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PubMed2002

Expression of cAMP-responsive element binding protein and inducible cAMP early repressor in hyperfunctioning thyroid adenomas.

OBJECTIVE: The pathogenesis of thyroid hyperfunctioning adenomas is still only partially understood and controversy exists about the frequency of gain-of-function mutations of the TSH receptor or G(s)alpha gene, which activate the cAMP pathway. The nuclear transcription factors cAMP-responsive element binding protein (CREB) and inducible cAMP early repressor (ICER) are among the final targets of this signalling cascade. DESIGN: In our study we focused on the expression of CREB and ICER genes in the nodular as well as in the extranodular tissue of hyperfunctioning tumours of the thyroid. METHODS: RT-PCR and Western blot analysis were performed in a series of 14 patients. The presence of an activating mutation of the TSH receptor or of the G(s)alpha gene was ascertained by direct sequencing. RESULTS: The levels of CREB transcripts did not significantly differ in the adenomas and in the normal tissues (CREB/GAPDH, mean optical density+/-s.e.: 0.98+/-0.18 vs 0.88+/-0.27 respectively, P = not significant (N.S.)), although case-to-case variability was observed. The absence of a significant difference between the adenoma and the surrounding normal tissue was maintained after dividing the patients into two groups, according to TSH receptor status. Accordingly, no significant difference in the levels of CREB protein (total and Ser(133)-phosphorylated) was observed between the nodular and the extranodular tissue. In addition, no difference was found in the levels of ICER transcripts (ICER/GAPDH, mean optical density+/-s.e.: 0.52+/-0.11, nodule vs 0.36+/-0.11, normal thyroid, P=N.S.), independently of the TSH receptor gene status (i.e. wild-type or mutated). CONCLUSIONS: Our results support the recent hypothesis that the activation of the cAMP pathway in hyperfunctioning adenomas of the thyroid might be counteracted by opposite events and suggest that complex molecular mechanisms might take part in the pathogenesis of hyperfunctioning tumours.

Activating Transcription Factor 1↗
PubMed2004

Contribution of the hormone-response elements of the proximal ApoA-I promoter, ApoCIII enhancer, and C/EBP binding site of the proximal ApoA-I promoter to the hepatic and intestinal expression of the ApoA-I and ApoCIII genes in transgenic mice.

We have generated and studied the pattern of expression of transgenic mouse lines carrying the human apoA-I and apoCIII gene cluster mutated at different sites. In two lines, we have either mutated the hormone-response element (HRE) of element G of the apoCIII enhancer or the C/EBP binding site of the proximal apoA-I promoter. In a third line, we have mutated the two HREs of the apoA-I promoter and the HRE of the apoCIII enhancer. Mutations in the HRE of element G reduced the hepatic and intestinal expressions of the reporter chloramphenicol acetyltransferase (CAT) gene (which substituted the apoCIII gene) to 4 and 13% of the wild-type (WT) control, whereas the hepatic and intestinal expressions of the apoA-I gene were reduced to 92 and 25% of the WT control, respectively. A mutation in the C/EBP site increased the hepatic and intestinal expressions of the apoA-I gene approximately 1.25- and 1.6-fold, respectively, and did not affect the expression of the CAT gene. The mutation in the three HNF-4 binding sites of the apoA-I promoter/apoCIII enhancer nearly abolished the expression of apoA-I and the reporter CAT gene in all tissues. These findings establish the importance of the HREs for the hepatic and intestinal expressions of the apoA-I and apoCIII genes and suggest that C/EBP does not play a central role in the expression of the apoA-I gene.

Animals↗
PubMed2003

Integration of multiple signal transducing pathways on Fgf response elements of the Xenopus caudal homologue Xcad3.

Early neural patterning along the anteroposterior (AP) axis appears to involve a number of signal transducing pathways, but the precise role of each of these pathways for AP patterning and how they are integrated with signals that govern neural induction step is not well understood. We investigate the nature of Fgf response element (FRE) in a posterior neural gene, Xcad3 (Xenopus caudal homologue) that plays a crucial role of posterior neural development. We provide evidence that FREs of Xcad3 are widely dispersed in its intronic sequence and that these multiple FREs comprise Ets-binding and Tcf/Lef-binding motifs that lie in juxtaposition. Functional and physical analyses indicate that signaling pathways of Fgf, Bmp and Wnt are integrated on these FREs to regulate the expression of Xcad3 in the posterior neural tube through positively acting Ets and Sox family transcription factors and negatively acting Tcf family transcription factor(s).

Animals↗
PubMed2005

Activation of interferon-stimulated response element in huh-7 cells replicating hepatitis C virus subgenomic RNA.

Interferon-alpha (IFN(alpha)) binds to receptors on the cell surface, which initiate a cascade of signal transduction pathways that leads to transcription of selected genes. This transduction pathway involves binding of transcription factors to a common cis-acting DNA sequence called IFN-stimulated response element (ISRE). To test whether these signaling pathways are functional in hepatitis C virus (HCV)-replicating cells, we studied the regulation of ISRE-mediated transcription of firefly luciferase gene in stable replicon cell lines. A plasmid construct was prepared (pISRELuc) which contains four tandem repeats of 9-27 ISRE sequences positioned directly upstream of the herpes virus 1 thymidine kinase promoter TATA box that drives the expression of firefly luciferase. Regulation of ISRE-mediated expression of firefly luciferase by IFN(alpha) was studied by transfecting this clone into Huh-7 cells replicating HCV subgenomic HCV RNA. The significance of ISRE-mediated transcriptional activation was studied in a replicon cell line by pretreatment of cells with actinomycin D, which inhibits cellular DNA-dependent RNA transcription. IFN treatment activates ISRE-mediated expression of luciferase, indicating that this pathway is functional in Huh-7 cells. Activation of ISRE-mediated transcription of luciferase is relatively high in two Huh-7 stable cell lines replicating HCV subgenomic RNA. Inhibition of ISRE-mediated transcription of luciferase by actinomycin D also makes HCV replication totally resistant to IFN(alpha). These in vitro studies suggest that activation of IFN-inducible genes is important in mounting a successful antiviral response against HCV.

Cell Line, Tumor↗
PubMed1999

The mammalian endoplasmic reticulum stress response element consists of an evolutionarily conserved tripartite structure and interacts with a novel stress-inducible complex.

When mammalian cells are subjected to calcium depletion stress or protein glycosylation block, the transcription of a family of glucose-regulated protein (GRP) genes encoding endoplasmic reticulum (ER) chaperones is induced to high levels. The consensus mammalian ER stress response element (ERSE) conserved among grp promoters consists of a tripartite structure CCAAT(N9)CCACG, with N being a strikingly GC-rich region of 9 bp. The ERSE, in duplicate copies, can confer full stress inducibility to a heterologous promoter in a sequence-specific but orientation-independent manner. In addition to CBF/NF-Y and YY1 binding to the CCAAT and CCACG motifs, respectively, we further discovered that an ER stress-inducible complex (ERSF) from HeLa nuclear extract binds specifically to the ERSE. Strikingly, the interaction of the ERSF with the ERSE requires a conserved GGC motif within the 9 bp region. Since mutation of the GGC triplet sequence also results in loss of stress inducibility, specific sequence within the 9 bp region is an integral part of the tripartite structure. Finally, correlation of factor binding with stress inducibility reveals that ERSF binding to the ERSE alone is not sufficient; full stress inducibility requires integrity of the CCAAT, GGC and CCACG sequence motifs, as well as precise spacing among these sites.

Adaptation, Biological↗
PubMed1996

Identification of a novel retinoic acid response element in the promoter region of the retinol-binding protein gene.

We have previously demonstrated that the retinol-binding protein (RBP) gene is induced by retinoids in hepatoma cells. In this report, we define in greater detail the region that mediates the retinoic acid response of the gene. It consists of two degenerate retinoic acid response elements, separated by 30 nucleotides that encompass a GC-rich Sp1 consensus-like sequence. We demonstrate that the entire region, as well as each element taken singly, can bind the retinoic acid receptors as homo- and heterodimers with low affinity. However, only the entire region is able to confer retinoic acid inducibility to a heterologous promoter. We also show that the correct phasing of the DNA segment is necessary to achieve full responsiveness. Site-directed mutants in each element retained partial induction after transfection, while the double mutant was no longer responsive, suggesting that the two elements act synergistically. Mutational analysis of the Sp1 binding site and cotransfection experiments revealed that Sp1 or a related protein plays an important role in the transcription of the gene. Thus, the retinoic acid induction of the RBP gene is mediated by a novel and complex responsive unit formed by two distinct elements located in a specific sequence context and the interplay of the retinoid receptors with Sp1 is required for induction.

Animals↗
PubMed1999

Potential role of the gene transcription factor cyclic AMP-responsive element binding protein in ethanol withdrawal-related anxiety.

This investigation examined the effects of acute and chronic ethanol exposure and its withdrawal on the cAMP-responsive element binding protein (CREB) and the activator protein-1 (AP-1) gene transcription factors in the rat brain. The anxiogenic effects of ethanol withdrawal after acute or protracted ethanol treatment of rats were measured by the elevated plus-maze (EPM) test. It was observed that ethanol withdrawal after acute ethanol treatment has no effect on open-arm activity (percent of open-arm entries and the mean percent of time spent on the open arms) of rats on the EPM test. On the other hand, the time course studies of the development of anxiety during ethanol withdrawal (0, 12, 24, and 72 h) after 15 days of ethanol treatment indicate that peak anxiety (significant decrease in open-arm activity) occurred at 24 h of ethanol withdrawal in rats. It was observed that acute ethanol treatment and its withdrawal (24 h) had no effect on CRE- or AP-1 DNA-binding activities in the rat cortex as determined by the electrophoretic gel-mobility shift assay. It was also found that chronic ethanol treatment and its withdrawal (24 h) had no effect on AP-1 DNA-binding activity in the rat cortex. Investigation of the time course studies of changes in CRE-DNA-binding activity during ethanol withdrawal (0, 12, 24, and 72 h) after 15 days of ethanol treatment indicated that the peak reduction of CRE-DNA-binding activity occurred at 24 h of ethanol withdrawal. The changes in the immunolabeling of the CREB-related target, that is, brain-derived neurotrophic factor (BDNF), in the rat cortex during chronic ethanol treatment and its withdrawal (24 h) were examined using western blotting. It was found that 24 h but not 0 h of ethanol withdrawal after 15 days of ethanol treatment caused a significant decrease in the immunolabeling of BDNF in the rat cortex. Fluoxetine (alone) treatment of rats for 1 or 15 days had no effect on open-arm activity and cortical CRE-DNA-binding activity. However, when fluoxetine was administered concurrently with ethanol treatment for 15 days, it caused a reversal of the anxiogenic effects of ethanol withdrawal and antagonized the down-regulation of CRE-DNA-binding activity and of the decrease in immunolabeling of BDNF in the cortices of ethanol-withdrawn rats. On the other hand, acute fluoxetine treatment produced normalization of the reduction of cortical CRE-DNA binding in ethanol-withdrawn rats (24 h) but did not reach the level of significance compared with normal control rats. Acute fluoxetine treatment had no effect on anxiety in ethanol-withdrawn rats. Taken together, these results suggest the possibility that decreased CRE-DNA-binding activity in the rat cortex may be associated with the molecular mechanisms of ethanol dependence (i.e., ethanol withdrawal-related anxiety).

Animals↗
PubMed2001

Identification of TFII-I as the endoplasmic reticulum stress response element binding factor ERSF: its autoregulation by stress and interaction with ATF6.

When mammalian cells are subjected to stress targeted to the endoplasmic reticulum (ER), such as depletion of the ER Ca(2+) store, the transcription of a family of glucose-regulated protein (GRP) genes encoding ER chaperones is induced. The GRP promoters contain multiple copies of the ER stress response element (ERSE), consisting of a unique tripartite structure, CCAAT(N(9))CCACG. Within a subset of mammalian ERSEs, N(9) represents a GC-rich sequence of 9 bp that is conserved across species. A novel complex (termed ERSF) exhibits enhanced binding to the ERSE of the grp78 and ERp72 promoters using HeLa nuclear extracts prepared from ER-stressed cells. Optimal binding of ERSF to ERSE and maximal ERSE-mediated stress inducibility require the conserved GGC motif within the 9-bp region. Through chromatographic purification and subsequent microsequencing, we have identified ERSF as TFII-I. Whereas TFII-I remains predominantly nuclear in both nontreated NIH 3T3 cells and cells treated with thapsigargin (Tg), a potent inducer of the GRP stress response through depletion of the ER Ca(2+) store, the level of TFII-I transcript was elevated in Tg-stressed cells, correlating with an increase in TFII-I protein level in the nuclei of Tg-stressed cells. Purified recombinant TFII-I isoforms bind directly to the ERSEs of grp78 and ERp72 promoters. The stimulation of ERSE-mediated transcription by TFII-I requires the consensus tyrosine phosphorylation site of TFII-I and the GGC sequence motif of the ERSE. We further discovered that TFII-I is an interactive protein partner of ATF6 and that optimal stimulation of ERSE by ATF6 requires TFII-I.

3T3 Cells↗
PubMed2001

Characterization of a putative insulin-responsive element and its binding protein(s) in rat angiotensinogen gene promoter: regulation by glucose and insulin.

We previously demonstrated that high glucose activates angiotensinogen (ANG) expression and that insulin inhibits this activation. The present studies aim to investigate whether insulin regulates ANG gene expression in kidney proximal tubular cells at the transcription level via interaction of the putative insulin-response element (IRE) with its binding protein(s) in the 5'-flanking region of the ANG gene. Fusion genes containing various lengths of the 5'-flanking region of the rat ANG gene fused to a human GH (hGH) gene as reporter were constructed and transiently introduced into rat immortalized renal proximal tubular cells (IRPTCs). The expression of the fusion genes was monitored by the amount of immunoreactive hGH secreted into the medium as assayed by a specific RIA for hGH. Insulin inhibited the expression of pOGH (rANG N-1498/+18), pOGH (rANG N-1120/+18) and pOGH (rANG N-882/+18) but not pOGH (rANG N-854/+18), pOGH (rANG N-820/+18), pOGH (rANG N-688/+18) and pOGH (rANG N-53/+18) in high-glucose (i.e. 25 mM) medium. Site-directed mutagenesis of nucleotides N-874 to N-867 (5' CCC GCC CT 3') in the 5'-flanking region of the rat ANG gene abolished the response to insulin. Insulin also inhibited the expression of the fusion gene containing the DNA fragment ANG N-882 to N-855 inserted upstream of the ANG gene promoter (N-53/+18), but had no effect on a mutant of N-882 to N-855. Gel mobility shift assays revealed that the labeled putative rat ANG-IRE motif (N-878 to N-864, 5' CCT TCC CGC CCT TCA 3') was bound to the nuclear proteins of IRPTCS: This binding was displaced by unlabeled ANG-IRE and IRE of human glyceraldehyde phosphate dehydrogenase but not by mutants of ANG-IRE and IRE of the rat glucagon gene. Southwestern blotting analysis revealed that the labeled putative ANG-IRE motif bound to a major nuclear protein with an apparent molecular mass of 48 kDA: Finally, high glucose levels enhanced 48-kDa nuclear protein expression and induced an additional 70-kDa nuclear protein expression in IRPTCs, as revealed by Southwestern blotting. Insulin inhibited both 48- and 70-kDa nuclear proteins expression induced by high glucose levels. Its inhibitory effect was reversed by the presence of PD98059 (an inhibitor of mitogen-activated protein kinase, MAPK) but not by wortmannin (an inhibitor of phosphatidylinositol 3- kinase). These studies demonstrate that insulin action on ANG gene expression is at the transcriptional level. The molecular mechanism (s) of insulin action is mediated, at least in part, via interaction of the functional IRE with unidentified 48- and 70- kDa nuclear proteins in the rat ANG gene and is MAPK dependent.

Angiotensinogen↗
PubMed1994

Characterization of a corticotropin-releasing hormone-responsive element in the rat proopiomelanocortin gene promoter and molecular cloning of its binding protein.

A corticotropin-releasing hormone (CRH) and cAMP-responsive region (-236/-133) in the rat POMC gene promoter previously reported to confer CRH/cAMP responsiveness to heterologous reporter constructs has been characterized. DNAse footprint analysis revealed that multiple elements in this region were bound by nuclear proteins from the POMC expressing AtT20 cells. When these individual DNA elements were separately tested in heterologous reporter constructs for CRH induction, only one element, designated PCRH-RE (POMC CRH responsive element, -171/-160) was found to give strong CRH stimulation (5- to 7-fold). This element appears novel as to the possible binding factors, although it has homology to the mouse metallothionein metal regulatory element. Gel shift analyses of the PCRH-RE with AtT20 cell nuclear extracts showed marked stimulation of retarded nucleoproteins following CRH stimulation, suggesting that the possible binding factor(s) may mediate transcriptional regulation at this site. The activity of PCRH-RE binding protein was inhibited by divalent cations, with Cu2+ and Cd2+ being most effective; Zn2+ had no effect, indicating that this binding factor(s) is functionally distinct from the metallothionein metal regulatory element binding protein. A 2.6 kilobase cDNA clone encoding a protein (PCRH-REB-1) binding to this element was isolated by Southwestern screening of an AtT20 expression library with radiolabeled PCRH-RE oligonucleotides. This clone was used to isolate several other cDNA clones to determine the sequence corresponding to the entire coding region of the protein (PCRH-REB), which proved to be identical to a recently described DNA binding protein of the replication factor C complex, mRFC140/Mouse Southwestern. Primer extension and Northern blot analysis revealed that the size of the full length mRNA is about 4.9 kilobases. PCRH-REB mRNA expression is not restricted to corticotrophs but is present in a broad tissue distribution as evaluated by reverse transcription polymerase chain reaction analysis. A bacterially expressed beta-galactosidase-PCRH-REB-1 fusion protein was shown to bind PCRH-RE efficiently. Furthermore, binding of the PCRH-REB-1 fusion protein to the POMC CRH-responsive element was inhibited by divalent cations with similar sensitivities to those observed using AtT20 nuclear extracts. The predicted PCHR-REB protein sequence presents several interesting motifs: one p-Loop motif (ATP binding site), nine protein kinase A phosphorylation sites (implying a possible role in responding to the CRH-induced cAMP signal), and regions of homology to proteins involved in DNA replication and repair. PCRH-REB is, therefore, a potential transacting factor binding to a major CRH-responsive element in the POMC promoter.

Animals↗
PubMed2004

Identification of a functional vitamin D response element in the human insulin-like growth factor binding protein-3 promoter.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] plays a critical role in maintaining calcium and phosphate homeostasis and bone formation but also exhibits antiproliferative activity on many cancer cells, including prostate cancer. We have shown that the antiproliferative actions of 1,25-(OH)2D3 in the LNCaP human prostate cancer cell line are mediated in part by induction of IGF binding protein-3 (IGFBP-3). The purpose of this study was to determine the molecular mechanism involved in 1,25-(OH)2D3 regulation of IGFBP-3 expression and to identify the putative vitamin D response element (VDRE) in the IGFBP-3 promoter. We cloned approximately 6 kb of the IGFBP-3 promoter sequence and demonstrated its responsiveness to 1,25-(OH)2D3 in transactivation assays. Computer analysis identified a putative VDRE between -3296/-3282 containing the direct repeat motif GGTTCA ccg GGTGCA that is 92% identical with the rat 24-hydroxylase distal VDRE. In EMSAs, the vitamin D receptor (VDR) showed strong binding to the putative IGFBP-3 VDRE in the presence of 1,25-(OH)2D3. Supershift assays confirmed the presence of VDR in the IGFBP-3 VDRE complex. Chromatin immunoprecipitation assay demonstrated that 1,25-(OH)2D3 recruited the VDR/retinoid X receptor heterodimer to the VDRE site in the natural IGFBP-3 promoter in intact cells. In transactivation assays, the putative VDRE coupled to a heterologous simian virus 40 promoter construct was induced 2-fold by 1,25-(OH)2D3. Mutations in the VDRE resulted in a loss of inducibility confirming the critical hexameric sequence. In conclusion, we have identified a functional VDRE in the distal region of the human IGFBP-3 promoter. The induction of IGFBP-3 by 1,25-(OH)2D3 appears to be directly mediated via VDR interaction with this VDRE.

Animals↗
PubMed1990

Thyroid hormone receptors form distinct nuclear protein-dependent and independent complexes with a thyroid hormone response element.

We have examined the binding of nuclear proteins and recombinant thyroid hormone receptors (TRs) to the palindromic thyroid hormone responsive element AGGTCATGACCT (TREp) using a gel electrophoretic mobility shift assay. Four specific protein-DNA complexes were detected after incubation of nuclear extracts (NE) from T3-responsive pituitary (GH3) cells with a TREp-containing DNA fragment. This was compared with the TREp binding of reticulocyte lysate-synthesized TRs. TR alpha 1 and TR beta 2 each formed a single major TR:TREp complex which comigrated with the least retarded complex formed by GH3 NE, while TR beta 1 formed multiple complexes suggesting that it can bind to TREp as an oligomer. Interestingly, coincubation of 35S-TR alpha 1, GH3 NE, and unlabeled TREp resulted in not only the 35S-TR:TREp complex, but in two additional more greatly retarded complexes containing 35S-TR alpha 1 and comigrating with those formed by GH3 extract alone. Incubation of each of the TRs with NE from COS-7 cells, which do not possess sufficient endogenous TRs to mediate T3-responses, resulted in formation of a new, more greatly shifted complex. A similar, heat labile activity which altered mobility of the TR:TRE complex was also present in NE from T3-unresponsive JEG-3 cells. At high concentration of NE, all of the TR bound to TREp was more greatly retarded than in the absence of NE. Truncation of TR alpha 1 at amino acid 210 prevented additional complex formation in the presence of NE without affecting DNA binding, suggesting that the carboxyl-terminus of the TRs is essential for interaction with nuclear proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1999

Up-regulation of the human gamma-glutamylcysteine synthetase regulatory subunit gene involves binding of Nrf-2 to an electrophile responsive element.

The rate-limiting step in the de novo synthesis of the cellular protectant glutathione is catalyzed by gamma-glutamylcysteine synthetase (GCS; also known as glutamine-L-cysteine ligase, GLCL), a heterodimer consisting of catalytic (GCS(h)) and regulatory (GCS(l)) subunits. Regulation of expression of the human gamma-glutamylcysteine synthetase regulatory subunit gene in response to beta-NF is mediated by an Electrophile Responsive Element (EpRE) [Moinova, H., and Mulcahy, R. T. (1998) J. Biol. Chem. 273, 14683-14689]. Oligonucleotide probes corresponding to wild-type and mutant EpRE sequences were used in gel-shift and super-shift analyses to identify proteins binding. Four protein:DNA complexes (a-d) with distinct mobilities were detected when the wild-type EpRE probe was incubated with nuclear extracts from control or beta-NF-treated HepG2 cells. Following beta-NF treatment, there was an increase in the intensity of a single band, band b. This band was eliminated in gel shifts employing mutant EpRE probes which abolish beta-NF inducibility, demonstrating a correlation between band b and transactivation. Super-shift analysis identified JunD, Nrf1, and Nrf2 in the EpRE-binding complexes. Antibodies to Nrf2 completely super-shifted the band b protein:DNA complex. These studies demonstrate that Nrf2 proteins recognize and bind the GCS(l) EpRE sequence to affect transactivation of the gene.

Base Sequence↗
PubMed1999

Transformation blocks differentiation-induced inhibition of serum response factor interactions with serum response elements.

The differentiation of nontransformed 3T3T mesenchymal stem cells is a multistep process that is associated with the progressive repression of mitogenic responsiveness to serum growth factors that ultimately results in expression of the terminally differentiated adipocyte phenotype. The repression of serum-induced mitogenesis by differentiation correlates with repression of the serum-inducible transcription of junB and c-fos. In contrast, the differentiation of neoplastically transformed cells does not repress mitogenic responsiveness or junB or c-fos inducibility. Because the junB and c-fos promoters both contain serum response elements (SREs), the current studies tested the possibility that differentiation might repress the ability of serum response factor (SRF) to bind to the SRE in normal cells but not in transformed cells. We now report that differentiation represses SRE serum inducibility using nontransformed cells transiently transfected with pjunB SRE thymidine kinase/chloroamphenicol acetyltransferase (SREtk/CAT) or pc-fos SREtk/CAT containing an intact SRF-binding domain. Adipocyte differentiation of nontransformed cells also markedly represses the ability of SRF to bind to the junB SRE, the c-fos SRE, and other SREs, as determined by mobility shift and gel supershift assays, without affecting the DNA binding characteristics of the nuclear protein SP-1. By comparison, the ability of SRF to bind SRE is not repressed by the differentiation of SV40 large T antigen-transformed 3T3T cells. The results further establish that adipocyte differentiation blocks the nuclear localization of SRF, thus preventing its interaction with SREs in nontransformed cells but not in transformed cells.

Adipocytes↗
PubMed2003

Functional specificity of two hormone response elements present on the human apoA-II promoter that bind retinoid X receptor alpha/thyroid receptor beta heterodimers for retinoids and thyroids: synergistic interactions between thyroid receptor beta and upstream stimulatory factor 2a.

DNA binding and mutagenesis in vitro established that the -67/-55 region of the apoA-II (apolipoprotein A-II) promoter contains a thyroid HRE (hormone response element), which strongly binds RXRalpha (retinoid X receptor alpha)/T(3)Rbeta (thyroid receptor beta) heterodimers and weakly T(3)Rbeta homodimers, but does not bind other homo- or heterodimers of RXRalpha or orphan nuclear receptors. Transactivation was abolished by point mutations in the thyroid HRE. In co-transfection experiments of HEK-293 (human embryonic kidney 293) cells, the -911/+29 human apoA-II promoter was transactivated strongly by RXRalpha/T(3)Rbeta heterodimers in the presence of RA (9- cis retinoic acid) or T(3) (tri-iodothyronine). Homopolymeric promoters containing either three copies of the -73/-40 (element AIIAB) or four copies of the -738/-712 (element AIIJ) apoA-II promoter could be transactivated by RXRalpha/T(3)Rbeta heterodimers in COS-7 cells only in the presence of T(3) or RA respectively. RXRalpha/T(3)Rbeta heterodimers and USF2a (upstream stimulatory factor 2a) synergistically transactivated the -911/+29 apoA-II promoter in the presence of T(3). USF2a also enhanced the activity of a GAL4-T(3)Rbeta fusion protein in the presence of T(3) and suppressed the activity of a GAL4-RXRalpha fusion protein in the presence of RA. These findings suggest a functional specificity of the two HREs of the apoA-II promoter for retinoids and thyroids, which is modulated by synergistic or antagonistic interactions between RXRalpha/T(3)Rbeta heterodimers and the ubiquitous transcription factor USF2a.

Alitretinoin↗
PubMed1993

Dominant negative inhibition by mutant thyroid hormone receptors is thyroid hormone response element and receptor isoform specific.

The heterogeneity of tissue-specific manifestations of generalized resistance to thyroid hormone (GRTH) could result from differential interactions between the mutant thyroid hormone (T3) receptor-beta (TR beta) on T3 response elements (TREs) in different T3-responsive genes. To explore this hypothesis, the mutant TR beta associated with kindred A, P448H; a TR beta mutant, P448L; and a comparable TR alpha mutant (P398H) were tested for intrinsic function and for inhibition of wild-type TR alpha- and -beta-induced expression from four structurally distinct TREs, the rGH ABC*, the rGH palindrome (PAL), the rat malic enzyme (ME), and the chicken lysozyme silencer F2 (F2). The relative function of the mutants was similarly reduced on the four TREs studied and was T3 concentration dependent. The TR alpha mutant retained the intrinsically greater potency characteristic of this isoform, but remained impaired with respect to wild-type TR alpha even at 500 nM T3. In general, dominant negative inhibition of wild-type TR alpha and -beta function was dependent upon the T3 concentration, as expected from the decreased affinity for ligand conferred by this mutation. A T3 concentration sufficient to relieve the inhibition of wild-type TR function on the ABC*, PAL, and ME TREs (50 nM) had no effect on inhibition of the F2 TRE by the mutant TRs. Receptor isoform preferential inhibition was observed on the ABC*, PAL, and ME TREs by the mutant TRs. Thus, both TRE structure and the isoform of endogenously active receptor could determine the degree of inhibition of a specific gene in GRTH individuals. Further, the lack of dominant negative potentials does not explain the absence of TR alpha mutations in GRTH kindreds.

Animals↗
PubMed2001

An mRNA loop/bulge in the ferritin iron-responsive element forms in vivo and Was detected by radical probing with Cu-1,10-phenantholine and iron regulatory protein footprinting.

Messenger RNA (mRNA) regulatory elements often form helices specifically distorted by loops or bulges, which control protein synthesis rates in vitro. Do such three-dimensional RNA structures form in vivo? We now observe formation of the internal loop/bulge (IL/B structure) in the IRE (iron-responsive element) of ferritin mRNA expressed in HeLa cells, using radical cleavage with Cu-phen (Cu-1,10-phenantholine), and protection of the loop/bulge by the regulatory protein (IRP), expressed by cotransfection. Cu-phen, a metal coordination complex (MC) selected because of binding and cleavage at the IL/B in solution, recognized the same site in mRNA in HeLa cells. Endogenous reductants apparently substituted for the sulfhydryl activation of Cu-phen cleavage in solution. Selective RNA IL/B recognition by Cu-phen in vivo is emphasized by resistance to cleavage of a mutated, IL/B IRE in ferritin mRNA. Development of small MCs even more selective than Cu-phen can exploit three-dimensional mRNA or viral RNA structures in vivo to manipulate RNA function. Formation in vivo of the IL/B in the ferritin IRE, which is associated in vitro with greater repression than single IRE structures in other mRNAs, likely contributes to larger derepression of ferritin synthesis in vivo triggered by signals for the IRE/IRP system.

Binding Sites↗
PubMed1997

Expression of testis angiotensin-converting enzyme is mediated by a cyclic AMP responsive element.

Testis angiotensin-converting enzyme (testis ACE), an ACE isozyme that plays an important role in male fertility, is transcribed from a unique promotor active only in developing spermatids. In vitro analysis suggests the importance of a cyclic AMP response element (CRE)-like region within the testis ACE promoter, and similar DNA motifs are important in the expression of a variety of testis-specific genes. In the present study, we examined the effects of mutations in the CRE-like element on testis ACE promoter activity in vivo using transgenic mice. Disruption of this element reduced reporter gene expression to near background levels. In contrast, conversion of the CRE-like element to a consensus CRE-binding site resulted in high level expression of the reporter gene specifically in the testis. These experiments prove that the CRE-like element is essential for testis ACE promoter activity, although it does not appear to be responsible for its tissue specificity. These data provide insight into how a phenotypically differentiated tissue, ie, male gem cells, regulate tissue-specific gene expression.

Animals↗