Role of PSF-TFE3 oncoprotein in the development of papillary renal cell carcinomas.

A subset of papillary renal cell carcinomas (RCC) is characterized by the expression of a TFE3 fusion protein, where the fusion partner can be any of the several proteins identified so far such as PSF (PTB associated splicing factor), NonO, PRCC, CLTC and ASPL. These proteins result from chromosomal translocations involving the TFE3 gene located on the X chromosome. Our present study documents the central role of PSF-TFE3 in oncogenic transformation. We show that the inhibition of PSF-TFE3 expression through siRNA or shRNA leads to impaired growth, proliferation, invasion potential and long-term survival of UOK-145 papillary renal carcinoma-derived cells, which endogenously express PSF-TFE3. The oncogenic potential of PSF-TFE3 became evident by stable expression of PSF-TFE3 in NIH-3T3 mouse fibroblast cells, which leads to the acquisition of anchorage-independent growth as revealed by soft agar assay. In addition, the expression of PSF-TFE3 in normal renal proximal tubular epithelial cells from where such tumors originate leads to dedifferentiation and loss of some key functional proteins, which may reflect an initial step in the multistep process of tumor development. This suggests that the expression of PSF-TFE3 in renal epithelial cells plays an important role in the initiation and maintenance of oncogenic phenotype in papillary RCC.

Domain structure of the NRIF3 family of coregulators suggests potential dual roles in transcriptional regulation.

The identification of a novel coregulator for nuclear hormone receptors, designated NRIF3, was recently reported (D. Li et al., Mol. Cell. Biol. 19:7191-7202, 1999). Unlike most known coactivators, NRIF3 exhibits a distinct receptor specificity in interacting with and potentiating the activity of only TRs and RXRs but not other examined nuclear receptors. However, the molecular basis underlying such specificity is unclear. In this report, we extended our study of NRIF3-receptor interactions. Our results suggest a bivalent interaction model, where a single NRIF3 molecule utilizes both the C-terminal LXXIL (receptor-interacting domain 1 [RID1]) and the N-terminal LXXLL (RID2) modules to cooperatively interact with TR or RXR (presumably a receptor dimer), with the spacing between RID1 and RID2 playing an important role in influencing the affinity of the interactions. During the course of these studies, we also uncovered an NRIF3-NRIF3 interaction domain. Deletion and mutagenesis analyses mapped the dimerization domain to a region in the middle of NRIF3 (residues 84 to 112), which is predicted to form a coiled-coil structure and contains a putative leucine zipper-like motif. By using Gal4 fusion constructs, we identified an autonomous transactivation domain (AD1) at the C terminus of NRIF3. Somewhat surprisingly, full-length NRIF3 fused to the DNA-binding domain of Gal4 was found to repress transcription of a Gal4 reporter. Further analyses mapped a novel repression domain (RepD1) to a small region at the N-terminal portion of NRIF3 (residues 20 to 50). The NRIF3 gene encodes at least two additional isoforms due to alternative splicing. These two isoforms contain the same RepD1 region as NRIF3. Consistent with this, Gal4 fusions of these two isoforms were also found to repress transcription. Cotransfection of NRIF3 or its two isoforms did not relieve the transrepression function mediated by their corresponding Gal4 fusion proteins, suggesting that the repression involves a mechanism(s) other than the recruitment of a titratable corepressor. Interestingly, a single amino acid residue change of a potential phosphorylation site in RepD1 (Ser(28) to Ala) abolishes its transrepression function, suggesting that the coregulatory property of NRIF3 (or its isoforms) might be subjected to regulation by cellular signaling. Taken together, our results identify NRIF3 as an interesting coregulator that possesses both transactivation and transrepression domains and/or functions. Collectively, the NRIF3 family of coregulators (which includes NRIF3 and its other isoforms) may play dual roles in mediating both positive and negative regulatory effects on gene expression.

In silico discovery of novel retinoic acid receptor agonist structures.

BACKGROUND: Several Retinoic Acid Receptors (RAR) agonists have therapeutic activity against a variety of cancer types; however, unacceptable toxicity profiles have hindered the development of drugs. RAR agonists presenting novel structural and chemical features could therefore open new avenues for the discovery of leads against breast, lung and prostate cancer or leukemia. RESULTS: We have analysed the induced fit of the active site residues upon binding of a known ligand. The derived binding site models were used to dock over 150,000 molecules in silico (or virtually) to the structure of the receptor with the Internal Coordinates Mechanics (ICM) program. Thirty ligand candidates were tested in vitro. CONCLUSIONS: Two novel agonists resulting from the predicted receptor model were active at 50 nM. One of them displays novel structural features which may translate into the development of new ligands for cancer therapy.

PSF is a novel corepressor that mediates its effect through Sin3A and the DNA binding domain of nuclear hormone receptors.

Members of the type II nuclear hormone receptor subfamily (e.g., thyroid hormone receptors [TRs], retinoic acid receptors, retinoid X receptors [RXRs], vitamin D receptor, and the peroxisome proliferator-activated receptors) bind to their response sequences with or without ligand. In the absence of ligand, these DNA-bound receptors mediate different degrees of repression or silencing of gene expression which is thought to result from the association of their ligand binding domains (LBDs) with corepressors. Two related corepressors, N-CoR and SMRT, interact to various degrees with the LBDs of these type II receptors in the absence of their cognate ligands. N-CoR and SMRT have been proposed to act by recruiting class I histone deacetylases (HDAC I) through an association with Sin3, although they have also been shown to recruit class II HDACs through a Sin3-independent mechanism. In this study, we used a biochemical approach to identify novel nuclear factors that interact with unliganded full-length TR and RXR. We found that the DNA binding domains (DBDs) of TR and RXR associate with two proteins which we identified as PSF (polypyrimidine tract-binding protein-associated splicing factor) and NonO/p54(nrb). Our studies indicate that PSF is a novel repressor which interacts with Sin3A and mediates silencing through the recruitment of HDACs to the receptor DBD. In vivo studies with TR showed that although N-CoR fully dissociates in the presence of ligand, the levels of TR-bound PSF and Sin3A appear to remain unchanged, indicating that Sin3A can be recruited to the receptor independent of N-CoR or SMRT. RXR was not detected to bind N-CoR although it bound PSF and Sin3A as effectively as TR, and this association with RXR did not change with ligand. Our studies point to a novel PSF/Sin3-mediated pathway for nuclear hormone receptors, and possibly other transcription factors, which may fine-tune the transcriptional response as well as play an important role in mediating the repressive effects of those type II receptors which only weakly interact with N-CoR and SMRT.

A new family of nuclear receptor coregulators that integrate nuclear receptor signaling through CREB-binding protein.

We describe the cloning and characterization of a new family of nuclear receptor coregulators (NRCs) which modulate the function of nuclear hormone receptors in a ligand-dependent manner. NRCs are expressed as alternatively spliced isoforms which may exhibit different intrinsic activities and receptor specificities. The NRCs are organized into several modular structures and contain a single functional LXXLL motif which associates with members of the steroid hormone and thyroid hormone/retinoid receptor subfamilies with high affinity. Human NRC (hNRC) harbors a potent N-terminal activation domain (AD1), which is as active as the herpesvirus VP16 activation domain, and a second activation domain (AD2) which overlaps with the receptor-interacting LXXLL region. The C-terminal region of hNRC appears to function as an inhibitory domain which influences the overall transcriptional activity of the protein. Our results suggest that NRC binds to liganded receptors as a dimer and this association leads to a structural change in NRC resulting in activation. hNRC binds CREB-binding protein (CBP) with high affinity in vivo, suggesting that hNRC may be an important functional component of a CBP complex involved in mediating the transcriptional effects of nuclear hormone receptors.

Rational discovery of novel nuclear hormone receptor antagonists.

Nuclear hormone receptors (NRs) are potential targets for therapeutic approaches to many clinical conditions, including cancer, diabetes, and neurological diseases. The crystal structure of the ligand binding domain of agonist-bound NRs enables the design of compounds with agonist activity. However, with the exception of the human estrogen receptor-alpha, the lack of antagonist-bound "inactive" receptor structures hinders the rational design of receptor antagonists. In this study, we present a strategy for designing such antagonists. We constructed a model of the inactive conformation of human retinoic acid receptor-alpha by using information derived from antagonist-bound estrogen receptor-alpha and applied a computer-based virtual screening algorithm to identify retinoic acid receptor antagonists. Thus, the currently available crystal structures of NRs may be used for the rational design of antagonists, which could lead to the development of novel drugs for a variety of diseases.

NRIF3 is a novel coactivator mediating functional specificity of nuclear hormone receptors.

Many nuclear receptors are capable of recognizing similar DNA elements. The molecular event(s) underlying the functional specificities of these receptors (in regulating the expression of their native target genes) is a very important issue that remains poorly understood. Here we report the cloning and analysis of a novel nuclear receptor coactivator (designated NRIF3) that exhibits a distinct receptor specificity. Fluorescence microscopy shows that NRIF3 localizes to the cell nucleus. The yeast two-hybrid and/or in vitro binding assays indicated that NRIF3 specifically interacts with the thyroid hormone receptor (TR) and retinoid X receptor (RXR) in a ligand-dependent fashion but does not bind to the retinoic acid receptor, vitamin D receptor, progesterone receptor, glucocorticoid receptor, or estrogen receptor. Functional experiments showed that NRIF3 significantly potentiates TR- and RXR-mediated transactivation in vivo but has little effect on other examined nuclear receptors. Domain and mutagenesis analyses indicated that a novel C-terminal domain in NRIF3 plays an essential role in its specific interaction with liganded TR and RXR while the N-terminal LXXLL motif plays a minor role in allowing optimum interaction. Computer modeling and subsequent experimental analysis suggested that the C-terminal domain of NRIF3 directly mediates interaction with liganded receptors through an LXXIL (a variant of the canonical LXXLL) module while the other part of the NRIF3 protein may still play a role in conferring its receptor specificity. Identification of a coactivator with such a unique receptor specificity may provide new insight into the molecular mechanism(s) of receptor-mediated transcriptional activation as well as the functional specificities of nuclear receptors.

Regulation of the mdm2 oncogene by thyroid hormone receptor.

The mdm2 gene is positively regulated by p53 through a p53-responsive DNA element in the first intron of the mdm2 gene. mdm2 binds p53, thereby abrogating the ability of p53 to activate the mdm2 gene, and thus forming an autoregulatory loop of mdm2 gene regulation. Although the mdm2 gene is thought to act as an oncogene by blocking the activity of p53, recent studies indicate that mdm2 can act independently of p53 and block the G1 cell cycle arrest mediated by members of the retinoblastoma gene family and can activate E2F1/DP1 and the cyclin A gene promoter. In addition, factors other than p53 have recently been shown to regulate the mdm2 gene. In this article, we report that thyroid hormone (T3) receptors (T3Rs), but not the closely related members of the nuclear thyroid hormone/retinoid receptor gene family (retinoic acid receptor, vitamin D receptor, peroxisome proliferation activation receptor, or retinoid X receptor), regulate mdm2 through the same intron sequences that are modulated by p53. Chicken ovalbumin upstream promoter transcription factor I, an orphan nuclear receptor which normally acts as a transcriptional repressor, also activates mdm2 through the same intron region of the mdm2 gene. Two T3R-responsive DNA elements were identified and further mapped to sequences within each of the p53 binding sites of the mdm2 intron. A 10-amino-acid sequence in the N-terminal region of T3Ralpha that is important for transactivation and interaction with TFIIB was also found to be important for activation of the mdm2 gene response element. T3 was found to stimulate the endogenous mdm2 gene in GH4C1 cells. These cells are known to express T3Rs, and T3 is known to stimulate replication of these cells via an effect in the G1 phase of the cell cycle. Our findings, which indicate that T3Rs can regulate the mdm2 gene independently of p53, provide an explanation for certain known effects of T3 and T3Rs on cell proliferation. In addition, these findings provide further evidence for p53-independent regulation of mdm2 which could lead to the development of tumors from cells that express low levels of p53 or that express p53 mutants defective in binding to and activating the mdm2 gene.

Role of the conserved C-terminal region of thyroid hormone receptor-alpha in ligand-dependent transcriptional activation.

The ligand binding domain (LBD) of thyroid hormone (T3) receptors contains subdomains that participate in transcriptional activation, hormone-relieved repression and dimerization. A sequence conserved within the nuclear receptor superfamily is found at positions 397-405 of the 408-amino acid chicken T3 receptor-alpha (cTR alpha) and is deleted in the related avian v-erbA. Since v-erbA exhibits compromised ligand binding and transcriptional activation, this conserved region may play a role in ligand-dependent transcriptional activation. Transfections reveal that cTR alpha(1-392) and site-directed mutants cTR alpha(L398R) and cTR alpha(F399E) are inactive, while cTR alpha(1-403) displays reduced ligand-dependent transcriptional activity. The loss of transcriptional activity in cTR alpha(1-392) is not caused by impaired DNA binding or receptor dimer formation. Proteolytic protection assays reveal that both transcriptionally active and inactive cTR alpha derivatives undergo T3-mediated conformational changes. Gal4 chimeras containing the final 16, 35 or 44 amino acids of cTR alpha indicate that the conserved C-terminal region does not function as an independent transactivation domain. Our results are consistent with a model in which ligand plays a structural role to position the conserved C-terminal regions of cTR alpha and related receptors in a transcriptionally active conformation.

A novel multifunctional motif in the amino-terminal A/B domain of T3Ralpha modulates DNA binding and receptor dimerization.

We reported previously that deletion of the 50-amino acid NH2-terminal A/B domain of the chicken (c) or rat thyroid hormone (T3) receptor-alpha (T3Ralpha) decreased the T3-dependent stimulation of genes regulated by native thyroid hormone response elements (TREs). This requirement of the NH2-terminal A/B domain for transcriptional activation was mapped to amino acids 21-30 of cT3Ralpha. Expression of transcription factor IIB (TFIIB) in cells was shown to enhance T3-dependent transcriptional activation by cT3Ralpha, and this enhancement by TFIIB was dependent on the same 10-amino acid sequence. In vitro binding studies indicated that cT3Ralpha interacts efficiently with TFIIB, and this interaction requires amino acids 23KRKRK27 in the A/B domain. In this study we document the functional importance of these five basic residues in transcriptional activation by cT3Ralpha, further supporting the biological significance of these residues and their interaction with TFIIB. Interestingly, we also find that the same amino acids also affect DNA binding and dimerization of cT3Ralpha. Gel mobility shift assays reveal that a cT3Ralpha mutant that has all five basic amino acids changed from 23KRKRK27 to 23TITIT27 binds to a palindromic TRE predominantly as a homodimer, whereas cT3Ralpha with the wild-type 23KRKRK27 sequence binds predominantly as a monomer. This results from both a marked decrease in the ability of the cT3Ralpha mutant to bind as a monomer and from an enhanced ability to dimerize as reflected by an increase in DNA-bound T3R-retinoic X receptor heterodimers. These effects of 23KRKRK27 on DNA binding, dimerization, transcriptional activation, and the association of T3Ralpha with TFIIB support the notion that this basic amino acid motif may influence the overall structure and function of T3Ralpha and, thus, play a role in determining the distinct context-dependent transactivation potentials of the individual T3R isoforms.

TLS (translocated-in-liposarcoma) is a high-affinity interactor for steroid, thyroid hormone, and retinoid receptors.

Nuclear receptors for steroid hormones, thyroid hormone, retinoids, and vitamin D are thought to mediate their transcriptional effects in concert with coregulator proteins that modulate receptor interactions with components of the basal transcription complex. In an effort to identify potential coregulators, receptor fusions with glutathione-S-transferase were used to isolate proteins in nuclear extracts capable of binding nuclear hormone receptors. Glutathione-S-transferase fusions with mouse retinoid X receptor-alpha enabled the selective isolation of a 65-kDa protein (p65) from nuclear extracts of rat and human cells. Binding of p65 to mouse retinoid X receptor-alpha was centered around the DNA-binding domain. p65 also bound regions encompassing the DNA-binding domain in estrogen, thyroid hormone, and glucocorticoid receptors. p65 was identified as TLS (translocated-in-liposarcoma), a recently identified member of the RNP family of nuclear RNA-binding proteins whose members are thought to function in RNA processing. The N-terminal half of TLS bound to thyroid hormone receptor with high affinity while the receptor was bound to appropriate DNA target sites. Functional studies indicated that the N-terminal half of TLS can interact with thyroid hormone receptor in vivo. TLS was originally discovered as part of a fusion protein arising from a chromosomal translocation causing human myxoid liposarcomas. TLS contains a potent transactivation domain whose translocation-induced fusion with a DNA-binding protein (CHOP) yields a powerful transforming oncogene and transcription factor. The transactivation and RNA-binding properties of TLS and the nature of its interaction with nuclear receptors suggest a novel role in nuclear receptor function.

Constitutive activation of gene expression by thyroid hormone receptor results from reversal of p53-mediated repression.

Thyroid hormone receptor (T3R) is a member of the steroid hormone receptor gene family of nuclear hormone receptors. In most cells T3R activates gene expression only in the presence of its ligand, L-triiodothyronine (T3). However, in certain cell types (e.g., GH4C1 cells) expression of T3R leads to hormone-independent constitutive activation. This activation by unliganded T3R occurs with a variety of gene promoters and appears to be independent of the binding of T3R to specific thyroid hormone response elements (TREs). Previous studies indicate that this constitutive activation results from the titration of an inhibitor of transcription. Since the tumor suppresser p53 is capable of repressing a wide variety of gene promoters, we considered the possibility that the inhibitor is p53. Evidence to support this comes from studies indicating that expression of p53 blocks T3R-mediated constitutive activation in GH4C1 cells. In contrast with hormone-independent activation by T3R, p53 had little or no effect on T3-dependent stimulation which requires TREs. In addition, p53 mutants which oligomerize with wild-type p53 and interfere with its function also increase promoter activity. This enhancement is of similar magnitude to but is not additive with the stimulation mediated by unliganded T3R, suggesting that they target the same factor. Since p53 mutants are known to target wild-type p53 in the cell, this suggests that T3R also interacts with p53 in vivo and that endogenous levels of p53 act to suppress promoter activity. Evidence supporting both functional and physical interactions of T3R and p53 in the cell is presented. The DNA binding domain (DBD) of T3R is important in mediating constitutive activation, and the receptor DBD appears to functionally interact with the N terminus of p53 in the cell. In vitro binding studies indicate that the T3R DBD is important for interaction of T3R with p53 and that this interaction is reduced by T3. These findings are consistent with the in vivo studies indicating that p53 blocks constitutive activation but not ligand-dependent stimulation. These studies provide insight into mechanisms by which unliganded nuclear hormone receptors can modulate gene expression and may provide an explanation for the mechanism of action of the v-erbA oncoprotein, a retroviral homolog of chicken T3R alpha.

Hormone-dependent and -independent transcriptional activation by thyroid hormone receptors are mediated by different mechanisms.

Transcriptional activation by thyroid hormone (T3) receptor (T3R) generally requires the binding of its high affinity ligand. However, we reported previously that chicken T3R alpha (cTaR alpha) and human T3R beta 1 (hT3R beta 1) could activate transcription from several promoters containing T3R response elements (TREs) in a hormone-independent fashion when expressed in rat anterior pituitary GH4C1 cells. In this study we show that rat T3R alpha 1 also activates transcription without T3 in GH4C1 cells and that the oncoprotein v-erbA that is derived from cT3R alpha but does bind T3 is not a constitutive activator in these cells. Increased expression of T3R results in transcriptional activation of both native and minimal promoters, and this activation does not appear to require a defined TRE in the promoter. Because hormone-independent activity is not observed in several other cell lines, this activity may involve specific factors present in GH4C1 cells. Three mutants with single amino acid changes in a 20-amino acid region of the ligand-binding domain of cT3R alpha do not mediate hormone-independent activity. This region is highly conserved within the nuclear receptor family and has been implicated in interactions with other proteins, suggesting participation of other transcription or accessory factors in the hormone-independent activity of T3R. Two of these mutants mediate hormone-dependent transcriptional activation similar to wild-type cT3R alpha. All three mutants interact in vitro with retinoid X receptor beta similar to wild-type cT3R alpha. Our findings suggest that hormone-dependent and hormone-independent transactivation proceed by distinct mechanisms.

Novel regulation of keratin gene expression by thyroid hormone and retinoid receptors.

Expression of keratin proteins, markers of epidermal differentiation and pathology, is uniquely regulated by the nuclear receptors for retinoic acid (RAR) and thyroid hormone (T3R) and their ligands: it is constitutively activated by unliganded T3R, but it is suppressed by ligand-occupied T3R or RAR. This regulation was studied using gel mobility shift assays with purified receptors and transient transfection assays with vectors expressing various receptor mutants. Regulation of keratin gene expression by RAR and T3R occurs through direct binding of these receptors to receptor response elements of the keratin gene promoters. The DNA binding "C" domain of these receptors is essential for both ligand-dependent and -independent regulation. However, the NH2-terminal "A/B" domain of T3R is not required for either mode of regulation of keratin gene expression. Furthermore, v-ErbA, an oncogenic derivative of cT3R, also activates keratin gene expression. In contrast to the previously described mechanism of gene regulation by T3R, heterodimerization with the retinoid X receptor is not essential for activation of keratin gene expression by unliganded T3R. These findings indicate that the mechanism of regulation of keratin genes by RAR and T3R differs significantly from the mechanisms described for other genes modulated by these receptors.

Interactions of thyroid hormone receptor with the human immunodeficiency virus type 1 (HIV-1) long terminal repeat and the HIV-1 Tat transactivator.

Thyroid hormone (T3) receptor (T3R) regulates the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) by binding to and activating thyroid hormone response elements (TREs) embedded within the viral NF-kappa B and Sp1 motifs. The TREs within the NF-kappa B sites are necessary for activation by T3 in the absence of Tat, while those in the Sp1 motifs function as TREs only when Tat is expressed, suggesting that Tat and T3R interact in the cell. Transactivation of the HIV-1 LTR by T3R alpha and several receptor mutants revealed that the 50-amino-acid N-terminal A/B region of T3R alpha, known to interact with the basal transcription factor TFIIB, is critical for activation of both Tat-dependent and Tat-independent responsive sequences of the LTR. A single amino acid change in the highly conserved tau 1 region in the ligand-binding domain of T3R alpha eliminates Tat-independent but not Tat-dependent activation of the HIV-1 LTR by T3. Ro 5-3335 [7-chloro-5-(2-pyrryl)-3H-1,4-benzodiazepin-2(H)-one], which inhibits Tat-mediated transactivation of HIV-1, also inhibits the functional interaction between Tat and T3R alpha. Binding studies with glutathione-S-transferase fusion proteins and Western (immunoblot) analysis indicate that T3R alpha interacts with Tat through amino acids within the DNA-binding domain of T3R alpha. Mutational analysis revealed that amino acid residues in the basic and C-terminal regions of Tat are required for the binding of Tat to T3R alpha, while the N terminus of Tat is not required. These studies provide functional and physical evidence that stimulation of the HIV-1 LTR by T3 involves an interaction between T3R alpha and Tat. Our results also suggest a model in which multiple domains of T3R alpha interact with Tat and other factors to form transcriptionally important complexes.

A 10-amino-acid sequence in the N-terminal A/B domain of thyroid hormone receptor alpha is essential for transcriptional activation and interaction with the general transcription factor TFIIB.

The effects of the thyroid hormone (3,5,3'-triiodo-L-thyronine [T3]) on gene transcription are mediated by nuclear T3 receptors (T3Rs). alpha- and beta-isoform T3Rs (T3R alpha and -beta) are expressed from different genes and are members of a superfamily of ligand-dependent transcription factors that also includes the receptors for steroid hormones, vitamin D, and retinoids. Although T3 activates transcription by mediating a conformational change in the C-terminal approximately 220-amino-acid ligand-binding domain (LBD), the fundamental mechanisms of T3R-mediated transcriptional activation remain to be determined. We found that deletion of the 50-amino-acid N-terminal A/B domain of chicken T3R alpha (cT3R alpha) decreases T3-dependent stimulation of genes regulated by native thyroid hormone response elements about 10- to 20-fold. The requirement of the A/B region for transcriptional activation was mapped to amino acids 21 to 30, which contain a cluster of five basic amino acids. The A/B region of cT3R alpha is not required for T3 binding or for DNA binding of the receptor as a heterodimer with retinoid X receptor. In vitro binding studies indicate that the N-terminal region of cT3R alpha interacts efficiently with TFIIB and that this interaction requires amino acids 21 to 30 of the A/B region. In contrast, the LBD interacts poorly with TFIIB. The region of TFIIB primarily involved in the binding of cT3R alpha includes an amphipathic alpha helix contained within residues 178 to 201. Analysis using a fusion protein containing the DNA-binding domain of GAL4 and the entire A/B region of cT3R alpha suggests that this region does not contain an intrinsic activation domain. These and other studies indicate that cT3R alpha mediates at least some of its effects through TFIIB in vivo and that the N-terminal region of DNA-bound cT3R alpha acts to recruit and/or stabilize the binding of TFIIB to the transcription complex. T3 stimulation could then result from ligand-mediated changes in the LBD which may lead to the interaction of other factors with cT3R alpha, TFIIB, and/or other components involved in the initiation of transcription.

The ligand-binding domains of the thyroid hormone/retinoid receptor gene subfamily function in vivo to mediate heterodimerization, gene silencing, and transactivation.

The ligand-binding domains (LBDs) of the thyroid/retinoid receptor gene subfamily contain a series of heptad motifs important for dimeric interactions. This subfamily includes thyroid hormone receptors (T3Rs), all-trans retinoic acid (RA) receptors (RARs), 9-cis RA receptors (RARs and retinoid X receptors [RXRs]), the 1,25-dihydroxyvitamin D3 receptor (VDR), and the receptors that modulate the peroxisomal beta-oxidation pathway (PPARs). These receptors bind to their DNA response elements in vitro as heterodimers with the RXRs. Unliganded receptors in vivo, in particular the T3Rs, can mediate gene silencing and ligand converts these receptors into a transcriptionally active form. The in vivo interactions of these receptors with RXR were studied by using a GAL4-RXR chimera containing the yeast GAL4 DNA-binding domain and the LBD of RXR beta. GAL4-RXR activates transcription from GAL4 response elements in the presence of 9-cis RA. Unliganded T3R, which does not bind or activate GAL4 elements, represses the activation of GAL4-RXR by 9-cis RA in HeLa cells. However, addition of T3 alone leads to transcriptional activation. These findings suggest that T3R can repress or activate transcription while tethered to the LBD of GAL4-RXR and that heterodimerization can occur in vivo without stabilization by hormone response elements. Similar ligand-dependent activation was observed in HeLa cells expressing RAR, VDR, or PPAR and in GH4C1 cells from endogenous receptors. Replacement of the last 17 amino acids of the LBD of RXRbeta with the 90-amino-acid transactivating domain of the herpes simplex virus VP16 protein leads to a GAL4 constitutive activator that is repressed by wild-type T3R but not by a ninth heptad mutant that does not form heterodimers. This finding suggests that the ninth heptad or T3R is important for gene silencing and that the LBD of RXR does not exhibit silencing activity. This conclusion was verified with GAL4-LBD chimeras and with wild-type receptors in assays using appropriate response elements. These studies indicate that the LBD has diverse functional roles in gene regulation.

A sequence in the rat Pit-1 gene promoter confers synergistic activation by glucocorticoids and protein kinase-C.

The 5'-flanking region of the gene for Pit-1, a pituitary-specific transcription factor, was isolated from a rat liver genomic library and sequenced. Expression of a reporter construct containing Pit-1 promoter sequences linked to the bacterial chloramphenicol acetyltransferase (CAT) gene was assessed by transient transfection in rat pituitary GH4C1 cells. Treatment of transfected cells with either dexamethasone (DEX) for 48 h or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) for the final 20 h of the 48-h posttransfection period had minimal effects on CAT expression. However, CAT activity was elevated about 20-fold when transfected cells were treated with both DEX and TPA. This apparent synergistic activation was lost when DEX treatment was also limited to the final 20 h of the 48-h posttransfection period, suggesting that a time-dependent accumulation of a DEX-induced gene product might be involved. This putative DEX-induced product appeared to be relatively stable, because synergistic activation was observed in cells treated with DEX alone for 36 h, followed by a 10-h incubation without DEX before the addition of TPA. The Pit-1 gene promoter region between -210 and -142 from the transcription start site conferred synergistic regulation by DEX and TPA when placed upstream of position -105 in the herpes viral thymidine kinase promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional evidence for ligand-dependent dissociation of thyroid hormone and retinoic acid receptors from an inhibitory cellular factor.

The ligand-binding domains of thyroid hormone (L-triiodothyronine [T3]) receptors (T3Rs), all-trans retinoic acid (RA) receptors (RARs), and 9-cis RA receptors (RARs and RXRs) contain a series of heptad motifs thought to be important for dimeric interactions. Using a chimera containing amino acids 120 to 392 of chicken T3R alpha (cT3R alpha) positioned between the DNA-binding domain of the yeast GAL4 protein and the potent 90-amino-acid transactivating domain of the herpes simplex virus VP16 protein (GAL4-T3R-VP16), we provide functional evidence that binding of ligand releases T3Rs and RARs from an inhibitory cellular factor. GAL4-T3R-VP16 does not bind T3 and does not activate transcription from a GAL4 reporter when expressed alone but is able to activate transcription when coexpressed with unliganded T3R or RAR. This activation is reversed by T3 or RA, suggesting that these receptors compete with GAL4-T3R-VP16 for a cellular inhibitor and that ligand reverses this effect by dissociating T3R or RAR from the inhibitor. A chimera containing the entire ligand-binding domain of cT3R alpha (amino acids 120 to 408) linked to VP16 [GAL4-T3R(408)-VP16] is activated by unliganded receptor as well as by T3. In contrast, GAL4-T3R containing the amino acid 120 to 408 ligand-binding region without the VP16 domain is activated only by T3. The highly conserved ninth heptad, which is involved in heterodimerization, appears to participate in the receptor-inhibitor interaction, suggesting that the inhibitor is a related member of the receptor gene family. In striking contrast to T3R and RAR, RXR activates GAL4-T3R-VP16 only with its ligand, 9-cis RA, but unliganded RXR does not appear to be the inhibitor suggested by these studies. Further evidence that an orphan receptor may be the inhibitor comes from our finding that COUP-TF inhibits activation of GAL4-T3R-VP16 by unliganded T3R and the activation of GAL4-T3R by T3. These and other results suggest that an inhibitory factor suppresses transactivation by the T3Rs and RARs while these receptors are bound to DNA and that ligands act, in part, by inactivating or promoting dissociation of a receptor-inhibitor complex.

Divergent dimerization properties of mutant beta 1 thyroid hormone receptors are associated with different dominant negative activities.

Syndromes of resistance to thyroid hormones are caused by mutations in the T3-binding domain of the c-erbA beta thyroid hormone receptor gene. The S receptor (deletion of THR332) is a potent dominant negative protein cloned from a kindred with generalized resistance to thyroid hormones. The G-H receptor (ARG311HIS) has compromised dominant negative function and was found in both normal individuals and in a patient with severe pituitary resistance to thyroid hormones. We have investigated the mechanism responsible for the difference in receptor phenotypes by analyzing the binding of S and G-H receptors to thyroid hormone response elements with electrophoretic mobility shift analysis. Wild-type human c-erbA beta 1 (WT), S, and G-H receptors were synthesized in reticulocyte lysate, reacted with a thyroid hormone response element consisting of a direct repeat with 4 base pairs (DR+4; AGGTCA CAGG AGGTCA), and the products analyzed by gel shift. G-H receptor homodimerization was greatly impaired; G-H formed predominantly monomeric complex compared with monomeric and homodimeric WT complexes. The G-H receptor was able to form heterodimeric complexes with cellular thyroid hormone receptor auxiliary protein (TRAP) factors including the human retinoid X receptor-alpha. When TRAP was limiting, the levels of G-H heterodimeric complex were 2- to 3-fold reduced compared with WT receptor. In contrast to the WT and G-H receptors, the S receptor formed almost exclusively homodimeric complex with DR+4; the approximate ratio of S:WT:G-H homodimeric complexes at equivalent concentrations of receptors was 60:20:1. A measurable increase (1.2- to 2.6-fold) in heterodimeric complex formation was observed with the S receptor relative to WT when TRAP was at limiting concentration. As reported previously by others, thyroid hormone significantly reduced the WT homodimeric complex with DR+4. There was no effect on the S homodimeric complex. Finally, the WT, S, and G-H receptors formed different complexes with the element consisting of an inverted repeat with 5 base pairs (IR+5; AGGTCA ACAGT TGACCT) and the IR element (AGGTCA TGACCT), which were differently regulated by thyroid hormone. The S receptor bound as a homodimer with IR+5, whereas the WT receptor bound as a homodimer only with thyroid hormone. No homodimeric complex formed with IR+5 and the G-H receptor. Qualitatively similar results were observed with the IR element. We conclude that the ARG311HIS mutation severely perturbs the homodimerization and, to a much less degree, heterodimerization functions of the c-erbA beta 1 receptor. Furthermore, the THR332 deletion mutation augments homodimerization of the c-erbA beta 1 receptor. These results indicate that different mutations in the c-erbA beta 1 thyroid hormone receptor have divergently affected dimerization activities which seem to influence the level of dominant negative activity in man.