Characteristics of a Negative Thyroid Hormone Response Element*

Thyroid hormones may stimulate or repress tran- scriptional activity depending upon the specific gene. Whereas, a palindromic DNA sequence, TREpal, medi- ates positive regulation by thyroid hormone, the negative response element (nTSRE) remains undefined. Therefore, we have examined the DNA sequences that mediate the inhibitory effects of thyroid hormone on the transcription of the @-subunit gene of rat thyrotropin (rTSH@). In rat pituitary tumor cells (GH,), transient expression of plasmid constructs containing the puta-tive nTsRE of rTSH@ mediated negative regulation by L-triiodothyronine (T,). Since this nTsRE contained sequences which resembled a half-site motif of the consen- sus TsRE and the idealized palindrome (I'REpal), we tested a construct containing this half-site motif in the same cells. Ts decreased the activity of this plasmid. Co-transfection studies in Ts-receptor (T,R)-deficient cells indicated that either a or f3 isoforms of T,R were required for the inhibitory effects of the hormone. Both TsR isomers bind to DNA sequences containing the nTsRE from rTSHB DNA or the half-site motif of TREpal. In summary, our results show that the repressive properties of Ts are mediated by a nTSRE from rTSH@. Un- expectedly, this motif resembles a half-site component of TREpal which enhances promoter activity in response to TS.


Thyroid hormones may stimulate
or repress transcriptional activity depending upon the specific gene. Whereas, a palindromic DNA sequence, TREpal, mediates positive regulation by thyroid hormone, the negative response element (nTSRE) remains undefined. Therefore, we have examined the DNA sequences that mediate the inhibitory effects of thyroid hormone on the transcription of the @-subunit gene of rat thyrotropin (rTSH@). In rat pituitary tumor cells (GH,), transient expression of plasmid constructs containing the putative nTsRE of rTSH@ mediated negative regulation by L-triiodothyronine (T,). Since this nTsRE contained sequences which resembled a half-site motif of the consensus TsRE and the idealized palindrome (I'REpal), we tested a construct containing this half-site motif in the same cells. Ts decreased the activity of this plasmid. Cotransfection studies in Ts-receptor (T,R)-deficient cells indicated that either a or f3 isoforms of T,R were required for the inhibitory effects of the hormone. Both TsR isomers bind to DNA sequences containing the nTsRE from rTSHB DNA or the half-site motif of TREpal. In s u m m a r y , our results show that the repressive properties of Ts are mediated by a nTSRE from rTSH@. Unexpectedly, this motif resembles a half-site component of TREpal which enhances promoter activity in response to TS.
Thyroid hormone regulation of gene expression includes both strong inductive as well as repressive effects (1, 2). A consensus palindrome derived from the rat growth hormone (rGH)' gene mediates positive regulation by T, (3-5

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inverted repeat (TAGGAC GGACCG) referred to as sites "B" and "C," respectively, this element mediates the positive effects of T3 on rGH gene transcription (4).
Sites A, B, and C are considered to be "half-sites" of a positive T3RE. This observation gave rise to the notion that positive T3REs, such as found in the rGH gene and TREpal, are comprised of more than on repeat of a half-site, whereas a nT3RE is formed by a single copy of the half-site motif. In support of this hypothesis, recent studies of other genes which are negatively regulated by T3 such as thyrotropin-releasing hormone (7), epidermal growth factor receptor (81, as well as the a-subunit of rTSH (9, 10) have revealed 6-base pair motifs that resemble the nT3RE of rTSHP. Therefore, we have examined the possibility that differential regulation by T3 may be attributed to the presence of a single or multiple repeats of a core motif by measuring the ability of a nT3RE from rTSHp and the half-site motif of TREpal to confer negative regulation to the heterologous thymidine kinase (TK) promoter.
All plasmid constructs were confirmed by DNA sequence analysis.
Statistical Analysis-For hormonal treatments, statistical signilicance was determined by analysis of variance and multiple t test (Duncan's). Significant variation in basal expression of plasmids was determined by one-way analysis of variance.

RESULTS
Location of Negative T3REs in rTSHp Promoter DNA-We have shown previously that two regions (-17/+9 and +11/+27) in the rTSHp promoter mediate the negative actions of T3 on a heterologous promoter (6). To locate precisely the nucleotides responsible for the inhibition, we introduced mutations within these regions of the native TSHp promoter (Fig. 1). The activity of the wild type promoter was reduced by 67% following 24-h exposure to T3. Motifs (Fig. lA, thick horizontal arrows) that resembled the consensus T3RE half-site were targeted for the mutational analysis. Transverse mutations in -17/+9 region at positions -9 and -8 ( Fig. 1, M-17/+9) reduced the negative effect of T3 to 50%. In contrast, mutations within the +11/+27 region (+18, +23, +27) eliminated sensitivity to T3 and slightly decreased basal expression (Fig. 1, M+11/+27). Mutations at intron sites 5 and 13 (Fig. 1, I1 /Z13) failed to alter the inhibitory properties of the promoter in the presence of T3. Results arising from four separate transfections using each one of the constructs yielded data shown in thegraph of Fig. lB. Together these observations indicate that the +11/+27 region of the rTSHp gene is the prominent region that mediates the inhibitory effects of the hormone.
Halfsite Motifs of the T a E s Mediate Negative Regulation by T-The preceding results indicated that a limited region of the +11/+27 sequence mediated the negative actions of T3. To test this possibility single copies of both the +11/+27 sequence and a more limited region (+18/+27) were inserted in front of the viral TK promoter to yield TSHp(+11/+27)TKCAT and TSHp(+l8/+27)TKCAT. In transfected pituitary GH3 cells, T3 decreased the promoter activity of both TSHpTKCAT chimera ( Fig. 2). As shown previously (61, TKCAT activity (percent total countdmin converted per pg of protein) was not different in the absence or presence (13.7 2 2.3, 11.3 2 3.1, respectively) of added T3 (lo-' M, 24 h). This observation narrowed the inhibitory effects of T3 to the +18/+27 sequence of the rTSHj3 nT3RE and raised the possibility that a half-sib motif within this region mediated repressive actions of the hormone. To test the half-site hypothesis, we inserted the half-site motif of the idealized palindrome derived from the rGH T3RE (TREpal(U2)) in the 5"flanking region of TKCAT to yield TREpal(U2)TKCAT.
As shown previously by numerous investigators, including ourselves, the intact TREpal conferred positive T3 regulation to a heterologous promoter (6).
In GH3 cells transfected with TREpal(l/2)TKCAT, T3 decreased CAT activity in a dose-dependent manner (Fig. 2). Together these results support the idea that a half-site motif can mediate the inhibitory effects of

Presence of T$ Is Required to Mediate the Repressive Effects
of T-The hormone-dependent inhibition of CAT activity in the transfected cells presumes that the negative effects of T3 were mediated by endogenous T3Rs present in GH3 cells. To verify that T3fi were indeed involved in this process, we introduced the various reporter constructs into T3R-deficient COS cells. Although CAT activity in COS cells co-transfected with T3Rs, and the parent vector, TKCAT, was unresponsive to T3, the expected increase was observed in cells co-transfected with the TREpalTKCAT construct (Fig. 3, A and B, respectively).
T3 had no effect on TSHp(+lY+27)TKCAT, TSHp(+18/ +27)TKCAT, or TREpal(V2)TKCAT expression when transfected alone into the COS cells (Fig. 4). However, T3 suppression of CAT activity was restored when the templates were co-transfected with an expression vector (PMT2) containing the cDNA for either T3Ra or T3RP. Co-transfection of either T3Ra or T3Rp enhanced basal expression of the TSHpTKCATs (Fig.   4, A and B) and TREpal(U2)TKCAT (Fig. 4C) constructs without diminishing the negative response to T3. The ability of T3Ra or T3Rp to enhance basal expression has also been noted T3. on other TRE models (13). These results confirm that the inhibitory effects of the nT3RE required the presence of T3R.
Recent studies indicating that a receptor for the O-cis-retinoic acid (RXR) heterodimerizes with various T3R isoforms prompted us to ask whether such an interaction might aug-ment the function of nT3REs. We co-transfected RXRP with T3Ra or T3RP and the T3RE half-site templates into COS cells. Unexpectedly, we found that co-transfection with RXRp blunted or abolished the negative response to T3 (Fig. 4). T3R Isomers Bind to the T3RE Half-site Motifs-Finally, we examined whether both T3R isomers bind to the TSHp T3RE. Nuclear extracts of transfected COS cells were tested for binding to the motif. Both T3Ra and T3Rp bound to the radiolabeled nT3RE (Fig. 5). Competition with homologous DNA or TREpal(l/2) DNA abolished binding of the T3R isoforms to the radiolabeled probe, whereas addition of nonspecific DNA failed to compete. The major complex is a monomer (upper arrow) consistent with our previous studies on in vitro synthesized hT3Rp (6). Because we used COS cell extracts in this study, the possibility exists that the upper complex may be a heterodimer comprised of a single T3R and a TRAP protein. A faster migrating complex (lower arrow) represents TSHp-nT3RE binding activity present in untransfected COS cell extracts. Together these observations have shown that both T3Ra and T3RP bind to rTSHp nT3RE and that homologous sequence or TREpal( 1/2) serve as potent competitors.

DISCUSSION
In this report, we have presented evidence that clearly shows a nonpalindromic half-site motif mediated the negative actions of T3. Specific mutations to motifs resembling a consensus T3RE half-site present in the context of the native rTSHp promoter impaired or abolished the inhibitory effects of T3. These findings extend our previous observations in vitro that mutations in these regions impair or completely block binding to GH3 nuclear protein extracts containing T3Rs (6). That T3RE half-site motifs were important features for negative regulation by T3 was further demonstrated by T3 suppression of TK promoter activity only in the presence of the rTSHp DNA. Additional support for this hypothesis was obtained by examining the half-site from TREpal, a palindromic element that responds ;ffects of T3 positively to TS. In these studies, T3 decreased TREpal(l/ 2)TKCAT in GH3 cells.
The suppressive effects of T3 required the presence of either the a or p isoforms of T3R. Unexpectedly, we found that the additional presence of RXR blunts or abolishes T3 inhibitory effects. One plausible explanation for this observation is that the known ability of RXRP to heterodimerize with T3Ra or T3RP effectively sequesters T3R in a form that restricts its interaction with the nT3RE. In contrast, RXRP augments the stimulatory effects of T3 and enhances binding to T3REs that mediate positive effects of the hormone (14-161, as verified in results summarized in Fig. 3B. How does this information fit into the current concepts of thyroid hormone action? The activity of the T3RE in positively regulated genes or the canonical T3RE appears to be dependent on the orientation, spacing of the half-sites, and context within the promoter (13,(17)(18)(19). The transcriptionally active form of the T3R is considered to be a homodimer or heterodimer, as suggested by the presence of multiple half-site motifs and the loss of the T3 stimulatory activity with mutation of one of the half-sites (20-22). The stimulatory activity of T3 has been attributed to enhanced stability of heterodimer formation between T3R and T3R auxillary proteins (TRAPS) and derepression of the dominant negative homodimers of T3R (23-28).
Although the preceding model may account for inductive effects of T3 on gene activity, it does not explain the inhibitory effects of T3 nor can it accommodate the findings in this report. These studies indicate that the half-site composition of the nT3RE does not conform to the homo-or heterodimer model of T3 action. Both T3R isoforms bind to the various T3REs and mediate positive and negative regulation by T3. Current data suggest that positive regulation by T3 is mediated by a palindrome such as TREpal or imperfect repeats as present in rGH DNA. In contrast, negative regulation appears to be mediated by half-site motifs as reflected in TSHp and other genes inhibited by T3 (6-10, 13, 29-33). T3Rs bind as either homoor heterodimers to positive T3REs, but only a monomer or heterodimer can bind to the half-site nT3RE (13,(17)(18)(19)(23)(24)(25)(26)(27)(28). Based on these observations, we speculate that for nT3REs, in the absence of T3, T3R has a neutral or a positive effect on transcription. Addition of T3 alters the conformation of the T3R bound to DNA such that it cannot activate transcription. Another possibility is that exposure to T3 may stabilize the T2R TRAP interaction such that it is sequestered from interactions with the nT3RE. Such a model is supported by our observation that RXRp abolishes the inhibitory effects of T3R. Both models may be functional depending upon the specific sequence and context of individual T3REs.
In summary, positive regulation by T2 is mediated by an idealized T3REpal or imperfect repeats, whereas negative effects reside in a half-site motif found in TSHp and other genes suppressed by T3 (7-10). The binding of T3R homo-/ heterodimer to the positive elements and a monomer to nT2RE may account for the differential actions of T3. Based on these observations, we propose that monomer T3R binding to a halfsite motif in the absence of T3 exerts a neutral or a positive effect on transcription. Addition of T3 alters the conformation of the T3R bound to DNA, leading to a change in activity possibly by facilitating interaction with TRAP proteins (activator or derepressor) or by destabilizing the T3R-DNA complex and in turn disrupting the transcription initiation complex. These models are not mutually exclusive and may contribute to the overall process of negative regulation by T3, depending on the specific nT3RE and the surrounding elements in the context of the native gene.