Binding Site For Nuclear Factor Research Articles

NFI-Ski interactions mediate transforming growth factor beta modulation of human papillomavirus type 16 early gene expression.

Human papillomaviruses (HPVs) are present in virtually all cervical cancers. An important step in the development of malignant disease, including cervical cancer, involves a loss of sensitivity to transforming growth factor beta (TGF-beta). HPV type 16 (HPV16) early gene expression, including that of the E6 and E7 oncoprotein genes, is under the control of the upstream regulatory region (URR), and E6 and E7 expression in HPV16-immortalized human epithelial cells is inhibited at the transcriptional level by TGF-beta. While the URR contains a myriad of transcription factor binding sites, including seven binding sites for nuclear factor I (NFI), the specific sequences within the URR or the transcription factors responsible for TGF-beta modulation of the URR remain unknown. To identify potential transcription factors and binding sites involved in TGF-beta modulation of the URR, we performed DNase I footprint analysis on the HPV16 URR using nuclear extracts from TGF-beta-sensitive HPV16-immortalized human keratinocytes (HKc/HPV16) treated with and without TGF-beta. Differentially protected regions were found to be located around NFI binding sites. Electrophoretic mobility shift assays, using the NFI binding sites as probes, showed decreased binding upon TGF-beta treatment. This decrease in binding was not due to reduced NFI protein or NFI mRNA levels. Mutational analysis of individual and multiple NFI binding sites in the URR defined their role in TGF-beta sensitivity of the promoter. Overexpression of the NFI family members in HKc/HPV16 decreased the ability of TGF-beta to inhibit the URR. Since the oncoprotein Ski has been shown to interact with and increase the transcriptional activity of NFI and since cellular Ski levels are decreased by TGF-beta treatment, we explored the possibility that Ski may provide a link between TGF-beta signaling and NFI activity. Anti-NFI antibodies coimmunoprecipitated endogenous Ski in nuclear extracts from HKc/HPV16, confirming that NFI and Ski interact in these cells. Ski levels dramatically decreased upon TGF-beta treatment of HKc/HPV16, and overexpression of Ski eliminated the ability of TGF-beta to inhibit the URR. Based on these studies, we propose that TGF-beta inhibition of HPV16 early gene expression is mediated by a decrease in Ski levels, which in turn dramatically reduces NFI activity.

A binding site for germ cell nuclear factor within c-mos regulatory sequences.

The proto-oncogene c-mos is specifically expressed in male and female germ cells. Previous studies have shown that the orphan nuclear receptor chicken ovalbumin upstream promoter transcription factor (COUP-TF) contributes to the repression of c-mos in somatic cells by binding to an inverted hexamer repeat within the c-mos regulatory region. In the present study, we demonstrate that another nuclear receptor superfamily member, germ cell nuclear factor (GCNF), binds to a sequence overlapping the c-mos COUP-TF binding site. Electrophoretic mobility shift assays with recombinant GCNF and both wild-type and mutant c-mos oligonucleotides demonstrated the binding of GCNF to an extended half site, CCAAGTTCA, which overlaps the first hexamer of the COUP-TF binding site. Transient transfection assays in NIH 3T3 cells further demonstrated that GCNF fused to a VP16 activation domain stimulated transcription from reporter constructs containing the c-mos GCNF binding site. Since GCNF is expressed in male and female germ cells at the same stages of development at which c-mos is transcribed, these results suggest that GCNF may serve as a regulator of c-mos transcription. Mol. Reprod. Dev. 67: 55-64, 2004.

Inhibition of interleukin-4 production in CD4+ T cells by peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands: involvement of physical association between PPAR-gamma and the nuclear factor of activated T cells transcription factor.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) has been implicated in the regulation of multiple inflammatory processes. However, little is known of PPAR-gamma in the regulation of interleukin (IL)-4 expression in T cells. In this study, the effects of PPAR-gamma ligands on production of IL-4, a pro-inflammatory cytokine associated with the pathophysiology of allergic diseases, were investigated. 15-Deoxy-Delta12,14 prostaglandin J2 (15d-PGJ2) and ciglitazone, two representative PPAR-gamma ligands, significantly inhibited IL-4 production in both antigen-stimulated primary CD4+ T cells and the phorbol 12-myristate 13-acetate (PMA)/ionomycin-activated EL-4 T cell line. 15d-PGJ2 and ciglitazone inhibited the activation of IL-4 gene promoter in EL-4 T cells transiently transfected with IL-4 promoter/reporter constructs, and the repressive effect mapped to a region in the IL-4 promoter containing binding sites for nuclear factor of activated T cells (NF-AT). The activation of T cells by PMA/ionomycin resulted in a marked enhancement of the binding activities to the NF-AT site that was significantly inhibited by the addition of PPAR-gamma ligands. In cotransfected EL-4 T cells, PPAR-gamma also inhibited the activation of the IL-4 promoter at multiple NF-AT sites in a ligand-dependent manner. NF-ATc1 bound PPAR-gamma both in vivo and in vitro, and the interaction interfaces involved the Rel similarity domain of NF-ATc1. In cotransfections of HeLa cells, PPAR-gamma inhibited the NF-ATc1 transactivation in a ligand-dependent manner. Coexpression of p300 or AP-1 relieved the PPAR-gamma ligand-mediated inhibition of the NF-AT transactivation. From these results, we propose that PPAR-gamma ligand-mediated suppression of IL-4 production in CD4+ T cells may involve both inhibition of the NFAT-DNA interactions and competitive recruitment of transcription integrators between NF-AT and PPAR-gamma.

Enhanced interleukin-4 production in CD4+ T cells and elevated immunoglobulin E levels in antigen-primed mice by bisphenol A and nonylphenol, endocrine disruptors: involvement of nuclear factor-AT and Ca2+.

Bisphenol A (BPA) and p-nonylphenol (NP) are representative endocrine disruptors (EDs) that may have adverse effects on human health. The influence of these compounds on allergic immune responses remains unclear. In this study, we have examined the effects of BPA and NP on production of interleukin-4 (IL-4), a pro-inflammatory cytokine closely associated with allergic immune responses. Both BPA and NP significantly enhanced IL-4 production in keyhole limpet haemocyanin (KLH)-primed CD4+ T cells in a concentration-dependent manner. Treatment with BPA or NP in vivo resulted in significant increase of IL-4 production in CD4+ T cells and of antigen-specific immunoglobulin E (IgE) levels in the sera of KLH-primed mice. Furthermore, BPA and NP enhanced the activation of IL-4 gene promoter in EL4 T cells transiently transfected with IL-4 promoter/reporter constructs, and the enhancing effect mapped to a region in the IL-4 promoter containing binding sites for nuclear factor (NF)-AT. Activation of T lymphocytes by phorbol 12-myristate 13-acetate/ionomycin resulted in markedly enhanced binding activities to the NF-AT site, which significantly increased upon addition of BPA or NP, as demonstrated by the electrophoretic mobility shift assay, indicating that the transcription factor NF-AT was involved in the enhancing effect of BPA and NP on IL-4 production. The enhancement of IL-4 production by BPA or NP was significantly reduced by nitrendipine, a blocker of Ca2+ influx, and by FK506, a calcineurin inhibitor. FK506 inhibited the NF-AT-DNA binding activity and IL-4 gene promoter activity enhanced by BPA or NP. These results represent the first report describing possible enhancement of allergic response by EDs through increasing IL-4 production in CD4+ T cells and antigen-specific IgE levels in the sera via the stimulation of Ca2+/calcineurin-dependent NF-AT activation.

Overexpressed nuclear factor-kappaB can participate in endogenous C-reactive protein induction, and enhances the effects of C/EBPbeta and signal transducer and activator of transcription-3.

C-reactive protein (CRP), the prototypical human acute phase protein, is produced primarily by hepatocytes. Its expression is modestly induced by interleukin (IL)-6 in Hep3B cells while IL-1, which alone has no effect, synergistically enhances the effects of IL-6. In previous studies of the proximal CRP promoter, we found that signal transducer and activator of transcription-3 (STAT3) and C/EBPbeta -mediated IL-6-induced transcription and that Rel p50 acted synergistically with C/EBPbeta, in the absence of p65, to enhance CRP transcription. Neither a requirement nor a binding site for the classic nuclear factor (NF)-kappaB heterodimer p50/p65 were found. The current studies were undertaken to determine whether similar novel transcription factor interactions might regulate the endogenous CRP gene. Transiently overexpressed p50 or p65 induced CRP mRNA accumulation in Hep3B cells. The heterodimer p50/p65 was markedly more effective than p50 or p65 homodimers. Co-overexpression of p50 or p65 with C/EBPbeta or STAT3 synergistically enhanced CRP expression. Maximal expression was observed with overexpression of all four transcription factors; comparable effects were observed with IL-1beta treatment of cells overexpressing STAT3 + C/EBPbeta. Data from the Human Genome Project revealed 13 potential kappaB sites in the first 4000 bases of the CRP promoter, only one of which, centred at -2652, bound nuclear p50/p65 heterodimer activated by IL-1beta. Our findings indicate that classical NF-kappaB activation can participate in endogenous CRP induction, and that activated NF-kappaB may synergistically enhance the effects of C/EBPbeta and STAT3. They raise the possibility, not as yet established, that NF-kappaB activation may be responsible for the synergistic effect of IL-1beta on IL-6-induced CRP expression.

Histone H1 enhances synergistic activation of the MMTV promoter in chromatin.

Minichromosomes assembled on the mouse mammary tumor virus (MMTV) promoter in vitro exhibit positioned nucleosomes, one of which covers the binding sites for progesterone receptor (PR) and nuclear factor 1 (NF1). Incorporation of histone H1 into MMTV minichromosomes improves the stability of this nucleosome and decreases basal transcription from the MMTV promoter, as well as its response to either PR or NF1. However, histone H1-containing minichromosomes display better PR binding and support a more efficient synergism between PR and NF1, leading to enhanced transcription initiation. A mutant MMTV promoter lacking positioned nucleosomes does not display enhanced transcriptional synergism in the presence of H1. Binding of PR leads to phosphorylation of H1, which leaves the promoter upon transcription initiation. Thus, H1 assumes a complex and dynamic role in the regulation of the MMTV promoter.

Nuclear factor 1 is required for both hormone-dependent chromatin remodeling and transcriptional activation of the mouse mammary tumor virus promoter.

The mouse mammary tumor virus (MMTV) promoter has been used as a model to study how the glucocorticoid receptor (GR) remodels chromatin to allow other transcription factors to bind and activate transcription. To dissect the precise role of nuclear factor 1 (NF1) in chromatin remodeling and transcriptional activation, we used linker-scanning mutants of transcription factor binding sites on the MMTV promoter. We compared the NF1 mutant MMTV promoter in the context of transiently transfected templates (transient transfection) and templates organized as chromatin (stable transfection) to understand the effect of chromatin on factor binding and transcription. We show that on a transiently transfected template, mutation in the NF1 binding site reduces both basal and hormone-dependent transcription. This suggests that NF1 is required for transcription in the absence of organized chromatin. We also found that binding of NF1 on a transiently transfected template is independent of mutation in hormone response elements or the octamer transcription factor (OTF) binding site. In contrast, the binding of OTF proteins to a transiently transfected template was found to be dependent on the binding of NF1, which may imply that NF1 has a stabilizing effect on OTF binding. On a chromatin template, mutation in the NF1 binding site does not affect the positioning of nucleosomes on the promoter. We also show that in the absence of NF1 binding, GR-mediated chromatin remodeling of nucleosome B is reduced and hormone-dependent activation of transcription is abolished. Further, we demonstrate that NF1 is required for both the association of BRG1 chromatin remodeling complex and the GR on the promoter in vivo. These results suggest the novel possibility that NF1 may participate in chromatin remodeling activities in addition to directly enhancing transcription and that in the absence of its binding site the GR is unable to effectively bind the promoter and recruit the remodeling complex.

Identification of nuclear factor 1 (NF1) as a transcriptional modulator of rat A 2A adenosine receptor

By a combination of PCR and DNA walking technique, we isolated a 4.8-kb DNA fragment containing a 4.3 kb 5′-flanking region and a 0.5-kb 5′-untranslated region of the rat A 2A adenosine receptor (A 2A-R) gene. Various lengths of the 5′-flanking region of the A 2A-R gene were inserted into an expression vector and transfected into several different cell lines for promoter analysis. Our results reveal that a consensus NF1 element (designated as A 2A-R/NF1), located between bases −2846 and −2827 of the A 2A-R gene, functions as a repressor for A 2A-R promoters in the rat brain-derived type-2 astrocyte cell line (RBA2), which expresses no A 2A-R. Electrophoretic gel mobility shift assay (EMSA) revealed that two A 2A-R/NF1–protein complexes of RBA2 nuclear extract were formed. Supershift experiments using an anti-NF1 antibody suggest that NF1 proteins exist in both A 2A-R/NF1–protein complexes. Furthermore, mutations in the conserved NF1 binding site of this A 2A-R/NF1 element disturbed DNA–protein formation. Thus, NF1 proteins appear to mediate this cell line-specific suppression of A 2A-R promoters in RBA2 cells. The importance of NF1 proteins in regulating A 2A-R promoters was further confirmed in another cell line (Siha) which expresses no endogenous A 2A-R. Moreover, addition of the A 2A-R/NF1element upstream of an irrelevant thymidine kinase (TK) promoter suppressed its promoter activity in Siha cells, but not in RBA2 cells. Thus, the NF1-mediated inhibition of the A 2A-R promoter was promoter- and cell line-specific. In summary, we have defined a distal negative element (A 2A-R/NF1) that plays a functional role in modulating the expression of A 2A-R.

Liver glucokinase gene expression is controlled by the onecut transcription factor hepatocyte nuclear factor-6.

Glucokinase plays a key role in glucose homeostasis and the expression of its gene is differentially regulated in pancreatic beta cells and in the liver through distinct promoters. The factors that determine the tissue-specific expression of the glucokinase gene are not known. Putative binding sites for hepatocyte nuclear factor (HNF)-6, the prototype of the ONECUT family of transcription factors, are present in the hepatic promoter of the glucokinase gene and hnf6 knockout mice are diabetic [corrected]. We hypothesized that HNF-6 controls the activity of the hepatic glucokinase promoter. We tested the binding of recombinant HNF-6 to DNA sequences from the mouse hepatic glucokinase promoter in vitro and the effect of HNF-6 on promoter activity in transfected cells. We investigated in vivo the role of HNF-6 in mice by examining the effect of inactivating the hnf6 gene on glucokinase gene-specific deoxyribonuclease I hypersensitive sites in liver chromatin and on liver glucokinase mRNA concentration. HNF-6 bound to the hepatic promoter of the glucokinase gene and stimulated its activity. Inactivation of the hnf6 gene did not modify the pattern of deoxyribonuclease I hypersensitive sites but was associated with a decrease of liver glucokinase mRNA to half the control value. Although HNF-6 is not required to open chromatin of the hepatic promoter of the glucokinase gene, it stimulates transcription of the glucokinase gene in the liver. This could partly explain the diabetes observed in hnf6 knockout mice.

Nuclear factor I-B ( Nfib) deficient mice have severe lung hypoplasia

Binding sites for transcription factor nuclear factor one (NFI) proteins, encoded by four genes in the mouse, have been characterized from many tissue-specific genes. NFI genes are expressed in unique but overlapping patterns in embryonic and in adult tissues. Nfib is highly expressed in the embryonic lung. Here we show that Nfib null mutants die early postnatally and display severe lung hypoplasia. Heterozygotes do survive, but exhibit delayed pulmonary differentiation. Expression of transforming growth factor β 1 (TGF-β1) and sonic hedgehog ( Shh) is not down-regulated in mutant lung epithelium at late stages of morphogenesis, which may result in incomplete lung maturation. Our study demonstrates that Nfib is essential for normal lung development, and suggests that it could be involved in the pathogenesis of respiratory distress syndromes in humans.

A Region to the N-terminal Side of the CTCF Zinc Finger Domain Is Essential for Activating Transcription from the Amyloid Precursor Protein Promoter

Transcription from the amyloid precursor protein (APP) promoter is largely dependent on a nuclear factor binding site designated as APBbeta. The protein that binds to this site is the multifunctional transcription factor CTCF, which consists of 727 amino acids and contains a domain of 11 zinc finger motifs that is flanked by 267 amino acids on the N-terminal side and 150 amino acids on the C-terminal side. Depleting HeLa cell nuclear extract of endogenous CTCF specifically reduced transcriptional activity from the APP promoter. However, transcriptional activity was restored by replenishing the depleted extract with recombinant CTCF. Deleting 201 amino acids from the C-terminal end of CTCF had no detrimental effect on transcriptional activation, whereas deleting either 248 or 284 amino acids from the N-terminal end abolished transcriptional activation. Competing endogenous CTCF in vivo was accomplished by cotransfecting COS-1 cells with a plasmid overexpressing CTCF constructs and a reporter plasmid containing the APP promoter. Under these conditions, an N-terminal deletion of CTCF reduced expression from the APP promoter, whereas the C-terminal deletion had no effect. These results demonstrate that CTCF activates transcription from the APP promoter and that the activation domain is located on the N-terminal side of the zinc finger domain.

Identification of a novel nuclear factor-binding site in the Pisum sativum sad gene promoters

DNA fragments containing the 5′ promoter regions of the Pisum sativum sadA and sadC genes were amplified from genomic DNA, cloned and sequenced. These sequences contain a number of conserved cis-acting elements, which are potentially involved in stress-induced transcription of the sad genes. To determine whether any of the identified elements are active in binding nuclear factors in vitro, 11 60-bp overlapping (by 30 bp) DNA probe fragments covering the proximal sadC promoter sequence (360 bp) were used in electrophoretic mobility shift assays with competition. Binding activities were compared in nuclear extracts from control, UV-B-stressed and wounded pea leaves. The pattern of DNA binding was almost identical with all three extracts, with one 30-bp region being the predominant site for factor binding. Using overlapping sub-fragments of this region, the majority of the specific binding could be attributed to the novel 11-bp GC-rich sequence GTGGCGCCCAC. An almost identical sequence is conserved in the sadA promoter. This motif has features in common with a number of recognised cis-elements, which suggests a possible binding site for factors which play a role in regulating sad gene transcription.

HNF-3β, C/EBPβ, and HNF-4 Act in Synergy to Enhance Transcription of the Human Apolipoprotein B Gene in Intestinal Cells

Recently, we identified a 315-bp intestinal enhancer (IE), localized over 55 kb upstream from the transcriptional start of the human apolipoprotein B (apoB) gene, that confers expression of human apoB transgenes in the intestines of mice. Four functional binding sites for the intestine-enriched transcription factors hepatocyte nuclear factor (HNF)-3beta, CAAT enhancer binding protein (C/EBP)beta, and HNF-4 were demonstrated within the 315-bp IE. In this report, we extend these earlier studies and examine the relative contributions of these three transcription factors to the activity of the enhancer as well as their mechanism of interaction with one another. Cotransfection experiments with the expression vectors for HNF-3beta, C/EBPbeta, and HNF-4 revealed that HNF-3beta bound to Site 1, C/EBPbeta bound to Site 2, and HNF-4 bound to Site 3 within the 315-bp IE and that the sites act synergistically to enhance intestinal expression of apoB. Each one of these four binding sites was mutated, and mutant constructs were transfected into intestine-derived CaCo-2 cells to evaluate the role of each of these binding sites in enhancer activity. The results of the mutagenesis experiments confirmed that the HNF-3beta and HNF-4 sites are most important for the enhancer activity, followed by C/EBPbeta Site 2. All three factors bound to Sites 1, 2, and 3 must act synergistically for optimal activity of the apoB IE.

Normal TCRβ transcription and recombination in the absence of the Jβ2–Cβ2 intronic cis element

The developmental regulation of antigen receptor gene transcription and recombination are mediated by cis regulatory elements. At the T cell receptor β chain locus ( TCRβ), two DNase I hypersensitive sites within the Jβ2–Cβ2 intron contained binding sites for NF-κB and additional nuclear factors and were postulated to be involved in controlling TCRβ transcription and V(D)J recombination. To test this possibility, we deleted these elements from the mouse genome by homologous recombination and assayed the effect on transcription of both the germline and rearranged TCRβ locus, and on TCRβ rearrangement in T and B lymphocytes. We found that TCRβ transcription and V(D)J recombination and T cell development were normal in these mutant mice. Therefore, the Jβ2–Cβ2 intronic elements are dispensable for TCRβ assembly and function.

Isolation and Characterization of the Promoter of the Human Prostate Cancer-specific DD3 Gene

Recently, we have described a novel gene, DD3, which is one of the most prostate cancer-specific genes described to date (Bussemakers, M. J. G., van Bokhoven, A., Verhaegh, G. W., Smit, F. P., Karthaus, H. F. M., Schalken, J. A., Debruyne, F. M. J., Ru, N., and Isaacs, W. B. (1999) Cancer Res. 59, 5975-5979). The prostate cancer-specific expression of DD3 indicates that the DD3 gene promoter is a promising tool for the treatment of prostate cancer. To identify the promoter elements that are responsible for the prostate cancer-specific expression of DD3, we have isolated and characterized the DD3 promoter. Sequence analysis of the DD3 5'-flanking region was performed and several promoter-human growth hormone reporter constructs were prepared, which were transiently transfected in the DD3-positive cell line LNCaP and several DD3-negative cell lines. Using a 500-base pair DD3 promoter construct, we could detect promoter activity in LNCaP cells, which was not affected by increasing the size of the constructs. Truncated constructs, however, showed an increased transcriptional activity, suggesting the presence of a silencer that negatively regulates the expression of DD3. DNase-I footprint analysis, using nuclear extracts from LNCaP cells, revealed the presence of three DNase-I-protected areas within the DD3 proximal promoter. We show that the high mobility group I(Y) protein binds to one of the DNase-I-protected areas and recruits another, yet unidentified, protein to the DD3 promoter in LNCaP cells.

Role of FXR and FTF in bile acid-mediated suppression of cholesterol 7alpha-hydroxylase transcription.

Bile acid biosynthesis is subjected to feedback regulation whereby bile acids down-regulate their own synthesis. The major point of this regulation is at the level of cholesterol 7alpha-hydroxylase (7alpha-hydroxylase), which controls bile acid output from the classic pathway. This regulation is at the level of transcription of the gene. Two bile acid response elements have been localized within the 5'-flanking region of the rat gene and these elements overlap three nuclear receptor binding sites for hepatocyte nuclear factor (HNF-4), liver X receptor (LXR) and alpha(1)-fetoprotein transcription factor (FTF). Recently it has been shown that bile acids are physiological ligands for the farnesyl X receptor (FXR), which suggested that FXR could function by binding to one of the three nuclear receptor sites to mediate regulation of 7alpha-hydroxylase transcription by bile acids. In this study we show that FXR is indeed a crucial factor for bile acid-mediated regulation, but that it functions without binding to DNA. Furthermore, we also demonstrate that neither the LXR nor the HNF-4 sites are involved in bile acid-mediated regulation of 7alpha-hydroxylase transcription. Most importantly, we show that the FTF site is essential for regulation of 7alpha-hydroxylase by bile acids, similar to what we have recently demonstrated for another gene of the bile acid biosynthetic pathway, the sterol 12alpha-hydroxylase gene. These studies demonstrate the crucial role of FTF in the expression and regulation of a critical gene in the bile acid biosynthetic pathways.

Promoter Activity and Regulation of the CYP4F2 Leukotriene B4 ω-Hydroxylase Gene by Peroxisomal Proliferators and Retinoic Acid in HepG2 Cells

The human liver CYP4F2 gene (Accession No. AF221943) encodes a leukotriene B4 ω-hydroxylase that metabolizes leukotriene B4 (LTB4) to a less potent proinflammatory eicosanoid, 20-OH-LTB4. We sequenced a 6.7-kb genomic fragment of the human CYP4F2 gene that has the first five exons and 500 bp of the 5′-flanking region. The major transcription start site was found to be 49 bp upstream of the 3′ end of exon 1 and the ATG translation initiation codon was located in exon 2. Besides the TATA box at −39 bp and basal transcription factor binding sites, the promoter region and 412-bp intron 1 have several putative binding sites for nuclear factors that may mediate the inflammatory response and lipid homeostasis. We found two DR1 elements in the 5′ promoter, a DR2 element in intron 1, and RXR/RAR binding sites in both intron 1 and the 5′ promoter. DNase I footprinting revealed three protected sequences, with the region containing two CAATT boxes at −71 and −111 bp important in CYP4F2 gene expression. Luciferase reporter assays showed that the 500-bp upstream sequence has strong promoter activity. Transient transfection experiments identified two sites in the 5′ promoter and intron 1 that cooperate in gene transcription while exon 1 and a GC-rich region flanking exon 1 inhibit transcription. trans-Retinoic acid and 9-cis-retinoic acid stimulate promoter activity 3- and 6-fold, respectively, while cotransfection with RXRα or RAR/RXRα further enhanced activity. Peroxisome proliferators inhibit CYP4F2 gene promoter activity and cotransfection with PPARα or PPARα/RXRα can slightly attenuate this inhibition. Both saturated fatty acids and 12-hydroxydodecanoic acid (12-OH-C12) can stimulate CYP4F2 gene promoter activity. Therefore, the CYP4F2 gene is repressed by peroxisomal proliferators and induced by retinoic acid, with RAR/RXRα mediating the induction while PPARα/RXR functions neither in the repression nor in the induction by peroxisomal proliferators or retinoic acid.

Identification of Transacting Factors Responsible for the Tissue-specific Expression of Human Glucose Transporter Type 2 Isoform Gene: COOPERATIVE ROLE OF HEPATOCYTE NUCLEAR FACTORS 1α AND 3β

We investigated transacting factors binding to the cis-element important in tissue-specific expression of the human glucose transporter type 2 isoform (GLUT2) gene. By transient transfection assay, we determined that the 227-base pair fragment upstream of the ATG start site contained promoter activity and that the region from +87 to +132 (site C) was responsible for tissue-specific expression. DNase I footprinting and electrophoretic mobility shift assay indicated that site C contained one binding site for hepatocyte nuclear factor 1 (HNF1) and two binding sites for HNF3. The mutations at positions +101 and +103, which are considered to be critical in binding HNF1 and HNF3, resulted in a 53% decrease in promoter activity, whereas the mutation of the proximal HNF3 binding site (+115 and +117) reduced promoter activity by 28%. The mutations of these four sites resulted in marked decrease (70%) in promoter activity as well as diminished bindings of HNF1 and HNF3. A to G mutation, which causes conversion of the HNF1 and HNF3 binding sequence to the NF-Y binding site, resulted in a 22% decrease in promoter activity. We identified that both HNF1 and HNF3 function as transcriptional activators in GLUT2 gene expression. Coexpression of the pGL+74 (+74 to +301) construct with the HNF1alpha and HNF3beta expression vectors in NIH 3T3 cells showed the synergistic effect on GLUT2 promoter activity compared with the expression of HNF1alpha, HNF3beta, or a combination of HNF1beta and HNF3beta. These data suggest that HNF1alpha and HNF3beta may be the most important players in the tissue-specific expression of the human GLUT2 gene.

Involvement of two NF-kappa B binding elements in tumor necrosis factor alpha -, CD40-, and epstein-barr virus latent membrane protein 1-mediated induction of the cellular inhibitor of apoptosis protein 2 gene.

The antiapoptotic function of NF-kappaB is believed to be mediated through the induction of antiapoptotic genes. Among the antiapoptotic genes, cellular inhibitor of apoptosis protein 2 (c-IAP2/HIAP-1/MIHC) is originally identified as a molecule recruited to the tumor necrosis factor (TNF) receptor complex, and its expression is preferentially up-regulated by TNF and other stimuli activating NF-kappaB. However, direct evidence of transcriptional regulation of NF-kappaB on the c-IAP2 gene is still missing. Here, we have cloned and characterized the promoter region required for NF-kappaB-dependent transcription of the c-IAP2 gene. Sequencing of a 3.5-kilobase fragment of the 5'-flanking region of the c-IAP2 gene has identified a TATA-like sequence and potential binding sites for nuclear factor of activated T cells, interferon regulatory factor 1, activator protein 1, glucocorticoid response element, and three putative NF-kappaB binding elements. Deletion and mutational analysis of the 5'-flanking region linked to the luciferase gene revealed that transcriptional activation by TNF or interleukin 1 is mediated cooperatively by two NF-kappaB binding sites. Electrophoretic mobility shift assays characterized that the two NF-kappaB sites can be recognized and bound by the NF-kappaB p50/p65 heterodimer. In addition, the transcription of c-IAP2 promoter was strongly up-regulated when CD40 or Epstein-Barr virus latent membrane protein 1 was overexpressed.

The rabbit sex hormone-binding globulin gene: structural organization and characterization of its 5-flanking region.

Sex hormone-binding globulin (SHBG) transports sex steroids in the blood. In humans and rabbits, the gene encoding SHBG (shbg) is expressed primarily in the liver and testis, whereas the testis is the major site of shbg expression in rodents postnatally. Sequence analysis has revealed that rabbit shbg (rbshbg) spans 2.5 kb and comprises eight exons with consensus splice sites at all exon-intron junctions. The major transcription start site ofrbshbg is located 52 bp upstream from the translation initiation codon for the rabbit SHBG precursor. Unlike the situation in humans and rats, rbshbg transcripts contain no alternative exon 1 sequences in the liver or testis, and this suggests that the rbshbg 5'-flanking region plays an equally important role in controlling transcription of this gene in these tissues. Like the human and rat shbg promoter sequences, the rbshbg proximal promoter lacks a typical TATA box. It also contains several transcription factor-binding sites, but deoxyribonuclease I footprinting experiments indicated that the human and rabbit shbg proximal promoters interact quite differently with proteins extracted from rabbit liver nuclei. However, the predominant footprint on the rbshbg promoter is conserved at the same position within the human shbg (hshbg) promoter and includes consensus binding sites for the transcription factor nuclear factor- 1. Transient transfection studies of the rbshbg 5'-flanking sequence (893 bp) revealed regions that actively enhance and repress its activity in human hepatoblastoma and mouse Sertoli cells, but not in Chinese hamster ovary cells. Like the rat shbg proximal promoter, the rbshbg 5'-flanking sequence lacks a region that corresponds to a cis-element, designated footprinted region 4 in the hshbg proximal promoter. Furthermore, the hshbg promoter footprinted region 3 sequence is poorly conserved in rbshbg, and when mutated to resemble the corresponding human sequence it increased the transcriptional activity of the rbshbg promoter by 7-fold in hepatoblastoma cells. Thus, the rabbit and hshbg promoters appear to be controlled by a different set of transcriptional regulators. Further comparisons of their functional activities may shed light on species-specific differences in the spatial and temporal expression of this gene, the products of which play important roles in regulating sex steroid access to target cells.