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PubMed1994

Identification of a functional cyclic adenosine 3',5'-monophosphate response element in the 5'-flanking region of the gene for transition protein 1 (TP1), a basic chromosomal protein of mammalian spermatids.

Transition protein 1 (TP1) is a small basic chromosomal protein that appears in mammalian spermatids during the period of chromatin condensation. The gene for TP1 from several species contains an apparent cAMP response element (CRE) in the immediate 5'-flanking region. The recent identification of high expression of the novel CRE-activating protein (CREM tau) in advanced testicular germ cells provided a stimulus to ask whether or not the TP1 CRE is functional. To this end we show both by gel retardation and by footprint assays that TP1 CRE forms specific bound complexes with proteins in whole testis nuclear extracts and that these complexes involve CREM as evidenced by recognition by a specific antibody. In addition, the TP1 CRE forms specific bound complexes with bacterially expressed CREM tau. Finally, the TPI CRE conveys protein kinase A-dependent induction to a linked chloramphenicol acetyl transferase gene when transfected into JEG-3 cells. Accordingly, TP1 is a good candidate for a testis-specific gene subject to CREM tau regulation.

Animals↗
PubMed1998

Insulin stimulates cAMP-response element binding protein activity in HepG2 and 3T3-L1 cell lines.

Earlier studies from our laboratory demonstrated an insulin-mediated increase in cAMP-response element binding protein (CREB) phosphorylation. In this report, we show that insulin stimulates both CREB phosphorylation and transcriptional activation in HepG2 and 3T3-L1 cell lines, models of insulin-sensitive tissues. Insulin stimulated the phosphorylation of CREB at serine 133, the protein kinase A site, and mutation of serine 133 to alanine blocked the insulin effect. Many of the signaling pathways known to be activated by insulin have been implicated in CREB phosphorylation and activation. The ability of insulin to induce CREB phosphorylation and activity was efficiently blocked by PD98059, a potent inhibitor of mitogen-activated protein kinase kinase (MEK1), but not significantly by rapamycin or wortmannin. Likewise, expression of dominant negative forms of Ras or Raf-1 completely blocked insulin-stimulated CREB transcriptional activity. Finally, we demonstrate an essential role for CREB in insulin activation of fatty-acid synthase and fatty acid binding protein (FABP) indicating the potential physiologic relevance of insulin regulation of CREB. In summary, insulin regulates CREB transcriptional activity in insulin-sensitive tissues via the Raf --> MEK pathway and has an impact on physiologically relevant genes in these cells.

3T3 Cells↗
PubMed2006

Combinational application of surface plasmon resonance spectroscopy and quartz crystal microbalance for studying nuclear hormone receptor-response element interactions.

Conventional methodologies for studying protein-DNA complexes, such as electrophoretic mobility shift assays (EMSAs), lack the real-time sensitivity and precision to accurately characterize the complex dynamics of interactions between transcription factors and their binding sites. To better understand the interactions between estrogen receptor (ER) subtypes and the estrogen response elements (EREs), we employed surface plasmon resonance (SPR) spectroscopy and quartz crystal microbalance with dissipation measurement (QCM-D) and made the following observations: (1) base substitutions in ERE half-sites reduced binding affinity for both ERalpha and ERbeta, (2) ERalpha has a higher sequence specificity than ERbeta or there were more nonspecific interactions between ERbeta and control DNA, and (3) ERalpha bound ERE as dimers and ERbeta bound as tetramers. These findings highlight intrinsic differences in DNA-binding properties between receptor subtypes, which are not apparent based on the high degree of conservation (96% identity) in their DNA-binding domains and results from EMSA studies. With this study, we demonstrate the potential of utilizing SPR and QCM in combination for a comprehensive characterization of ER-DNA interactions, including sequence-dependent binding mechanisms and structural differences in ERalpha-DNA and ERbeta-DNA complexes.

DNA↗
PubMed2006

Identification of a cis-acting element responsible for negative regulation of the human UDP-glucose dehydrogenase gene expression.

The enzyme UDP-glucose dehydrogenase (UGDH) catalyzes the conversion of UDP-glucose to UDP-glucuronic acid, which is essential for the biosynthesis of complex carbohydrates such as hyaluronan in many cell types, and is required for detoxification of toxic compounds in the liver. We previously defined the 714 bp 5'-flanking region of the UGDH gene as the core promoter, with putative negative regulatory elements residing in the region upstream of it. In the present study, we delineated the region from nucleotide positions -1057 to -957 on the UGDH promoter to be responsible for the repression of promoter activity. A mutation at nucleotide -1003, which is contained within a motif predicted to be the response element for peroxisome proliferator receptor alpha (PPARalpha), abolished the suppression effect. DNA-protein interaction was observed at this motif by electrophoretic mobility shift assay. The proteins interacting with the PPRE-like repressor motif were purified by biotin-labeled DNA affinity chromatography. Subsequently, MALDI-TOF identified the purified proteins as a 62-kDa zinc finger and a 42-kDa beta-actin protein. Hence in this study we report the presence of an inhibitory cis-element in the distal region of the UGDH promoter that interacts with putative transcriptional repressors for the negative regulation of the UGDH gene.

Cell Line, Tumor↗
PubMed1994

Cloning of a cDNA encoding a human protein which binds a sequence in the c-myc gene similar to the interferon-stimulated response element.

A human cDNA clone encoding a c-myc promoter-binding protein (IRLB) was selected by screening a human fibroblast lambda gt11 phage library with the hexamer oligodeoxyribonucleotide (oligo) 5'-GGCGGGAAAAAGAACGGA, corresponding to the protein-binding element of human c-myc similar to the interferon-stimulated response element (ISRE). The lambda gt11 phage clone, encoding a fusion protein which bound the probe oligo, was used to create an strain of Escherichia coli. The deduced amino-acid sequence of the cloned protein contains a putative alpha-helix which is expected to act as the DNA-binding domain. DNase footprinting analysis and oligo-binding specificity assays showed that the cloned factor recognizes the ISRE-like element of the P2 promoter region of human c-myc.

Amino Acid Sequence↗
PubMed1990

Xenobiotic-inducible expression of murine glutathione S-transferase Ya subunit gene is controlled by an electrophile-responsive element.

Glutathione S-transferase (GST) Ya subunit gene expression is induced in mammalian tissues by two types of chemical agents: (i) planar aromatic compounds (e.g., 3-methylcholanthrene, beta-naphthoflavone, and 2,3,7,8-tetrachlorodibenzo-p- dioxin) and (ii) electrophiles (e.g., trans-4-phenyl-3-buten-2-one and dimethyl fumarate) or compounds easily oxidized to electrophiles (e.g., tert-butylhydroquinone). To study the mechanism of this induction, we have introduced deletions in the 5' flanking region of a mouse GST Ya subunit gene, fused it to the coding sequence for chloramphenicol acetyltransferase (CAT) activity, and transfected the Ya-CAT genes for expression into hepatoma cells. We show that a single cis-regulatory element, between nucleotides -754 and -713 from the start of transcription, is responsible for the induction by both planar aromatic and electrophilic compounds. Using murine hepatoma cell mutants defective in either the Ah-encoded aryl hydrocarbon receptor (BPrc1 mutant) or in cytochrome P1-450 gene (c1 mutant), we show that induction by planar aromatic but not by electrophilic inducers requires a functional Ah receptor and cytochrome P1-450 activity. From this it is concluded that Ya gene activation by planar aromatic compounds involves metabolism of these inducers by the phase I xenobiotic-metabolizing cytochrome P1-450 system into electrophilic compounds, which is consistent with a recently proposed model [Prochaska, H. J. & Talalay, P. (1988) Cancer Res. 48, 4776-4782]. Therefore, the regulatory sequence of the Ya gene should be considered an electrophile-responsive element (EpRE) activated exclusively by inducers containing an electrophilic center. An EpRE-containing 41-bp oligonucleotide ligated at the -187 site of the Ya gene promoter confers upon it an increase in basal activity and xenobiotic inducibility. The basal activity augments with the number of EpRE copies. DNase I protection patterns show the protection of the EpRE domain by a nuclear factor(s) that becomes more abundant upon exposure of Hepa 1c1c7 cells to tert-butylhydroquinone.

Animals↗
PubMed1998

Localization of xenobiotic-responsive element binding protein in rat hepatocyte nuclei after methylcholanthrene administration as revealed by in situ Southwestern hybridization.

Xenobiotic-responsive element binding protein (XRE-BP), a heterodimer of aryl hydrocarbon receptor (AhR) and its nuclear translocator (Arnt), regulates the transcription of cytochrome P-450 1A1 gene (CYP1A1) through XRE in response to xenobiotic inducers. For a better understanding of the regulatory mechanism of CYP1A1 through XRE, localization of XRE-BP was examined in liver sections or isolated hepatocyte nuclei from control and 3-methylcholanthrene (MC)-treated rats by in situ Southwestern hybridization, using synthetic XRE as a probe, and was observed by confocal laser scanning microscopy and electron microscopy. Gel mobility shift assay and competitive binding assay showed specificity of the synthetic XRE probe. XRE-BP was exclusively localized in hepatocyte nuclei in liver sections from animals 3 hr after MC injection, whereas the protein was absent in hepatocyte cytoplasm in MC-treated animals and in hepatocyte nuclei and cytoplasm in control animals. In isolated hepatocyte nuclei, XRE-BP began to accumulate in the central region between 0.5 and 3 hr, showed a peak between 3 and 6 hr, decreased gradually between 6 and 72 hr, and disappeared at 72 hr after MC injection. The protein was scarce in peripheral and nucleolar regions of the nucleus. Therefore, XRE-BP is formed in the nuclei of hepatocytes after MC stimulation. In addition, XRE-BP was found in isolated hepatocyte nuclei from control animals after preincubation with cytoplasmic lysate from MC-treated animals, although the protein was absent in the nuclei before the preincubation. These findings strongly suggest that AhR translocates from hepatocyte cytoplasm to the nucleus and forms XRE-BP in the nucleus after MC stimulation.

Animals↗
PubMed2004

The vitamin D response element in the distal osteocalcin promoter contributes to chromatin organization of the proximal regulatory domain.

Vitamin D receptor (VDR) and Runx2 are key regulators of tissue-specific gene transcription. Using the bone-related osteocalcin (OC) gene, we have previously shown that Runx2 is required for the extensive chromatin remodeling that accompanies gene activation. Here, we have addressed the direct contribution of the VDR to chromatin remodeling events necessary for regulation of OC transcription using mutational analysis. Our studies demonstrate that both the distal and proximal DNase I-hypersensitive sites characteristic of the transcriptionally active OC promoter are not enhanced in the absence of a functional vitamin D response element (VDRE). Furthermore, restriction enzyme accessibility studies reveal that nucleosomal reorganization of the proximal promoter occurs in response to vitamin D and this reorganization is abrogated by mutation of the VDRE. These findings indicate that binding of liganded VDR in the distal promoter directly impacts the chromatin structure of the proximal promoter. We find that, in the absence of functional Runx sites, the VDR cannot be recruited to the OC promoter and, therefore, the VDRE is not competent to mediate vitamin D responsiveness. On the other hand, chromatin immunoprecipitation assays show that Runx2 association with the OC promoter is not significantly impaired when the VDRE is mutated. Chromatin immunoprecipitation assays also demonstrate that basal levels of histone acetylation occur in the absence of Runx2 binding but that the VDRE and vitamin D are required for enhanced acetylation of histones H3 and H4 downstream of the VDRE. Together our results support a stepwise model for chromatin remodeling of the OC promoter and show that binding of the liganded VDR.retinoid X receptor directly impacts both the distal and proximal regulatory domains.

Animals↗
PubMed1991

Rapid downregulation of transcription mediated by the cAMP response element of c-fos promoter does not require new protein synthesis.

Induction of the c-fos gene is well known to be transient, due to autorepression of its transcription. While the dyad symmetry element (DSE) in the c-fos promoter has been shown to mediate transient c-fos transcription in response to serum, it is yet unknown whether the cAMP response elements (CREs) in the promoter mediate transient transcription in response to cAMP. We observed that forskolin induced transient expression of the c-fos gene in the presence of a calcium ionophore A23187 in NIH3T3 fibroblasts. We investigated transcriptional abilities of one of the CREs (-60 CRE) as well as the DSE. Both of the elements mediated the synergistic effects of forskolin and A23187 on transcription. Transcription driven by the -60 CRE was downregulated at around 2 hrs. after stimulation of forskolin plus A23187 with similar kinetics to that by the DSE. The downregulation with the -60 CRE was less sensitive to an inhibitor of protein synthesis comparing with that by the DSE. These results suggest that function of the -60 CRE activated by forskolin plus A23187 is downregulated by activation of pre-existing factors.

3T3 Cells↗
PubMed2006

The human kallikrein 10 promoter contains a functional retinoid response element.

Human kallikrein 10 (hK10) protein is expressed in normal breast but is significantly downregulated in a majority of invasive breast cancers. Thus, understanding how hK10 expression is regulated is of substantial significance. In this study, we analyzed the promoter region of hK10 using a website software (TRANSFAC 3.0), which predicted three possible retinoic acid response elements (RAREs), RARE1 at -1041 (TGACCTCGTGATCC), RARE2 at -859 (TGACCTCCTATGA) and RARE3 at -765 (TGACCTCCTGTGA), each with a half-site of a canonical sequence (TGACCT; reverse complement AGGTCA). Using electrophoretic mobility shift assays and nucleotide competition analysis, as well as chromatin immunoprecipitation of the native hK10 promoter, we demonstrated specific binding of RXR only to RARE1. The functional importance of RARE in the hK10 promoter was demonstrated by retinoid induction of hk10 promoter-reporters; furthermore, mutation of RARE1 but not of RARE2 or RARE3 abolished the induction of the reporter. Finally, we demonstrated the induction of hK10 mRNA and protein expression upon retinoid treatment of cells. In view of the correlation of the downregulation of hK10 mRNA and protein with breast cancer progression, these findings suggest a potential approach to restore hK10 expression in cancer patients.

Gene Expression Regulation, Neoplastic↗
PubMed2001

Low concentration N-methyl-N'-nitro-N-nitrosoguanidine activates DNA polymerase-beta expression via cyclic-AMP-protein kinase A-cAMP response element binding protein pathway.

Ultraviolet light (UV), ionizing-radiation or alkylating agents are known as carcinogens, mostly because of their ability to damage DNA directly. However, they may also play a diverse role in activating the signal pathways and altering the gene expression. We have shown previously that N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) of 0.2 microM can increase the transcription of DNA polymerase-beta gene, which has a cyclic AMP response element (CRE) motif in its promoter. Using the CRE report vector, we show here, such treatment can stimulate the CRE-driven gene expression by approximately 1.5-fold compared with control. Consistent with it, the proportion of ser-133 phosphorylated CRE binding protein (CREB), the related transcription factor was 2.08-fold higher versus control in vero cells after 60 min of MNNG treatment. Although CREB has many putative kinases for its phosphorylation, such as p38 mitogen-activated protein kinase (p38 MAPK), Ca(2+)/calmodulin-dependent protein kinase Pi (CaMK Pi) and protein kinase C (PKC), we found the protein kinase A (PKA) was activated and its activation peaked when cells were treated for 60 min (with arbitrary activity unit of 11.03+/-2.80 and 0.86+/-0.43 in treatment and control, respectively), this phasic character was similar to that of the CREB phosphorylation. We also determined the intracellular cyclic AMP (cAMP) levels and it was found that the cAMP concentration was elevated after 60 min treatment (1.53-fold higher). However, to our surprise, we did not find any accompanying cAMP elevation in cells treated by MNNG for 30 min, in which PKA was activated significantly. These findings, together with other observations, suggest that cAMP-PKA-CREB signaling pathway mediates the low concentration MNNG induced pol-beta expression. In addition to elevated cAMP, there might exist a cAMP-independent PKA activation manner in this course.

Animals↗
PubMed2001

Estrogen response element sequence impacts the conformation and transcriptional activity of estrogen receptor alpha.

Estrogens play a critical role in mammary gland development, bone homeostasis, reproduction, and the pathogenesis of breast cancer by activating estrogen receptors (ERs) alpha and beta. Ligand-activated ER stimulates the expression of target proteins by interacting with specific DNA sequences: estrogen response elements (EREs). We have demonstrated that the ERE sequence and the nucleotide sequences flanking the ERE impact ERalpha binding affinity and transcriptional activation. Here, we examined whether the sequence of the ERE modulates ERalpha conformation by measuring changes in sensitivity to protease digestion. ERalpha, occupied by estradiol (E2) or 4-hydroxytamoxifen (4-OHT), was incubated with select EREs and digested by chymotrypsin followed by a Western analysis with antibodies to ERalpha. ERE binding increased the sensitivity of ERalpha to chymotrypsin digestion. We found both ligand-specific and ERE-specific differences in ERalpha sensitivity to chymotrypsin digestion. The ERE-mediated increase in ERalpha sensitivity to chymotrypsin digestion correlates with E2-stimulated transcriptional activity from the same EREs in transiently transfected cells. Transcriptional activity also correlates with the affinity of ERalpha-ERE binding in vitro. Our results support the hypothesis that the ERE sequence acts as an allosteric effector, altering ER conformation. We speculate that ERE-induced alterations in ERalpha conformation modulate interaction with co-regulatory proteins.

Allosteric Regulation↗
PubMed2004

Nuclear factor-kappaB motif and interferon-alpha-stimulated response element co-operate in the activation of guanylate-binding protein-1 expression by inflammatory cytokines in endothelial cells.

The large GTPase GBP-1 (guanylate-binding protein-1) is a major IFN-gamma (interferon-gamma)-induced protein with potent anti-angiogenic activity in endothelial cells. An ISRE (IFN-alpha-stimulated response element) is necessary and sufficient for the induction of GBP-1 expression by IFN-gamma. Recently, we have shown that in vivo GBP-1 expression is strongly endothelial-cell-associated and is, in addition to IFN-gamma, also activated by interleukin-1beta and tumour necrosis factor-alpha, both in vitro and in vivo [Lubeseder-Martellato, Guenzi, Jörg, Töpolt, Naschberger, Kremmer, Zietz, Tschachler, Hutzler, Schwemmle et al. (2002) Am. J. Pathol. 161, 1749-1759; Guenzi, Töpolt, Cornali, Lubeseder-Martellato, Jörg, Matzen, Zietz, Kremmer, Nappi, Schwemmle et al. (2001) EMBO J. 20, 5568-5577]. In the present study, we identified a NF-kappaB (nuclear factor kappaB)-binding motif that, together with ISRE, is required for the induction of GBP-1 expression by interleukin-1beta and tumour necrosis factor-alpha. Deactivation of the NF-kappaB motif reduced the additive effects of combinations of these cytokines with IFN-gamma by more than 50%. Importantly, NF-kappaB p50 rather than p65 activated the GBP-1 promoter. The NF-kappaB motif and ISRE were detected in an almost identical spatial organization, as in the GBP-1 promoter, in the promoter regions of various inflammation-associated genes. Therefore both motifs may constitute a cooperative inflammatory cytokine response module that regulates GBP-1 expression. Our findings may open new perspectives for the use of NF-kappaB inhibitors to support angiogenesis in inflammatory diseases including ischaemia.

Base Sequence↗
PubMed2000

Puralpha, a single-stranded DNA binding protein, suppresses the enhancer activity of cAMP response element (CRE).

Puralpha, a single-stranded DNA binding protein, recognizes a PUR element (GGN repeat). We have reported that Puralpha binds to a single-stranded oligonucleotide probe containing the cAMP response element (CRE) of rat somatostatin gene using a gel mobility shift assay. Here, we showed that Puralpha binds to the probe only in the presence of a PUR element by a more detailed characterization. We also examined the effects of Puralpha on the enhancer activity of the somatostatin CRE in PC12 cells using the reporter gene assay. Transfected Puralpha suppressed the CRE enhancer activity stimulated by forskolin (which increases intracellular cAMP), but suppression was not observed when the PUR element was deleted. The neurite extension induced by forskolin was inhibited by the transfection of Puralpha, but that by NGF was not suppressed. The c-fos mRNA induced by forskolin, but not by NGF, was also suppressed by Puralpha transfection. These results indicate that Puralpha suppresses the biological activities induced by forskolin, but not by NGF, in PC12 cells and that Puralpha could interfere with a cAMP-CRE signal pathway.

Animals↗
PubMed2003

HER2/neu uses Akt to suppress retinoic acid response element binding activity in MDA-MB-453 breast cancer cells.

We previously demonstrated that HER2/neu prevents all trans-retinoic acid (ATRA) from inducing growth inhibition in MDA-MB-453 breast cancer cells. For ATRA to induce growth inhibition, it needs to bind to retinoic acid receptors and modulate gene transcription via retinoic acid response elements (RAREs). We hypothesized that HER2/neu suppresses RARE binding activity to prevent ATRA from inducing growth arrest in breast cancer cells. Electrophoretic mobility shift assays showed that when HER2/neu was inhibited by the trastuzumab antibody, RARE binding activity increased, indicating that HER2/neu suppresses RARE binding. Since trastuzumab also decreased Akt activity, we determined whether Akt regulates RARE binding activity. Compared to parental MDA-MB-453 cells, MDA-MB-453 cells transfected with a dominant negative Akt mutant (MDA-MB-453/DN-Akt) had higher RARE binding activity. However, trastuzumab did not further increase RARE binding activity in MDA-MB-453/DN-Akt cells. These data indicate that HER2/neu predominantly uses Akt to suppress RARE binding activity, which may be one mechanism by which HER2/neu induces ATRA resistance in breast cancer cells.

Antibodies, Monoclonal↗
PubMed2006

[Delayed disappearance of h-VEGF₁₆₅ mRNA and protein under regulation of hypoxic response element].

Transfer of vascular endothelial growth factor (VEGF) gene to ischemic myocardium may provide a useful approach for angiogenesis and improve cardiac performance. However, uncontrolled expression of VEGF in vivo may result in certain side effects, such as hemangioma formation, retinopathy, and tumor development. We investigated the feasibility of using the nine copies of hypoxic response element (HRE) to control the expression of human VEGF(165) (h-VEGF(165)) under anoxic condition at cell level and also observed the synchron of h-VEGF(165) mRNA and protein expressions. Recombinant adeno-associated viral (rAAV) vector was prepared by using the three-plasmid system and cotransfected to human embryo kidney 293T cells by the calcium phosphate precipitates method. The rAAV vector was purified by chloroform-PEG8000/NaCl-chloroform and added to cultured myocardiocytes. Myocardiocytes of Sprague-Dawley rat were cultured in serum-free medium and then randomly divided into eight groups. Group I: cultured under normoxic conditions (21% O2) for 8 h as control; Group II: cultured under anoxic conditions (1% O2) for 8 h; Group III: cultured under normoxic conditions (21% O2) for 8 h with gene transfer; Group IV: cultured under anoxic conditions (1% O2) for 8 h with gene transfer; Group V, VI, VII: cultured under anoxic conditions (1% O2) for 8 h with gene transfer and then tured to normoxic conditions (21% O2) for 4, 8 or 12 h, respectively; Group VIII: cultured under anoxic conditions (1% O2) for 20 h with gene transfer. After completion of cell culture, the amount of h-VEGF(165) protein in culture supernatant was quantified by using enzyme linked immunosorbent assay (ELISA). Expression of h-VEGF(165) protein in cultured cardiacmyocytes was also evaluated by immunofluorescence. RT-PCR was employed to detect the expression of h-VEGF(165) mRNA. The results revealed that there were no expressions of h-VEGF(165) mRNA and protein in groups I, II, III, VI and VII. After gene transfer, the expressions of h-VEGF(165) protein and mRNA were significantly higher in groups IV and VIII than those in other groups (P<0.01); Immunofluorescence positive cells were observed in groups IV, V and VIII. RT-PCR revealed that a 484-bp strip can be found in groups IV and VIII, but unavailable in other groups. We conclude that HRE is a promising regulator for h-VEGF(165) gene expression following the changes of oxygen environment. HRE can induce the expression of h-VEGF(165) gene after hypoxia, but in normal oxygen condition, the expression of h-VEGF(165) was inhibited. Although expression of h-VEGF(165) mRNA ceased in normal oxygen condition under the control of HRE, expression of h-VEGF(165) protein was hysteretic to h-VEGF(165) mRNA expression.

Animals↗
PubMed1989

Transcription from a murine T-cell receptor V beta promoter depends on a conserved decamer motif similar to the cyclic AMP response element.

We identified a regulatory region of the murine V beta promoter by both in vivo and in vitro analyses. The results of transient transfection assays indicated that the dominant transcription-activating element within the V beta 8.3 promoter is the palindromic motif identified previously as the conserved V beta decamer. Elimination of this element, by linear deletion or specific mutation, reduced transcriptional activity from this promoter by 10-fold. DNase I footprinting, gel mobility shift, and methylation interference assays confirmed that the palindrome acts as the binding site of a specific nuclear factor. In particular, the V beta promoter motif functioned in vitro as a high-affinity site for a previously characterized transcription activator, ATF. A consensus cyclic AMP response element (CRE) but not a consensus AP-1 site, can substitute for the decamer in vivo. These data suggest that cyclic AMP response element-binding protein (ATF/CREB) or related proteins activate V beta transcription.

Animals↗
PubMed2004

The pregnane X receptor binds to response elements in a genomic context-dependent manner, and PXR activator rifampicin selectively alters the binding among target genes.

The pregnane X receptor (PXR) is a key regulator of genes encoding several major types of cytochrome P450 enzymes and transporters (e.g., multidrug resistance-1, MDR1); therefore, PXR contributes significantly to drug-drug interactions. PXR binds to response elements and confers transactivation. Several target genes such as CYP3A4 and 3A7 contain two PXR elements (distant and proximal) that are separated by more than 7000 nucleotides in the genome. Disruption of the distant element causes a 73% decrease of the reporter activity, whereas inactivation of the proximal element decreases by only 53%. This study was undertaken to test the hypothesis that PXR differentially binds to the elements with the distant enhancer being bound to a higher extent. To test this hypothesis, a stable transfected line (hPXR-HRE) was prepared to constitutively express human PXR and harbor a chromatinized CYP3A4-ER6 reporter. This line responded to rifampicin and dexamethasone similarly as hepatocytes based on the relative potency and activation kinetics. Contrary to the hypothesis, chromatin immunoprecipitation experiments showed that the genomic fragment harboring the proximal element was preferably precipitated over the fragment containing the distant element in the CYP3A4 gene, but the opposite was true with the CYP3A7 gene. In addition, the promoters from the MDR1 and CYP2B6 genes were abundantly present in the PXR immunocomplexes from the vehicle-treated cells. However, such abundant interactions were markedly diminished when cells were treated with PXR activator rifampicin. These findings suggest that PXR binding is dependent on the genomic context and PXR activators modulate such bindings.

ATP Binding Cassette Transporter, Subfamily B, Mem↗