©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Interplay of Half-site Sequence and Spacing on the Activity of Direct Repeat Thyroid Hormone Response Elements (*)

(Received for publication, May 9, 1994; and in revised form, December 5, 1994)

Ronald W. Katz José S. Subauste Ronald J. Koenig (§)

From the University of Michigan Medical Center, Ann Arbor, Michigan 48109-0678

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Direct repeats of the hexamer AGGTCA can serve as response elements for vitamin D, thyroid hormone, or retinoic acid. The specificity of the response appears to reside in the spacing between the hexamers, with response elements for vitamin D restricted to direct repeats separated by a 3-base pair (bp) spacer, thyroid hormone a 4-bp spacer, and retinoic acid a 5-bp spacer (3-4-5 rule). Recently we have shown that the optimum thyroid hormone receptor binding site consists of an 8-bp sequence (TAAGGTCA), not a hexamer. Therefore we tested whether the 3-4-5 rule is valid for octamer sequence direct repeats. In transfection experiments octamer direct repeats with 3-, 4-, or 5-bp spacers conferred equivalently strong thyroid hormone responses, although a repeat with a 9-bp spacer was substantially weaker. For the 4- and 5-bp spacer constructs, the 5` half-site octamer had as strong an influence on thyroid hormone induction as did the 3` half-site octamer, although for the 3-bp spacer construct the 5` octamer was marginally less potent than the 3` octamer. Transfection and gel shift experiments did not suggest a simple correlation between the binding of thyroid hormone receptor-retinoid X receptor heterodimers and thyroid hormone induction from these response elements. We conclude that half-site sequence can override the effect of spacing in determining the hormone responsiveness of a direct repeat response element. In addition, the thyroid hormone response may not be due simply to the binding of thyroid hormone receptor-retinoid X receptor heterodimers to the DNA.


INTRODUCTION

Thyroid hormone (3,5,3`-triiodothyronine; T(3)) (^1)receptors (TRs) are members of a large family of zinc finger transcription factors which also includes the receptors for all known steroid hormones, retinoic acid (RA) and 1,25-dihydroxycholecalciferol (vitamin D(3); vitD). In the case of thyroid hormone-responsive genes, T(3) regulation is dependent on the binding of TRs to specific cis-acting sequences, or T(3) response elements (TREs)(1, 2, 3, 4, 5) . The well characterized positive TRE in the promoter of the rat growth hormone (GH) gene has been shown to consist of three copies of a loosely conserved hexamer motif, AGGTCA(6, 7) . This hexamer has also been characterized as a binding half-site for several other related members of this family of transcription factors, including retinoic acid receptors (RARs), retinoid X receptors (RXRs) and the vitamin D receptor (VDR)(8, 9, 10, 11) . It is recognized that these receptors can bind as dimers to direct repeats (DR) of this hexamer(8, 9, 10, 11) . Differential spacing between hexamer half-sites has been proposed to account for the specificity of the T(3)-, RA-, and vitD-dependent responses of otherwise equivalent DR response elements. Thus, a DR of the hexamer AGGTCA has been shown to function specifically as a vitD response element (VDRE) if the hexamers are separated by 3 base pairs (bp), as a TRE if the spacer is 4 bp, and as an RA response element (RARE) if the spacer is 5 bp (3-4-5 rule)(8, 9) .

Recently, we and others have shown that nucleotides surrounding the hexamer sequence can influence TR binding and function(12, 13, 14) . A protocol of sequential electrophoretic mobility shift assays (EMSAs) and polymerase chain reaction (PCR) amplifications allowed us to identify the optimal TRalpha1 monomer binding sequence from an original pool of random DNA sequences(12) . This optimal binding sequence, 5`-TAAGGTCA, is 2 bp longer than the consensus hexamer sequence. We have demonstrated that the 2 additional 5` bp are important for the binding and function of TRs (12) and that a single octamer sequence can function as a TRE but not as an RARE, VDRE, or RXR response element (RXRE)(15) .

The 3-4-5 rule was based on functional studies of the effect of variable spacing between hexamer sequences. In retrospect, the hexamer AGGTCA represents an incomplete TR binding sequence. Therefore we wanted to evaluate the effect of spacing on the ligand responses of reporter genes containing optimal octamer TRalpha1 binding sequences arranged as DR response elements. In the transfection studies described below, we demonstrate that octamer DRs with 3-, 4-, and 5-bp spacers (^2)all function as equally strong TREs. Thus, the two upstream nucleotides of the receptor binding site play a prominent role in determining the strength and specificity of T(3) DR response elements.


MATERIALS AND METHODS

DNA Construction

Various response element sequences were ligated as single copies into the unique BamHI site of pUTKAT3 located upstream of the basal herpes simplex virus thymidine kinase promoter directing chloramphenicol acetyltransferase (CAT) expression(16) . The top strands are indicated below, and the octamer or hexamer sequences are underlined. The reporter plasmid ptkMA contains a single copy of the octamer sequence inserted into pUTKAT3 (12) . In naming the complex reporter plasmids, the number preceding ``DR'' signifies whether the half-sites are octamers or hexamers, and the number following DR signifies the bp spacing between the core AGGTCA sequences. The plasmid ptk6DR4 was described previously as ptk6DR(17) .

In addition, constructs were made in which one of the two half-sites contained the octamer sequence (TAAGGTCA) while the other contained the hexamer (GCAGGTCA). For example, ptk86DR3 had the octamer in the upstream position, whereas ptk68DR3 had the octamer in the downstream position.

Cell Culture and Transfections

JEG-3 cells were grown in 90% modified Eagle's medium plus 10% fetal bovine serum and were transfected using a calcium phosphate precipitation protocol(18) . Mouse TRalpha1(19) , rat TRbeta1(18) , human RARalpha(20) , and human VDR (21) were expressed from the vector pCDM(18) . Transfections included 3 µg of receptor (or control pCDM) plus an additional 3 µg of pCDM vector as ``filler'' plasmid. Reporter plasmids (described under ``DNA Construction'') were transfected at a dose of 4 µg/Petri dish. To control for transfection efficiency, each transfection also included 0.25 µg of pRSVGH, in which the Rous sarcoma virus promoter directs expression of human GH. This reporter plasmid was not affected by the cotransfection of the different receptors or their respective ligands (data not shown). Cells in 60-mm Petri dishes were transfected in the presence of 90% modified Eagle's medium plus 10% charcoal-stripped fetal bovine serum supplemented with 100 nM dexamethasone and cultured for 2 days with or without the appropriate ligand. The ligand concentrations used were 10 nM T(3), 1 µM all-trans-RA, and 100 nM vitD. Cell lysates were assayed for CAT activity and media were assayed for human GH as described previously(18, 22) . Ligand responsiveness is defined as CAT activity/human GH for cells cultured in media with ligand divided by CAT activity/human GH for cells cultured in media without ligand. Results are presented as the mean ± S.E. for at least four transient transfections per condition. Ligand-induced CAT activity required the presence of the appropriate cotransfected receptor, except for a modest 2-3-fold RA induction of ptk8DR4 and ptk8DR5 in the absence of cotransfected RAR (data not shown).

Electrophoretic Mobility Shift Assays

The DNA probes were obtained by PCR with primers specific to regions flanking the BamHI site in pUTKAT3 (PCR primers span bp 2211-2225 and 2279-2299 (reverse complement); the BamHI site is located at bp 2251). The templates were pUTKAT3 derivatives described under ``DNA construction.'' The amplification products were digested with EcoRI (restriction cleavage site located at bp 2233), and the PCR fragments were purified by polyacrylamide gel electrophoresis. The fragments were labeled with [alpha-P]dTTP by Klenow fill-in. Recombinant mouse RXRalpha (23) and recombinant mouse TRalpha1 (19) were produced in Escherichia coli as described previously(17) .

The protein-DNA binding reactions were performed in 35 µl of 20 mM HEPES, pH 7.8, 50 mM KCl, 1 mM dithiothreitol, 0.1% Nonidet P-40, and 20% glycerol. The incubations included varying amounts of receptor(s), 1.4 µg of poly(dIbulletdC), and 80,000 cpm of labeled DNA. Reactions were incubated at room temperature for 40 min and then subjected to electrophoresis in 6% polyacrylamide gels (29:1 acrylamide:bisacrylamide) in 0.25 times TBE buffer (22 mM Tris base, 22 mM boric acid, 0.5 mM EDTA) at room temperature. The gels were fixed in 30% methanol/10% acetic acid, dried, and exposed to film with an intensifying screen for 1-6 h at -70 °C as indicated.


RESULTS

Octamer DR Elements with 3-, 4-, or 5-bp Spacers Show Equivalent T(3) Inductions in Transient Transfections

A series of transfections was performed examining the T(3) responsiveness of variably spaced octamer DR reporter plasmids. Fig. 1illustrates the T(3)-dependent increases in gene activation from a single octamer sequence (ptkMA) and DR octamer response elements with 3-, 4-, 5-, and 9-bp spacings (ptk8DR3, ptk8DR4, ptk8DR5, and ptk8DR9) in the presence of cotransfected TRalpha1. The 3-, 4-, and 5-bp spacer DR elements all showed equivalently robust T(3) responsiveness, indicating that T(3) induction from octamer DR response elements does not obey the 3-4-5 rule. In contrast, the T(3) induction of CAT activity conferred by the 9-bp spacer DR was relatively weak. The more potent T(3) responses on the 3-, 4-, and 5-bp DRs suggests that some type of dimeric T(3) complex regulated transcription from these elements, and that increasing the spacing to 9 bp presumably limited this receptor interaction by introducing an additional half-turn of the DNA helix between the half-sites. These experiments also were performed with TRbeta1 with similar findings (data not shown).


Figure 1: Octamer direct repeat response elements with 3-5-bp spacers have equivalent T(3) responses. The octamer 5`-TAAGGTCA was inserted as a single copy into the reporter plasmid pUTKAT3 in a forward orientation (MA) or as direct repeats with 3-, 4-, 5-, or 9-bp spacers (8DR3-8DR9) and transfected into JEG-3 cells along with the internal control plasmid pRSVGH and an expression vector for TRalpha1. Cells were cultured for 2 days ± T(3), and then the cell lysates were assayed for CAT activity and the media for human GH. CAT activity was normalized using GH and presented in arbitrary CAT/human growth hormone units. Results are the mean ± S.E. for at least four independent transfections.



Variably Spaced Hexamer DRs Display T(3) Responses Consistent with the 3-4-5 Rule

A series of experiments was performed on the reporter plasmids containing variably spaced arrangements of the hexamer motif (5`-GCAGGTCA) to determine whether our laboratory could replicate the original observations that DR hexamers require a 4-bp spacer to function as TREs. As can be observed in Fig. 2, the reporter plasmid with a 4-bp spacer between the hexamer repeats (ptk6DR4) was the most responsive to T(3) in the presence of TRalpha1, although a very modest T(3) response was noted with the 3- and 5-bp spacer reporter plasmids. Thus, in contrast to the DR octamer data in Fig. 1, the T(3) response of suboptimal hexamer sites obeys the 3-4-5 rule. In addition, increasing the spacing to 9 bp caused the hexamer DR element to become resistant to T(3) induction. A similar lack of T(3) response was previously noted with a response element composed of a single copy of the 5`-GCAGGTCA sequence(12) . The modest T(3) response of 6DR4 relative to that of 8DR4 (6-fold versus 12-fold) indicates that suboptimal sites arranged as DRs do not act as potently as DRs of optimal sites. The requirement for appropriate spacing and the lack of T(3) response by the 9-bp spacer element (ptk6DR9) also suggest that suboptimal TR binding sites require dimeric receptor interactions for positive T(3) regulation. Experiments performed with TRbeta1 had similar results (data not shown).


Figure 2: Hexamer direct repeat response elements conform to the 3-4-5 rule. The sequence 5`-GCAGGTCA was arranged as a direct repeat with a 3-, 4-, 5-, or 9-bp spacer in the reporter plasmid pUTKAT3 (6DR3-6DR9). Transfections were performed as described in Fig. 1. Ligand responsiveness (-fold CAT induction) is defined as CAT/human GH for cells cultured with T(3) divided by CAT/human GH for cells cultured in the absence of T(3). Results are the mean ± S.E. for at least four independent transfections. Basal CAT activities were similar for all 4 reporter plasmids.



T(3) Induction Is Enhanced with the Octamer in Either the 5` or 3` Half-site

Current evidence suggests that under most circumstances T(3) induction of gene expression is mediated by TRbulletRXR heterodimers(23, 24, 25, 26, 27, 28) . When these heterodimers bind to traditional DR4 response elements, RXR occupies the 5` half-site and TR occupies the 3` half-site(29, 30) . Thus, if TRbulletRXR heterodimers account for gene activation on the 8DR3-5 response elements, it might be expected that only the 3` half-site would require the octamer sequence. This idea was tested in a series of transfections using TREs in which one half-site was an octamer and the other a hexamer (for example, 86DR4 has an octamer as the 5` half-site and a hexamer as the 3` half-site, whereas the opposite holds for 68DR4). In contrast to the above expectations, 86DR4 and 68DR4 were equally potent TREs, and in fact were as potent as 8DR4 (104 ± 5% and 115 ± 8% [mean ± SE] the T(3) induction of 8DR4, respectively; n = 6). The results with the DR5 series again showed that the 5` octamer was as potent as the 3` octamer, although the DR5's with one octamer and one hexamer were slightly weaker TREs than the two octamer DR5. Thus, 86DR5 and 68DR5 showed 71 ± 8% and 70 ± 7% the T(3) induction of 8DR5, respectively (n = 8). Results with the DR3 series were similar to those with the DR5's, except that there was a trend for the 5` octamer to be marginally less potent than the 3` octamer. Thus, 86DR3 showed 57 ± 12%, and 68DR3 70 ± 13%, the T(3) induction of 8DR3 (n = 4). Overall, the results demonstrate that there is little if any difference in the effect of having the octamer in the 5` versus 3` half-site, suggesting that perhaps the T(3) induction from these response elements cannot be accounted for solely by the binding of TRbulletRXR heterodimers.

Octamer DRs Obey the 3-4-5 Rule for RA and vitD Inductions

Since the 3-4-5 rule is not essential for T(3) induction from octamer DR elements, we wished to determine whether it was important for RA or vitD induction from these same elements. The cotransfection of RARalpha with 8DR3, 8DR4 and 8DR5 showed that the 5-bp spacer response element had the greatest RA response, as predicted by the 3-4-5 rule (Fig. 3). Experiments in which VDR was cotransfected with the same reporter constructs revealed that 8DR3 had the greatest vitD response (Fig. 3), again consistent with the 3-4-5 rule. Thus, octamer half-sites relax the spacing requirement for T(3) induction, but not for RA or vitD induction.


Figure 3: Octamer direct repeat response elements obey the 3-4-5 rule as VDREs and RAREs. Transfections were performed as described in Fig. 1. Octamer direct repeat response elements with 3-5-bp spacers (8DR3-8DR5) were cotransfected with either the VDR or RAR expression plasmid, and CAT induction by the appropriate ligand was determined. Results are the mean ± S.E. for at least four independent transfections. CAT activities in the absence of ligand were similar for all three reporter plasmids.



TRbulletRXR Heterodimers Bind to Octamer DRs, but Overall the Magnitude of Binding Correlates Poorly with Reporter Gene Induction

The surprising result that 8DR3, 8DR4 and 8DR5 all are stronger TREs than 6DR4 led us to examine the nature of the TR-DNA complexes that form with these elements. Previous studies with hexamer DRs have shown that TR homodimers and TRbulletRXR heterodimers have a strong preference for DR elements with a 4-bp spacer and that the heterodimer forms a higher affinity complex than does the homodimer (10) , especially in the presence of T(3)(17, 27, 31) . Therefore we examined the binding of TRbulletRXR heterodimers to the octamer DRs and 6DR4 by EMSA (Fig. 4). These experiments utilized a relatively low dose of TRalpha1 such that detectable monomer complexes formed with 8DR3, -4, and -5, but not with 6DR4, and homodimers did not form on any element (middle panel). The addition of RXR caused the formation of a more slowly migrating TRbulletRXR heterodimer complex with each element (right panel). The heterodimer-DNA complex was of comparable intensity on all DRs, even though 6DR4 is weaker as a TRE. Furthermore, both a single octamer and 8DR9 bound TRbulletRXR heterodimers nearly as well as did 8DR3-5 (data not shown), yet these also are weaker response elements (Fig. 1). Thus, the strength of 8DR3-5 as TREs cannot be explained simply by enhanced TRbulletRXR binding, at least as measured by EMSA. Although it may seem surprising that TRbulletRXR heterodimers can bind to a single octamer site, this probably reflects the fact that these receptors form stable heterodimers in solution off DNA(32) , and that heterodimerization does not substantially inhibit TR binding to the octamer. A similar observation has been published previously(15) .


Figure 4: TRbulletRXR heterodimers bind equivalently to 6DR4 and the 8DR3-5 elements. EMSAs were performed in the presence of T(3) with P-labeled DNA probes derived from reporter plasmids containing the elements 6DR4, 8DR3, 8DR4, or 8DR5. The probe labeled C is derived from the empty reporter plasmid vector (pUTKAT3). Incubation with RXRalpha alone failed to yield detectable complexes with any of the probes (left panel). Incubation with TRalpha1 alone yielded monomer-DNA complexes only with the 8DR probes (middlepanel), consistent with their enhanced affinity relative to the hexamer TRE sequence for TR monomers. Incubation with TRalpha1 plus RXRalpha yielded heterodimer-DNA complexes with 6DR4 and all three 8DR probes (right panel).



Since TR homodimers might contribute to the T(3) responsiveness of DR TREs, we also evaluated TR homodimer formation on the 8DRs by performing EMSAs with a 10-fold increased dose of TRalpha1. TR homodimers did not form on the single octamer element, but did form on 8DR3-8DR5 and 8DR9 with approximately equal intensity (data not shown). Since 8DR9 is a weak TRE, the strength of 8DR3-8DR5 as TREs cannot be directly correlated with the strength of TR homodimer binding as measured by EMSA. Although the EMSA data do not allow one to conclude whether TR monomers, TR homodimers, or TRbulletRXR heterodimers actually are involved in gene activation from the 8DR elements, they do indicate that all of these complexes could potentially contribute to the T(3) response.


DISCUSSION

Despite much progress over the last several years, the molecular basis of hormone-specific regulation of gene expression is still unclear. An essential component of a hormone response is the binding of the appropriate receptors to the regulatory regions of responsive genes. In order to understand this process relative to the ErbA superfamily of hormone receptors, it is necessary to determine the optimum binding sequence of each particular receptor, as well as the degree of cross-binding possible with other closely related members of this gene family.

Previous investigators did not know a priori the exact size or sequence of the optimal TR binding site, so a consensus binding sequence was derived from limited mutagenesis of a complex TRE in the rat growth hormone promoter and sequence comparisons of the positive and negative TREs of a limited number of T(3)-responsive genes. The result was an initial characterization of the TR binding site as containing 6 nucleotides (5`-AGGTCA) and the observation that positive TREs consisted of complex arrangements of loosely conserved sites. In the case of DR elements, the effect of spacing was believed to be essential in conferring ligand-specific responses, with vitD responses being restricted to elements with a 3-bp spacer, T(3) responses to elements with a 4-bp spacer, and RA responses to elements with a 5-bp spacer (3-4-5 rule). However, these studies were based on suboptimal TR binding sites.

In the present report we demonstrate that DRs of the optimal (octamer) TR binding sequence are less affected by alterations in spacing than had been previously proposed according to the 3-4-5 rule. Thus, 8DR3, 8DR4, and 8DR5 all are capable of eliciting an equally strong T(3) response. However, interposing an additional half-turn of DNA helix between the receptor binding sites (8DR9) weakens the magnitude of T(3) induction. This suggests that the induction from 8DR3, 8DR4, and 8DR5 involves a protein-protein interaction in addition to DNA binding. This interaction can occur with half-site spacings of 3, 4, and 5 bp, but not 9 bp. It is perhaps surprising that 8DR9 is no more active than a single octamer sequence as a TRE. The explanation for this observation is not known. Although it could reflect an adverse effect of phasing on T(3) induction, this TRE is more than 100 bp from the transcriptional start site and we are unaware of published data to demonstrate such effects for comparably positioned TREs. Alternatively, perhaps the number of TR monomers bound to DNA is not the rate-limiting factor for determining the level of T(3) induction. In any case, it is clear that the 3-4-5 rule cannot be used to predict the specificity of hormone induction from these octamer DR response elements. Although the 3-4-5 rule represents a major advance in our understanding of a variable (spacing) that restricts the specificity of hormone induction from hexamer DR elements, this rule is less influential when optimal TR binding sequences are present. We conclude that the affinity of the DNA half-site sequence for TR has at least as great an influence on conferring ligand response as does the spacing of DRs (at least for spacings of 3-5 bp).

TRs can bind to direct repeat TREs as monomers, homodimers, or heterodimers with a variety of other ErbA superfamily members such as RXRs(23, 24, 25, 26) , RARs(33) , VDR(34) , and chicken ovalbumin upstream promoter-transcription factor(35) . It is not known which of these complexes are important transcriptional regulators in vivo. However, indirect evidence has led to the notion that TRbulletRXR heterodimers may be the most important functional complex within the cell, at least for traditional hexameric DR4 TREs(23, 24, 25, 26, 27, 28) . TRbulletRXR heterodimers have been shown to bind DNA with the 5` half-site occupied by RXR and the 3` half-site by TR(29, 30) . Thus, if the transcriptional response from 8DR3-5 were due entirely to the binding of TRbulletRXR heterodimers, one would expect that the octamer would be required only for the 3` half-site. This is especially the case since the optimal half-site sequence for RXRalpha binding is GGGGTCA, not TAAGGTCA(36) . However, a direct test using reporters comprising one octamer and one hexamer half-site failed to substantiate this prediction. Indeed, 86DR4 was as potent as 68DR4, and also as potent as 8DR4. Although neither 86DR5 nor 68DR5 was quite as potent as 8DR5, the 5` octamer was again as potent as the 3` octamer. These data argue against TRbulletRXR heterodimers as being the sole activators of transcription from the 8DR elements. This conclusion is further supported by EMSA data (Fig. 4), which demonstrate that 6DR4 binds TRbulletRXR heterodimers as well as the 8DRs, even though it is weaker as a TRE. It is likely that part of the T(3) induction from the 8DR elements is accounted for by TR monomers(12, 15) , and it is possible that TR homodimers or TR heterodimers with other co-regulators also may account for some of the induction.

When the octamer elements were tested for RA and vitD induction, the 5-bp spacer (8DR5) was found to be the most potent RARE, and the 3-bp spacer (8DR3) had the greatest vitD response. Thus, in contrast to the above data for TRs, the specificity of induction by RAR and VDR on these sequences does conform to the 3-4-5 rule. However, it appears that TAAGGTCA does not represent the optimal binding site for RAR (37) or VDR(38, 39) . It is possible that variably spaced DR response elements composed of the optimal binding sequences for RAR or VDR will show RA and vitD responses that do not conform to the 3-4-5 rule, analogous to what is shown here for T(3) and 8DR3-5.

As noted above, it appears that T(3)-responsive cells may contain a diverse population of TR-containing complexes ranging from TR monomers to several different TR dimers and potentially larger complexes. The ability of a particular DNA sequence to confer a T(3) response in a particular cell or tissue may depend on the affinity of different specific TR-containing complexes for that sequence. Since a T(3) response requires the binding of a TR-containing complex, TREs appear to require at least one TR binding site. Genes whose regulatory regions contain one or more optimal TR binding sites may have their T(3) response transduced by TR monomers, homodimers, heterodimers, and/or larger order complexes. In those cases where the individual half-sites are suboptimal for TR binding, a TR homo- or heterodimer may still possess sufficient cooperative affinity such that it may confer T(3) responsiveness. In order to understand the relative contribution of each TR-containing complex to conferring T(3) responsiveness in various tissues, it will be necessary to understand how TRs bind to different arrangements of optimal and suboptimal sites. In addition it will be necessary to understand how each of the potential dimerization partners of TRs bind to their own optimal and suboptimal sites.

Since many members of this receptor family are closely related, a particular DNA sequence may function as a high affinity site for one receptor complex and a modest affinity site for several other receptor complexes. With the close proximity of two sites, some sequences can function as response elements for multiple receptors (e.g. a palindromic sequence can function as a TRE and an RARE; (40) ) and other arrangements will be more restricted in their potential receptor interactions (e.g. TAAGGTCA can function as a TRE but not an RXRE, RARE, or VDRE; (15) ). We believe it is reasonable to propose that a T(3)-responsive cell contains a limited variety of TR complexes (monomers, specific heterodimers, etc.) and that each TR complex will confer the greatest ligand response to a set of regulatory elements that bind a particular complex with the highest affinity and in a conformation supportive of trans-activation. For example, TR monomers will confer the greatest T(3) response from a single octamer sequence 5`-TAAGGTCA placed in a forward orientation relative to the transcriptional start site(12) . In this view, TR homodimers will confer maximal T(3) responses to sites composed of two copies of 5`-TAAGGTCA. Based on studies showing that RXR binds 5` to TR in TRbulletRXR heterodimers(29, 30) , we hypothesize that TRbulletRXR heterodimers will confer the greatest T(3) response from an element composed of an optimal TR binding site in close proximity and downstream of an optimal RXR binding site. Thus, changes in the composition of the pool of TR complexes could alter the genetic response of a cell to T(3). It is possible that the presence or absence of a particular TR complex may account for the tissue-specific effects of T(3). Before one can understand physiologic and pathologic hormone or vitamin regulation at the transcriptional level, it will be necessary to characterize the optimal binding sites for a large group of related zinc finger receptors (such as RARs, RXRs, and chicken ovalbumin upstream promoter-transcription factor), determine how different combinations of sequences confer ligand-restricted changes in gene expression through specific receptor combinations, and identify these sequences as the actual regulatory elements of specific sets of genes modulated by the relevant hormone receptors.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants DK44155 and DE00301. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: University of Michigan Medical Center, 5560 MSRB II, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0678. Tel.: 313-763-3056; Fax: 313-936-6684.

(^1)
The abbreviations used are: T(3), 3,5,3`-triiodothyronine; bp, base pair(s); CAT, chloramphenicol acetyltransferase; DR, direct repeat; EMSA, electrophoretic mobility shift assay; GH, growth hormone; PCR, polymerase chain reaction; RA, retinoic acid; RAR, retinoic acid receptor; RARE, retinoic acid response element; RXR, retinoid X receptor; RXRE, retinoid X response element; TR, thyroid hormone receptor; TRE, thyroid hormone response element; VDR, vitamin D receptor; VDRE, vitamin D response element; vitD, 1,25-dihydroxycholecalciferol.

(^2)
In order to maintain continuity with the original description of spacing effects on ligand-dependent response elements, all spacings of DR response elements denote the nucleotide distances between the traditional core hexamer motifs.


ACKNOWLEDGEMENTS

We thank Eric Beninghof, David Olson, and Rajal Patel for their assistance in these experiments. P. Chambon kindly provided the mouse RXRalpha cDNA, R. Evans the human RARalpha cDNA, and J. W. Pike the human VDR cDNA.


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