T3 Response Elements Research Articles

Cell-Specific Regulation of Transcription of the Malic Enzyme Gene: Characterization of cis-Acting Elements That Modulate Nuclear T3 Receptor Activity

Stimulation of malic enzyme transcription by triiodothyronine (T3) is robust (>60-fold) in chick embryo hepatocytes, weak (5-fold) in chick embryo fibroblasts that stably overexpress the nuclear T3 receptor-α, and still weaker (1-fold) in chick embryo fibroblasts which contain nuclear T3 receptor levels that are similar to those of chick embryo hepatocytes. Using DNase I hypersensitivity, functional transfection, andin vitroDNA-binding analyses, four cis-acting elements were identified in the malic enzyme 5′-flanking DNA that conferred differences in nuclear T3 receptor activity between chick embryo hepatocytes and chick embryo fibroblasts. These cell-specific regulatory elements are located at −3895/−3890, −3761/−3744, −3703/−3686, and −3474/−2715 bp and overlap with DNase I hypersensitive sites that are observed in chromatin of chick embryo hepatocytes. Each element enhances T3 responsiveness of the malic enzyme promoter in chick embryo hepatocytes but has no effect on T3 responsiveness in chick embryo fibroblasts. Three of the cell-specific regulatory elements flank a previously identified DNA fragment (−3889 to −3769 bp; Hodnettet al., Arch. Biochem. Biophys.334, 309–324, 1996) that contains one major and four minor T3 response elements. The cell-specific regulatory element at −3703/−3686 bp binds to the liver-enriched factor, CCAAT/enhancer-binding protein-α, whereas cell-specific regulatory elements at −3895/−3890 and −3761/−3744 bp bind proteins of unknown identity. While the cell-specific regulatory element at −3761/−3744 bp contains sequences that resemble binding sites for CCAAT/enhancer-binding protein, activator protein-1, cyclic AMP response element binding protein, and NF-1, none of these proteins appear to bind to this DNA fragment. These data suggest that cell-specific differences in T3 responsiveness of the malic enzyme gene are mediated in large part by nonreceptor proteins that augment the transcriptional activity of the nuclear T3 receptor in hepatocytes.

The thyroid hormone down-regulates the mouse α-foetoprotein promoter

The thyroid hormone (T3) was shown to down regulate the level of α-foetoprotein (AFP) mRNA in hepatoma cells HepG2. Recombinant plasmids containing segments from the mouse AFP gene promoter were transfected in HepG2 cells and transient expression assays showed that the T3 inhibitory effect depends on the sequence limited by positions −80 and −38, upstream from the TATA box. This sequence is able to confer T3 sensitivity to a heterologous promoter and contains a putative T3-responsive element, as well as likely CEBP- and HNF1-responsive elements. These observations suggest that T3 is a good candidate for hormonal control of the AFP gene expression and especially for the neonatal shut off of the gene.

Isolation and Characterization of a Novel Ligand-dependent Thyroid Hormone Receptor-coactivating Protein

The thyroid hormone receptor (TR) regulates the expression of target genes upon binding to triiodothyronine (T3) response elements. In the presence of T3, the TR recruits coactivating proteins that both modulate and integrate the ligand response. We report here the cloning of a novel protein using the TR ligand-binding domain as bait in the yeast two-hybrid system. Analysis of a putative full-length clone demonstrates a cDNA sequence that encodes a protein of 920 amino acids with a size of 120 kDa (p120). Alignment with known sequences shows homology to a previously identified protein of unknown function, termed skeletal muscle abundant protein. Interaction studies demonstrate that p120 interacts with the TR AF-2 domain in the presence of ligand through a 111-amino acid region. Northern analysis demonstrates widespread expression in human tissues. Cotransfection assays in CV-1 cells demonstrate that p120 enhances TR-mediated transactivation on multiple T3 response elements in the presence of T3. In addition, CREB-binding protein synergizes with p120 to enhance this effect. When linked to the GAL4 DNA-binding domain, p120 is an activator of transcription alone. Thus, p120 satisfies a number of important criteria as a nuclear receptor coactivator.

3,5-Diiodo-L-thyronine (T2) has selective thyromimetic effects in vivo and in vitro.

Recent data have suggested that the iodothyronine, 3,5-diiodo-l-thyronine (T2), has selective thyromimetic activity. In vivo, T2 has been shown to suppress TSH levels at doses that do not produce significant peripheral manifestations of thyroid hormone activity. Furthermore, T2 has been shown to produce smaller increments in peripheral indices of thyroid status than does T3, when doses resulting in equivalent suppression of circulating TSH are compared. We have assessed the selective thyromimetic activity of T2 in vivo and in vitro, and performed in vitro studies to assess the potential molecular basis for these selective properties. T2 was 100-fold less potent than T3 in stimulating GH mRNA levels in GH3 cells. In contrast, the iodothyronines were almost equivalent in their ability to downregulate TRbeta2 mRNA levels in this cell line. Both 3,3'-diiodo-L-thyronine and thyronine exhibited no significant thyromimetic effects on either process. In vivo, doses of T2 and T3 that were equivalent in their induction of hepatic malic enzyme (ME) mRNA did not produce equivalent suppression of circulating TSH, with T2 being only 27% as effective as T3. T2 was up to 500-fold less potent than T3 in displacing [125I]-T3 from in vitro translated specific nuclear receptors (TRs) and GH3 cell nuclear extracts. Electrophoretic mobility shift assays, assessing the ability of T2 to produce dissociation of TRbeta1 homodimers from inverted palindrome T3 response elements, indicated that T2 was also 1000-fold less potent than T3 in this respect. These data confirm that T2 has significant thyromimetic activity, and that this activity is selective both in vivo and in vitro. However, there are no data to support a selective central effect, T2 being relatively more potent in stimulating hepatic ME mRNA than in suppression of TSH in vivo. The basis for this differential thyromimetic activity is not selective affinity of the different TR isoforms for T2, or divergent properties of T2 in competitive binding and functional assays in vitro.

The major transcription initiation site of the SV40 late promoter is a potent thyroid hormone response element.

Thyroid hormone receptors (TRs) are members of the nuclear hormone receptor superfamily, which act as transcription factors upon binding to specific DNA sequences called thyroid hormone (T3) response elements (TREs). Such elements are found in the upstream regulatory region of promoters as well as in intragenic sequences of T3-responsive genes. In this report, we demonstrate that SV40 late (SVL) promoter activity is strongly down-regulated by TR in the absence of ligand. Addition of T3 releases this repression, but does not further induce SVL promoter activity. Electrophoretic mobility shift analyses reveal a TR binding element that overlaps with the SV40 major late transcription initiation site. This element closely fits the consensus TRE, formed of two hexanucleotides organized in a tandem repeat separated by 4 nt, and is able to confer T3 responsiveness on a heterologous promoter. We further show that, although the presence of TR leads to quantitatively modified expression of an SVL-driven reporter gene, neither displacement of the site of transcription initiation nor modification of the splicing pattern of the primary transcripts occur.

Molecular cloning and functional characterization of the human cytosolic malic enzyme promoter: thyroid hormone responsiveness.

We report the structural and functional features of the 5'-flanking region of the human cytosolic malic enzyme (ME) gene. A 2.2-kb subclone, comprising 1.5 kb upstream of the translation initiation codon, the first exon, and 0.7 kb of flanking intronic region, was sequenced and mapped to chromosome 6. The proximal promoter region is rich in G + C, lacks TATA or CCAAT boxes, and shows multiple transcription start sites, the major one 106 nucleotides upstream the ATG codon. Sequences -59/-13 and -137/-103 conferred maximal promoter activity. Deletional analysis revealed the presence of two regions positively regulated by 3,5,3'-triiodo-L-thyronine (T3). The proximal region confers the strongest T3 inducibility to the human ME as well as to a heterologous promoter. Thyroid hormone receptor beta (TRbeta) binds to an inverted palindromic T3 response element (TRE) at position -105/-87 in a manner that is prevented by T3. Nuclear extracts or in vitro-translated retinoid acid receptor alpha (RXR alpha) shifted the TRbeta retarded band to slower-mobility complexes, which are unaffected by T3. In the absence of T3, overexpression of TRbeta repressed the ME promoter activity, most probably, through binding of TRbeta homodimers to the TRE. Thus, T3 seems to control ME transcription by inducing the dissociation of TRbeta homodimers and the functional activation of liganded heterodimers.

The function of retinoid X receptors on negative thyroid hormone response elements

Retinoid X receptors (RXRs) form heterodimers with thyroid hormone receptors (TRs). RXRs increase DNA binding affinity of TRs and T3-mediated transactivation on positive T3 response elements (TREs). However, the role of RXRs on negative TREs, and the relation of RXRs to the dominant negative effect of mutant TRs, are not defined. To clarify the function of RXRs on negative TREs, we performed transient cotransfection studies using the rat glycoprotein hormone α promoter fused to luciferase gene ( αLuc), and human TRH promoter fused to luciferase gene (TRH-Luc) as reporters. We found that the JEG-3 cell- αLuc system was very sensitive to TR regulation. Using TR β1 wild-type (WT) expression vector, 6.2 ng/well (170 ng/10 cm dish), and 0.2 ng/well (11 ng/10 cm dish) caused maximal, and half maximal, inhibition of Luc activities in the presence of 1 nM T3. A T3 dose dependent inhibition study was also performed. From these studies, we determined that the appropriate conditions in which to study αLuc transactivation, in a linear portion of the dose response curve, was using 0.8 ng/well TR β1 expression vector and 0.1 nM T3. Under these conditions, TR β1 mutant R316H (GH), but not G345R (Mf), showed a weak dominant negative effect at a 1:1 ratio in the presence of 0.1 nM T3 although neither mutant had detectable T3 binding affinity. Moreover this dominant negative effect of R316H on the αLuc reporter was enhanced in the presence of RXR γ. Mutant G345R showed a stronger dominant negative effect than did R316H when using a double palindromic TRE fused to herpes simplex thymidine kinase-Luc reporter as a positive TRE. These results conform to the clinical features of R316H which is associated with apparent pituitary resistance of thyroid hormone (PRTH). Mutant R316H also showed a weak dominant negative effect with TRH-Luc at a 1:1 ratio in the absence or presence of RXR γ. However RXR γ did not enhance the dominant negative effect as it did using αLuc reporter gene. Electrophoretic gel mobility shift assay (EMSA) showed that RXR α augmented the DNA binding affinity of wild type and R316H TRs as heterodimers on the previously reported negative TREs of glycoprotein hormone α promoter, suggesting that RXR does not produce its response by removing TRs from these TREs. RXR α augmented DNA binding affinity of TR β1WT, and R316H showed a weaker heterodimer band than did the wild type in EMSA. Using the TRH-Luc reporter, basal activity was increased by wild type TR β1. However a TR β1 DNA binding domain mutant, (C127S) which can not bind to DNA, did not increase the basal activity. This indicates that DNA binding of the TR is required for increasing basal activity of TRH promoter. These results indicate that (1) RXR-TR heterodimers play a role in basal transactivation and T3 suppression of negatively regulated genes, and (2) RXRs increase the dominant negative effect of some mutant TRs on specific negative TREs. (3) This effect occurs without removing TRs from the TRE. (4) The differential dominant negative effect of mutant R316H (negative TRE>positive TRE) may explain, at least in part, the presentation of R316H as PRTH. (5) Augmentation of basal activity by wild type TRs on a negative TRE requires DNA binding.

Transient stimulation of myelin basic protein gene expression in differentiating cultured oligodendrocytes: a model for 3,5,3'-triiodothyronine-induced brain development.

We compared the regulation of myelin basic protein (MBP) gene expression by T3 in differentiating oligodendrocytes in culture with that previously observed by us in the neonatal rat brain. As in intact brain, expression of the T3R alpha gene preceded that of the T3R beta gene. Although the absence of T3 retarded the rate of accumulation of MBP messenger RNA, the level ultimately attained was similar to that reached in the presence of T3. This relationship mirrored the pattern observed in the neonatal brain. Transient transfection experiments showed that T3 regulates MBP expression at the transcriptional level, but only for a limited period during differentiation. These observations imply that the early rise of MBP messenger RNA is T3 dependent, whereas the terminal levels are maintained independently of T3. Both the T3-dependent and, surprisingly, the T3-independent expression of MBP require the presence of an intact T3 response element. T3 receptor may regulate MBP expression in a ligand-independent manner, or a nuclear factor other than T3 receptor may bind to the T3 response element of MBP to regulate terminal gene expression. These findings support the use of differentiating oligodendrocytes as a model of T3-induced brain development.

Introducing a point mutation identified in a patient with pituitary resistance to thyroid hormone (Arg 338 to Trp) into other mutant thyroid hormone receptors weakens their dominant negative activities.

Clinical resistance to thyroid hormone (RTH) has been classified into generalized resistance to thyroid hormone (GRTH) and pituitary resistance to thyroid hormone (PRTH) types. Since similar mutations have been identified in tri-iodothyronine (T3) receptor (TR) beta gene in GRTH and PRTH, and since considerable overlap has been seen in the clinical manifestations in patients with GRTH and PRTH, two subtypes of RTH are now considered to be a continuous spectrum with the same genetic defect. A point mutation at amino acid Arg 338 to Trp (R338W) which we identified in a patient with PRTH is very interesting, since R338W has been found in several other patients with PRTH, raising the possibility that this mutation may tend to associate with a phenotype of PRTH. In our previous study, we found that R338W had relatively less impaired transcriptional potency, weaker dominant negative activity on various T3 response elements and poor homodimer formation, as compared with another GRTH mutant. In this study, to investigate the functional properties of R338W further, especially in terms of the relation between transcriptional activity and dimer formations, we introduced the R338W mutation into the mutant receptors, K443E and F451X, constructing the double mutants, R338W/K443E and R338W/ F451X. Both R338W/K443E and R338W/F451X showed negligible T3 binding and transcriptional activities. The dominant negative activities of K443E and F451X were, however, significantly weakened by introducing the R338W mutation. As a control, a double mutant G345R/K443E was constructed by introducing a point mutation, G345R, located in the same exon 9 as R338W, into the K443E mutant. Dominant negative activity did not differ between G345R/K443E and K443E. Homodimer formation was significantly reduced in the double mutants containing R338W, but not G345R. In summary, introducing the R338W mutation, but not G345R, into the mutant TR significantly weakened the dominant negative activity, despite further impairment of the T3 binding and transcriptional activities.

Negative Regulation of the Gene for the Preprothyrotropin-releasing Hormone from the Mouse by Thyroid Hormone Requires Additional Factors in Conjunction with Thyroid Hormone Receptors

To gain additional insights into the negative gene regulatory action by triiodothyronine (T3), we isolated a 2-kilobase pair 5'-flanking region of the mouse preprothyrotropin-releasing hormone (ppTRH) gene and characterized the DNA elements mediating inhibitory regulation by T3 in the promoter region. In GH4C1 cells, the expression of the 2-kilobase pair mouse ppTRH 5'-flanking region fused to the luciferase reporter gene occurred by transfection and was significantly suppressed by T3. In contrast, T3 suppression was not observed in T3 receptor (T3R)-deficient CV-1 cells, suggesting that T3Rs were required for the negative regulation. Cotransfected mouse T3R alpha1, beta1, and beta2 possessed indistinguishable potency for the negative regulation. Deletion analysis localized the element mediating the negative regulation to the region between -83 and +46, and the sequence downstream of the transcription start site (TSS) between +12 and +46 was found to be essential for the inhibitory regulation. In mobility shift assays, only T3R monomers bound to the element containing a T3 response element half-site at -57. No apparent T3R binding was observed to the element downstream of TSS. Neither the T3 response element half-site nor the element downstream of the TSS confer T3 suppression individually in heterologous promoters. These results indicate that the negative regulation of murine ppTRH gene by T3 might be mediated by the cooperation of T3R monomers with unknown factor(s) interacting with the element downstream of the TSS.

Thyroid hormone and estrogen interact to regulate behavior.

Environmental perturbations that increase plasma thyroid hormone (T3) concentrations also profoundly affect female reproductive behavior and physiology. We explored whether these effects were mediated by interactions between T3 receptor (TR) and estrogen receptor (ER). This hypothesis was of interest because the half-site of a consensus T3 response element DNA sequence is identical to an ER response element (ERE), and TRs bind to a consensus ERE. Molecular data presented in the accompanying paper [Zhu, Y.-S., Yen, P.M., Chin, W.W.& Pfaff, D.W. (1996) Proc. Natl. Acad. Sci. USA 93, 12587-12592] demonstrate that TRs and ERs are both present in rat hypothalamic nuclear extracts and that both can bind to the promoter the hypothalamic gene preproenkephalin and that interations between liganded TRs and ERs affect preproenkephalin transcription. In this paper, we show that molecular interactions between TRs and ERs are sufficient to mediate environmental effects on estrogen-controlled reproductive behavior. Ovariectomized (OVX) rats treated with high doses of T3 showed significantly lower levels of lordosis behavior in response to estradiol benzoate (EB) compared with OVX females treated with EB alone. Conversely, thyroidectomized/OVX females treated with EB showed significantly greater levels of lordosis behavior compared with OVX females treated with EB, showing the effect of endogenous T3. Thyroid hormone interference with EB-induced behavior could not be explained by a reduction in plasma E2 concentrations or by a general reduction in responsiveness of EB-sensitive tissues. Moreover, numbers of hypothalamic ER-immunoreactive cells increased dramatically following T3 treatment. These data suggest that T3 may reduce EB-dependent sexual behavior through interactions between TR and ER in the nuclei of behaviorally relevant hypothalamic neurons, envisioning for the first time a functional consequence of interactions between two nuclear hormone receptors in brain. These results also open up the possibility of molecular interactions on DNA encoding environmental signals, a new field for the study of neuronal integration.

The Chicken Malic Enzyme Gene: Structural Organization and Identification of Triiodothyronine Response Elements in the 5′-Flanking DNA

In vivo,feeding stimulates and starvation inhibits transcription of the malic enzyme gene. In chick-embryo hepatocytes in culture, triiodothyronine (T3) stimulates and glucagon inhibits transcription of this gene. As a first step in the characterization of the involved regulatory mechanisms, fragments of genomic DNA spanning the structural and 5′-flanking regions of the chicken malic enzyme gene were cloned. The coding region of the gene is organized into 14 exons and 13 introns and is greater than 106 kb in length. The size of the gene, the number and lengths of the exons, and positions at which introns are inserted into the coding regions are virtually identical in the chicken and rat genes. When transiently transfected into chick-embryo hepatocytes, 5800 bp of 5′-flanking DNA conferred T3 responsiveness to a linked chloramphenicol acetyltransferase (CAT) reporter gene. Using deletion and site-specific mutations of 5′-flanking DNA, we identified a complex T3 response unit that contains one major T3 response element (T3RE) and several minor ones. The major element contains two degenerate copies of the hexamer, RGGWMA, separated by 4 bp and was a strong repressor in the absence of ligand. Endogenous levels of T3 receptor are sufficient to allow the T3 response elements in the upstream region of the malic enzyme gene to confer responsiveness to T3, suggesting that they are physiologically relevant.

Malic enzyme gene in chick embryo hepatocytes in culture: clofibrate regulates responsiveness to triiodothyronine

In chick embryo hepatocytes, triiodothyronine (T3) causes a 30- to 40-fold increase in malic enzyme activity when added between 1 and 3 days, but has no effect when added between 5 and 7 days in culture. This transcription-mediated decline in T3 responsiveness is partially reversed by corticosterone (Roncero, C. and A. G. Goodridge, 1992. Arch. Biochem. Biophys. 295: 258-267). Clofibrate also reversed the decline in responsiveness to T3, and did so in the absence of an increase in binding of T3 to nuclear receptors. The effects of clofibrate and corticosterone were additive, suggesting different mechanisms. The responsiveness of a gene to a specific agent depends on specific regulatory sequences of DNA in that gene. When 5.8 kb of the 5'-flanking DNA of the malic enzyme gene was linked to the chloramphenicol acetyltransferase (CAT) gene and transfected into hepatocytes, T3 stimulated CAT activity. Responsiveness of CAT activity to T3 decreased with time, and this decrease was partially reversed by clofibrate. The T3 responses of cells transfected with various chimeric DNAs that contained T3 response elements (T3REs) of the malic enzyme gene or synthetic consensus T3REs also were increased by clofibrate. The results suggest that clofibrate regulates expression of a metabolite or a protein factor which, in turn, influences function of the T3 receptor.

Thyroid Hormone Activation of Transcription Is Potentiated by Activators of cAMP-dependent Protein Kinase

We characterized the cross-talk between activators of protein kinase A (PKA) and thyroid hormone (T3) in T3 receptor (TR)-mediated transcription. U937 cells were cotransfected with a plasmid expressing the TR and a reporter plasmid containing a T3 response element (TRE) oriented either as a direct repeat or as a palindrome upstream of the thymidine kinase promoter linked to the chloramphenicol acetyltransferase gene. T3 activated transcription by 10-fold. T3 response was potentiated 2.5-3-fold by activators of PKA, but an activator of protein kinase C or of guanylate kinase was ineffective. In the absence of T3, activators of PKA had no effect on transcription. TR heterodimerization with the retinoid X receptor may facilitate T3/PKA cross-talk because coexpression of the retinoid X receptor potentiated cross-talk. Synergy was not observed in JEG-3, F9, CV-1, HeLa, L929, and HTC cells, indicating that it may require cell-specific factors. Synergy required the DNA- and ligand-binding domains, but not the amino-terminal domain, indicating that T3- and TRE-induced conformational changes on the TR are essential for cross-talk. PKA phosphorylated the TR in vitro, suggesting that, like other nuclear receptors, the TR is a target for PKA. These results imply that PKA cross-talks with T3 at the level of the TRE-bound TR, enhancing its transcriptional activity in a cell-specific manner.

Ligand induction of a transcriptionally active thyroid hormone receptor coactivator complex.

Transcriptional regulation by nuclear hormone receptors is thought to involve interactions with putative cofactors that may potentiate receptor function. Here we show that human thyroid hormone receptor alpha purified from HeLa cells grown in the presence of thyroid hormone (T3) is associated with a group of distinct nuclear proteins termed thyroid hormone receptor-associated proteins (TRAPs). In an in vitro system reconstituted with general initiation factors and cofactors (and in the absence of added T3), the "liganded" thyroid hormone receptor (TR)/TRAP complex markedly activates transcription from a promoter template containing T3-response elements. Moreover, whereas the retinoid X receptor is not detected in the TR/TRAP complex, its presence is required for the function of the complex. In contrast, human thyroid hormone receptor alpha purified from cells grown in the absence of T3 lacks the TRAPs and effects only a low level of activation that is dependent on added ligand. These findings demonstrate the ligand-dependent in vivo formation of a transcriptionally active TR-multisubunit protein complex and suggest a role for TRAPs as positive coactivators for gene-specific transcriptional activation.

In vivo genomic footprinting of thyroid hormone-responsive genes in pituitary tumor cell lines.

We studied the effects of thyroid hormone (T3) on nuclear protein-DNA interactions by using dimethyl sulfate (DMS) and DNase I ligation-mediated PCR footprinting. We examined an endogenous gene the growth hormone (GH) gene, and a stably transfected plasmid containing the chicken lysozyme silencer (F2) T3 response element (TRE) gene, F2-TRE-TK-CAT, both in pituitary tumor (GC) cells. The 235-1 cell line, which expresses prolactin (PRL) and Pit-1, but not the T3 receptor (TR) or GH, was used as a control. DMS and DNase I footprinting identified protected G residues in the Pit-1, Sp1, and Zn-15 binding sites of the GH gene in GC, but not in 235-1, cells. There was no specific protection of the tripartite GH TRE at -180 bp against either DMS or DNase I in the absence or presence of T3 in either cell line. However, T3 increased protection of the Pit-1 and Sp1 binding sites against DMS in GC cells. In GC cells stably transfected with a plasmid containing F2-TRE-TK-CAT or TRalpha, chloramphenicol acetyltransferase expression was T3 inducible and DMS footprinting revealed both F2 TRE TR-binding half sites in a pattern suggesting the binding of TR homodimers before and during T3 exposure. We conclude that the GH gene is accessible to specific nuclear proteins in GC, but not in 235-1, cells and that T3 enhances this interaction, although there is no evidence of TR binding to the low-affinity rat GH TRE. The presence of TR binding to the high-affinity F2 TRE before and during T3 exposure suggests that reversible interaction of T3 with DNA-bound TRs, rather than transient T3-TR contact with TREs, determines the level of T3-stimulated transcriptional activation.

Alternative splicing gives rise to two isoforms of Zfhep, a zinc finger/homeodomain protein that binds T3-response elements.

We have previously isolated a cDNA for a transcription factor referred to as Zfhep (zinc finger homeodomain enhancer-binding protein) containing two separate zinc finger domains, ZD1 and ZD2, each of which binds DNA, and a homeodomain. The rat Zfhep cDNA lacks a 5'-methionine codon, present in some homologs from other species. Hence, the aim of this work was to isolate the 5'-end of the rat Zfhep cDNA. Zfhep-2 cDNA was isolated, having a total length of 2.5 kbp, including more than 1.1 kbp of novel sequence followed by 1.4 kbp identical to the Zfhep-1 clone. The 1.1 kbp of novel sequence contains multiple stop codons in all reading frames, suggesting that it represents the 5'-untranslated (5'-UT) region of the rat Zfhep-2 mRNA. However, the Zfhep-2 clone does not contain the extreme 5'-exon(s) of the Zfhep-1 coding sequence, possibly due to alternative splicing of Zfhep RNA. To distinguish between a splice junction versus an intron-exon junction, the polymerase chain reaction (PCR) with rat genomic DNA and junction-flanking primers from the Zfhep-2 sequence was conducted. No bands were amplified from the genomic DNA by two different pairs of primers, indicating that the Zfhep-2-specific sequence is not intronic. Ribonuclease protection assays were performed to investigate the expression of multiple Zfhep mRNAs. Two protected bands were detected, and both were identified in total RNA or mRNA of rat ovary, hindbrain, forebrain, heart, kidney, small intestine, and GH4C1 cells. Zfhep-2 represents about 20% of the Zfhep RNA in each tissue. Hence, two mRNAs are expressed in these tissues, confirming the alternative splicing. To confirm independently the presence of both Zfhep-2 and Zfhep-1 mRNAs, reverse transcriptase (RT)-PCR was done using primers that span the Zfhep splice site. Specific bands representing both RNAs were obtained. The Zfhep-2 PCR product was subcloned and DNA sequence analysis confirmed the absence of ATG codons near the 5'-end of the open reading frame. The theoretical translation of the Zfhep-2 clone predicts a smaller protein than Zfhep-1. In vitro translation in reticulocyte lysates showed that Zfhep-2 is about 40 kD smaller than Zfhep-1. Hence, Zfhep-2 apparently lacks most of the first zinc finger domain (ZD1) of Zfhep-1. Because the two zinc finger domains bind different DNA sequences, Zfhep-2 is predicted to bind to only a subset of genes recognized by Zfhep-1.

Peroxisome Proliferator-activated Receptor α Inhibits Hepatic S14 Gene Transcription: EVIDENCE AGAINST THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR α AS THE MEDIATOR OF POLYUNSATURATED FATTY ACID REGULATION OF S14 GENE TRANSCRIPTION

The peroxisome proliferator-activated receptor (PPARalpha) has been implicated in fatty acid regulation of gene transcription. Lipogenic gene transcription is inhibited by polyunsaturated fatty acids (PUFA). We have used the PUFA-sensitive rat liver S14 gene as a model to examine the role PPARalpha plays in fatty acid regulation of hepatic lipogenic gene transcription. Both PPARalpha and the potent peroxisome proliferator, WY14643, inhibit S14CAT activity in transfected primary hepatocytes. WY14643 and PPARalpha target the S14 T3 regulatory region (TRR, -2.8 to -2.5 kilobases), a region containing 3 T3 response elements (TRE). Transfer of the TRR to the thymidine kinase (TK) promoter conferred negative control to the TKCAT gene following WY14643 and PPARalpha treatment. Gel shift analysis showed that PPARalpha, either alone or with RXRalpha, did not bind the S14TRR. However, PPARalpha interfered with TRbeta/RXRalpha binding to a TRE (DR+4). Functional studies showed that co-transfected RXRalpha, but not T3 receptor beta1 (TRbeta1), abrogated the inhibitory effect of PPARalpha on S14 gene transcription. These results suggest that WY14643 and PPARalpha functionally interfere with T3 regulation of S14 gene transcription by inhibiting TRbeta1/RXR binding to S14 TREs. Previous studies had established that the cis-regulatory targets of PUFA control were located within the proximal promoter region of the S14 gene, i.e. between -220 and -80 bp. Finding that the cis-regulatory elements for WY14643/PPARalpha and PUFA are functionally and spatially distinct argues against PPARalpha as the mediator of PUFA suppression of S14 gene transcription.

A Unique Thyroid Hormone Response Element in the Human Immunodeficiency Virus Type 1 Long Terminal Repeat That Overlaps the Sp1 Binding Sites

Long terminal repeat (LTR) of human immunodeficiency virus (HIV) type 1 is activated by thyroid hormone (T3) receptor alpha (T3R alpha) in the absence of ligand. Addition of T3 reverses this effect. This activity is mediated by a high affinity T3 response element (T3RE) within the HIV-1 LTR, termed the HIV-T3RE (bases -74 to -50), which coincides with the Sp1 element as demonstrated by mobility shift, DNaseI footprinting, and methylation interference analyses. HIV-T3RE mediates ligand-independent activation of transcription by T3R alpha when linked to a heterologous promoter. In addition, the viral transactivator Tat synergizes with T3R alpha to activate the HIV-1 LTR in the absence of T3, which is relieved in its presence. These findings have implications for the possible control of HIV-1 LTR activity by T3.

Thyroid hormone receptor-beta mutants associated with generalized resistance to thyroid hormone show defects in their ligand-sensitive repression function.

Thyroid hormone (T3) responses are mediated by two receptors, TR alpha and TR beta, that have been shown to require heterodimer formation with the retinoid-X receptors for effective interaction with most T3-responsive elements (TRE). In addition, it has been shown recently that one type of TRE, an inverted palindrome (IP) with a 4-, 5-, or 6-base pair spacer, can also bind TR homodimers with high affinity. This binding, however, is sensitive to T3, which suggests that TR homodimers could have important biological roles as T3-sensitive repressors. Here we have analyzed eight natural TR beta mutants associated with the syndrome of generalized resistance to thyroid hormone (GRTH). These receptor mutants are characterized by a variably decreased affinity for T3. We show here that their homodimer binding characteristics are altered. For example, kindred GH binds as a homodimer more weakly to DNA than wild-type (WT), whereas mutant PV forms clearly stronger homodimer complexes than WT even in the presence of TREs that bind WT receptor homodimers poorly. Although other mutants were able to bind IP-6 elements efficiently as homodimers, these homodimers showed a decreased sensitivity to T3 in accordance with their reduced affinities for the ligand. In vivo, six of the eight mutants were able to function as strong repressors on IP sites located 3' of the TATA box. Although T3 released repression by WT TR beta, the hormone did not release repression by some of the mutant receptors, and elevated concentrations of T3 were required to release repression by other mutants. Importantly, most of the GRTH-associated mutants were able to function as potent dominant negative repressors of WT in the homodimer pathway, whereas they showed little dominant negative activity in the heterodimer-dependent transcriptional activation pathway. Only one of the eight GRTH mutants, a deletion of the carboxy-terminus, was found to have a strong dominant negative activity on both T3 response pathways. Our data suggest a dominant negative mechanism of action for GRTH mutants that is consistent with their homodimer binding characteristics to IP-TREs and correlates well with T3 resistance in patients.