remains somewhat controversial (21, 70, 72, 109, 161). Most of us probably have accumulated some TCDD in our cells (123); however, it is not clear that this consti- tutes any m easurable risk to the well-being of the general population. However, we cannot rule out the possibility that certain individuals are relatively susceptible to the effects of T'CDD, either because of a genetic predisposi- tion (34, 94, 141) and/or because of exposure to an additionat: environmental chemical(s). 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