Sites For Nuclear Factor Research Articles

NF1, Sp1 and HSF1 are synergistically involved in sulfide-induced sqr activation in echiuran worm Urechis unicinctus

BackgroundSulfide is a well-known environmental toxic substance. Mitochondrial sulfide oxidation is a main mechanism of sulfide detoxification in organisms, and sulfide: quinone oxidoreductase (SQR) is a key enzyme which is involved in transferring electrons from sulfide to ubiquinone and converting sulfide into thiosulfate. Previous studies have revealed the SQR-mediated mitochondrial sulfide oxidation exists in the echiuran worm Urechis unicinctus, and its sqr mRNA level increased significantly when the worm is exposed to sulfide. In this study, we attempt to reveal the synergistic regulation of transcription factors on sulfide-induced sqr transcription in U. unicinctus. MethodsChIP and EMSA were used to identify the interactions between sqr proximal promoter (from −391 to +194bp) and transcription factors NF1 (nuclear factor 1) and Sp1 (specificity protein 1). Site-directed mutation and transfection assays further revealed their binding sites and synergistic roles of HSF1, NF1 and Sp1 in the sqr transcription. When U. unicinctus were exposed to 150μM sulfide, the expression levels and nuclear contents of NF1 and Sp1 were examined by Western blotting, and the binding contents between NF1 or Sp1 and the sqr promoter were also detected by ChIP. ResultsTranscription factors NF1 and Sp1 were confirmed to interact with the sqr proximal promoter, and their binding sites were identified in −75 to −69bp for NF1 and −210 to −201bp for Sp1. Transfection assays showed mutation of NF1 or Sp1 binding site significantly decreased the sqr promoter activity by 50% or 73%, respectively. Moreover, we demonstrated three transcription factors NF1, Sp1 and HSF1 enhanced synergistically the activity of sqr transcription. Furthermore, contents of NF1 or Sp1 binding to the sqr proximal region increased significantly in the hindgut when the worms were exposed to 150μM sulfide. Similar changes of NF1 or Sp1 levels and nuclear NF1 or Sp1 levels were also presented. ConclusionTranscription factors NF1, Sp1 and HSF1 are all involved in sulfide-induced sqr transcription. Sulfide can activate sqr transcription by not only increasing their expression levels, but also promoting them entering nucleus and binding to the sqr promoter. NF1 and Sp1 participate in both basal and sulfide-induced sqr transcription, while HSF1 functions mainly in sulfide-induced sqr transcription.

Expression of ADAMTS1 in endothelial cells is induced by shear stress and suppressed in sprouting capillaries

ADAMTS1 inhibits capillary sprouting, and since capillary sprouts do not experience the shear stress caused by blood flow, this study undertook to clarify the relationship between shear stress and ADAMTS1. It was found that endothelial cells exposed to shear stress displayed a strong upregulation of ADAMTS1, dependent upon both the magnitude and duration of their exposure. Investigation of the underlying pathways demonstrated involvement of phospholipase C, phosphoinositide 3-kinase, and nitric oxide. Forkhead box protein O1 was identified as a likely inhibitor of the system, as its knockdown was followed by a slight increase in ADAMTS1 expression. In silico prediction displayed a transcriptional binding site for Forkhead box protein O1 in the promotor region of the ADAMTS1 gene, as well as sites for nuclear factor 1, SP1, and AP-1. The anti-angiogenic effects of ADAMTS1 were attributed to its cleavage of thrombospondin 1 into a 70-kDa fragment, and a significant enhancement of this fragment was indeed demonstrated by immunoblotting shear stress-treated cells. Accordingly, scratch wound closure displayed a slowdown in conditioned medium from shear stress-treated endothelial cells, an effect that could be completely blocked by a knockdown of thrombospondin 1 and partially blocked by a knockdown of ADAMTS1. Non-perfused capillary sprouts in rat mesenteries stained negative for ADAMTS1, while vessels in the microcirculation that had already experienced blood flow yielded the opposite results. The shear stress-dependent expression of ADAMTS1 in vitro was therefore also demonstrated in vivo and thereby confirmed as a mechanism connecting blood flow with the regulation of angiogenesis.

Nuclear factor I-A represses expression of the cell adhesion molecule L1

BackgroundThe neural cell adhesion molecule L1 plays a crucial role in development and plasticity of the nervous system. Neural cells thus require precise control of L1 expression.ResultsWe identified a full binding site for nuclear factor I (NFI) transcription factors in the regulatory region of the mouse L1 gene. Electrophoretic mobility shift assay (EMSA) showed binding of nuclear factor I-A (NFI-A) to this site. Moreover, for a brain-specific isoform of NFI-A (NFI-A bs), we confirmed the interaction in vivo using chromatin immunoprecipitation (ChIP). Reporter gene assays showed that in neuroblastoma cells, overexpression of NFI-A bs repressed L1 expression threefold.ConclusionOur findings suggest that NFI-A, in particular its brain-specific isoform, represses L1 gene expression, and might act as a second silencer of L1 in addition to the neural restrictive silencer factor (NRSF).

Variations in the Ghrelin Receptor Gene Associate with Obesity and Glucose Metabolism in Individuals with Impaired Glucose Tolerance

BackgroundGhrelin may influence the development of obesity through its role in the control of energy balance, food intake, and regulation of body weight. The effects of ghrelin are mediated via the growth hormone secretagogue receptor (GHSR).Methodology/Principal FindingsWe genotyped 7 single nucleotide polymorphisms (SNPs) in the GHSR gene and assessed the association between those SNPs and obesity and type 2 diabetes-related phenotypes from 507 middle-aged overweight persons with impaired glucose tolerance participating in the Finnish Diabetes Prevention Study (DPS). Additionally, we performed in silico screening of the 5′-regulatory region of GHSR and evaluated SNPs disrupting putative transcription factor (TF) binding sites in vitro with gelshift assays to determine differences in protein binding between different alleles of SNPs. Rs9819506 in the promoter region of GHSR was associated with body weight (p = 0.036); persons with rs9819506-AA genotype having the lowest body weight. Individuals with rs490683-CC genotype displayed highest weight loss in the whole study population (p = 0.032). The false discovery rate for these results was <10%. Rs490683 and rs509035 were associated with several measures of glucose and insulin metabolism during the follow-up. Rs490683 may be a functional SNP, since gelshift experiments showed differential protein binding between the alleles, with higher binding to the G-allele. Rs490683-C may disrupt a putative binding site for the TF nuclear factor 1 (NF-1), thus rs4906863-GG genotype where the NF-1 site is intact may lead to a higher GHSR gene expression.Conclusion/SignificancePolymorphisms in the GHSR promoter may modify changes in body weight during long-term lifestyle intervention and affect ghrelin receptor signalling through modulation of GHSR gene expression.

Regulated Expression of ADAM8 (a Disintegrin and Metalloprotease Domain 8) in the Mouse Ovary: Evidence for a Regulatory Role of Luteinizing Hormone, Progesterone Receptor, and Epidermal Growth Factor-Like Growth Factors1

ADAM8 (a disintegrin and metalloprotease domain 8) is expressed in immune, neuronal, and bone progenitor cells and is thought to be involved in the tissue-remodeling process. Microarray analyses indicate that Adam8 is a potential target of the progesterone receptor (Pgr) in murine ovary. Further studies document that Adam8 mRNA and protein are expressed in granulosa cells and cumulus cells of periovulatory follicles whereas expression is significantly reduced in Pgr null mice that fail to ovulate. There is a reduced expression in granulosa cells from cultured, in vitro ovulated follicles exposed to inhibitors of progesterone or epidermal growth factor signaling while epiregulin induced its expression in the absence of hCG. In vitro studies with primary mouse granulosa cells document that Adam8 is induced in response to forskolin (Fo) and phorbol ester (PMA) or Fo and Amphiregulin treatment. To understand the transcriptional regulation of the Adam8, we amplified 1 kb of the mouse Adam8 promoter by PCR and subcloned it into a pGL3-luciferase reporter construct. The Adam8 promoter-luciferase constructs are induced by Fo and PMA treatment after transfection into rat granulosa cells, and cotransfection with a PGR-A expression vector further augment basal and Fo/PMA inducibility. Site-specific mutations within the -615/+50 promoter document that a GC-rich region, NF-1 (nuclear factor-1) site, and putative TATA box are critical for Adam8 promoter activation by Fo/PMA. Thus, ADAM8 is expressed in a stage-specific manner and is hormonally regulated in ovulating follicles by the coordinate actions of LH and PGR. To our knowledge, ADAM8 is the first member of the ADAM family shown to be hormonally regulated.

Mutational Analysis of the Varicella-Zoster Virus ORF62/63 Intergenic Region

The varicella-zoster virus (VZV) ORF62/63 intergenic region was cloned between the Renilla and firefly luciferase genes, which acted as reporters of ORF62 and ORF63 transcription, and recombinant viruses were generated that carried these reporter cassettes along with the intact native sequences in the repeat regions of the VZV genome. In order to investigate the potential contributions of cellular transregulatory proteins to ORF62 and ORF63 transcription, recombinant reporter viruses with mutations of consensus binding sites for six proteins within the intergenic region were also created. The reporter viruses were used to evaluate ORF62 and ORF63 transcription during VZV replication in cultured fibroblasts and in skin xenografts in SCIDhu mice in vivo. Mutations in putative binding sites for heat shock factor 1 (HSF-1), nuclear factor 1 (NF-1), and one of two cyclic AMP-responsive elements (CRE) reduced ORF62 reporter transcription in fibroblasts, while mutations in binding sites for HSF-1, NF-1, and octamer binding proteins (Oct-1) increased ORF62 reporter transcription in skin. Mutations in one CRE and the NF-1 site altered ORF63 transcription in fibroblasts, while mutation of the Oct-1 binding site increased ORF63 reporter transcription in skin. The effect of each of these mutations implies that the intact binding site sequence regulates native ORF62 and ORF63 transcription. Mutation of the only NF-kappaB/Rel binding site had no effect on ORF62 or ORF63 transcription in vitro or in vivo. The segment of the ORF62/63 intergenic region proximal to ORF63 was most important for ORF63 transcription, but mutagenesis also altered ORF62 transcription, indicating that this region functions as a bidirectional promoter. This first analysis of the ORF62/63 intergenic region in the context of VZV replication indicates that it is a dual promoter and that cellular transregulatory factors affect the transcription of these key VZV regulatory genes.

225. A Novel Mechanism between Diabetes Mellitus Complications and Procalcitonin Gene Expression

Introduction: Procalcitonin (PCT) was originally described in 1984 as a 116 aminoacid protein with a molecular weight of 14.5 kDa. The PCT gene, referred to as Calc-1, is located on chromosome 11p15.4 and was sequenced in 1989. The promoter has sites for basal transcription factors but more interestingly, also has sites for Nuclear factor |[kappa]||[beta]| (NF|[kappa]||[beta]|) and activator protein -1 (AP-1), factors induced under inflammatory conditions. Diabetes Mellitus is associated with oxidative stress and elevation of advanced glycation end products (AGEs). AGEs are produced by a non-enzymatic, maillard reaction between reducing sugars and either proteins or lipids. AGEs interacts the receptor for advanced glycation end products (RAGE) and RAGE activation is caused by elevation of transcriptional factors NF|[kappa]||[beta]| and AP-1. These factors induce procalcitonin gene expression. The aim of this study was to determine whether or not procalcitonin is a specific marker in patients with diabetic complications.

The CYP2B2 phenobarbital response unit contains binding sites for hepatocyte nuclear factor 4, PBX–PREP1, the thyroid hormone receptor β and the liver X receptor

A 163 bp enhancer in the CYP2B2 5' flank confers PB (phenobarbital) inducibility and constitutes a PBRU (PB response unit). The PBRU contains several transcription factor binding sites, including NR1, NR2 and NR3, which are direct repeats separated by 4 bp of the nuclear receptor consensus half-site AGGTCA, as well as an ER (everted repeat) separated by 7 bp (ER-7). Constitutive androstane receptor (CAR)-RXR (retinoic X receptor) heterodimers are known to bind to NR1, NR2 and NR3. Electrophoretic mobility-shift analysis using nuclear extracts from livers of untreated or PB-treated rats revealed binding of several other proteins to different PBRU elements. Using supershift analysis and in vitro coupled transcription and translation, the proteins present in four retarded complexes were identified as TRbeta (thyroid hormone receptor beta), LXR (liver X receptor), HNF-4 (hepatocyte nuclear factor 4) and heterodimers of PBX-PREP1 (pre-B cell homoeobox-Pbx regulatory protein 1). LXR-RXR heterodimers bound to NR3 and TRbeta bound to NR3, NR1 and ER-7, whereas the PBX-PREP1 site is contained within NR2. The HNF-4 site overlaps with NR1. A mutation described previously, GRE1m1, which decreases PB responsiveness, increased the affinity of this site for HNF-4. The PBRU also contains a site for nuclear factor 1. The PBRU thus contains a plethora of transcription factor binding sites. The profiles of transcription factor binding to NR1 and NR3 were quite similar, although strikingly different from, and more complex than, that of NR2. This parallels the functional differences in conferring PB responsiveness between NR1 and NR3 on the one hand, and NR2 on the other.

Regulation of Cytochrome b5 Gene Transcription by Sp3, GATA-6, and Steroidogenic Factor 1 in Human Adrenal NCI-H295A Cells

Sex steroid synthesis requires the 17,20 lyase activity of P450c17, which is enhanced by cytochrome b5, acting as an allosteric factor to promote association of P450c17 with its electron donor, P450 oxidoreductase. Cytochrome b5 is preferentially expressed in the fetal adrenal and postadrenarchal adrenal zona reticularis; the basis of this tissue-specific, developmentally regulated transcription of the b5 gene is unknown. We found b5 expression in all cell lines tested, including human adrenal NCI-H295A cells, where its mRNA is reduced by cAMP and phorbol ester. Multiple sites, between -83 and -122 bp upstream from the first ATG, initiate transcription. Deletional mutagenesis localized all detectable promoter activity within -327/+15, and deoxyribonuclease I footprinting identified protein binding at -72/-107 and -157/-197. DNA segments -65/-40, -114/-70 and -270/-245 fused to TK32/Luc yielded significant activity, and mutations in their Sp sites abolished that activity; electrophoretic mobility shift assay (EMSA) showed that Sp3, but not Sp1, binds to these Sp sites. Nuclear factor 1 (NF-1) and GATA-6, but not GATA-4 bind to the NF-1 and GATA sites in -157/-197. In Drosophila S2 cells, Sp3 increased -327/Luc activity 58-fold, but Sp1 and NF-1 isoforms were inactive. Mutating the three Sp sites ablated activity without or with cotransfection of Sp1/Sp3. In NCI-H295A cells, mutating the three Sp sites reduced activity to 39%; mutating the Sp, GATA, and NF-1 sites abolished activity. In JEG-3 cells, GATA-4 was inactive, GATA-6 augmented -327/Luc activity to 231% over the control, and steroidogenic factor 1 augmented activity to 655% over the control; these activities required the Sp and NF-1 sites. Transcription of cytochrome b5 shares many features with the regulation of P450c17, whose activity it enhances.

Distinct roles of enhancer nuclear factor 1 (NF1) sites in plasmacytoma and osteopetrosis induction by Akv1-99 murine leukemia virus

Murine leukemia viruses (MLVs) can be lymphomagenic and bone pathogenic. In this work, the possible roles of two distinct proviral enhancer nuclear factor 1 (NF1) binding sites in osteopetrosis and tumor induction by B-lymphomagenic Akv1-99 MLV were investigated. Akv1-99 and mutants either with NF1 site 1, NF1 site 2 or both sites disrupted induced tumors (plasma cell proliferations by histopathology) with remarkably similar incidence and mean latency in inbred NMRI mice. Clonal immunoglobulin gene rearrangement detection, by Southern analysis, confirmed approximately half of the tumors induced by each virus to be plasmacytomas while the remaining lacked detectable clonally rearranged Ig genes and were considered polyclonal; a demonstration that enhancer NF1 sites are dispensable for plasmacytoma induction by Akv1-99. In contrast, X-ray analysis revealed significant differences in osteopetrosis induction by the four viruses strongly indicating that NF1 site 2 is critical for viral bone pathogenicity, whereas NF1 site 1 is neutral or moderately inhibitory. In conclusion, enhancer NF1 sites are major determinants of osteopetrosis induction by Akv1-99 without significant influence on viral oncogenicity.

Molecular mechanisms of hereditary persistence of α-fetoprotein (AFP) in two Japanese familiesA hepatocyte nuclear factor-1 site mutation leads to induction of the AFP gene expression in adult livers

alpha-Fetoprotein (AFP) is produced abundantly in fetal liver but is hardly detectable in adults. In this study, we investigated two unrelated Japanese families with hereditary persistence of AFP. A g-->a substitution at nucleotide -119 (-119g-->a) in the hepatocyte nuclear factor (HNF)-1 binding site of the AFP promoter was identified in both families. The activity of the wild- or variant-type human AFP promoter was evaluated by in vitro and in vivo transfection experiments. This substitution in the AFP promoter significantly stimulated its transcriptional activity in human hepatoma cells, regardless of their prior AFP production. The variant-type AFP promoter was also active in adult mouse livers in vivo. Additionally, overexpression of HNF-1alpha stimulated the activity of both the wild- and variant-type AFP promoters in hepatoma cells. HNF-1alpha expression also activated both AFP promoters even in nonhepatoma cells, and this activation was suppressed by nuclear factor (NF)-I overexpression. These results indicate that an HNF-1 binding site mutation leads to induction of the AFP gene expression in adult liver, and suggest that competition between HNF-1 and NF-I in this region is involved in transcriptional regulation of the AFP gene during hepatic development.

Chromatin-Mediated Restriction of Nuclear Factor 1/CTF Binding in a Repressed and Hormone-Activated Promoter In Vivo

Mouse mammary tumor virus (MMTV) promoter-driven transcription is induced by glucocorticoid hormone via binding of the glucocorticoid receptor (GR). The MMTV promoter also harbors a binding site for nuclear factor 1 (NF1). NF1 and GR were expressed in Xenopus oocytes; this revealed GR-NF1 cooperativity both in terms of DNA binding and chromatin remodeling but not transcription. A fraction of NF1 sites were occupied in a hormone-dependent fashion, but a significant and NF1 concentration-dependent fraction were constitutively bound. Activation of the MMTV promoter resulted in an approximately 50-fold increase in the NF1 accessibility for its DNA site. The hormone-dependent component of NF1 binding was dissociated by addition of a GR antagonist; however, the antagonist RU486, which supports partial GR-DNA binding, also maintained partial NF1 binding. Hence GR-NF1 cooperativity is independent of agonist-driven chromatin remodeling. NF1 induced the formation of a micrococcal-nuclease-resistant protein-DNA complex containing the DNA segment from -185 to -55, the MMTV enhanceosome. Coexpression of NF1 and Oct1 resulted in a significant stimulation of hormone-induced MMTV transcription and also in increased basal transcription. We propose that hormone-independent NF1 binding may be involved in maintaining transcriptional competence and establishment of tissue-specific gene networks.

Hereditary persistence of α-fetoprotein is due to both proximal and distal hepatocyte nuclear factor-1 site mutations

Background & Aims: The molecular mechanism of hereditary persistence of α-fetoprotein (HPAFP) has been previously described in a large Scottish family, consisting of a −119G>A substitution in the distal hepatocyte nuclear factor 1 (HNF-1) binding site of the α-fetoprotein (AFP) gene promoter. We report here the molecular mechanisms of HPAFP in 2 new unrelated families. Methods: Family 1 was of Bengali origin, and family 2 was Italian. Four of 5 subjects (family 1) and 3 of 9 (family 2) showed HPAFP. The AFP gene promoter was studied in all available family members. Results: All subjects with high AFP levels had mutated promoter sequences. Family 1 showed the reported −119G>A substitution. Family 2 showed −55C>A and −65C>T substitutions in the proximal putative HNF-1 binding region of the promoter. The −55C>A mutation increased the similarity of the proximal HNF-1 binding region to a consensus binding region. Gel shift assays confirmed its increased affinity toward HNF-1, and transfection experiments revealed an increased level of gene transcription. The −65C>T substitution theoretically created a CCAAT box. However, gel shift and transfection experiments failed to show any biological effect of this substitution that is associated with the −55C>A mutation. Conclusions: Two different mutations localized in either HNF-1 binding sites of the AFP gene promoter may result in HPAFP. This highlights the importance of HNF-1 in AFP gene expression. Unexplained persistent AFP should lead to family study and/or AFP gene promoter sequencing to avoid inappropriate explorations and treatment decisions.

NF-1C, Sp1, and Sp3 are essential for transcription of the human gene for P450c17 (steroid 17alpha-hydroxylase/17,20 lyase) in human adrenal NCI-H295A cells.

Cytochrome P450c17 catalyzes steroid 17alpha-hydroxylase and 17,20 lyase activities, which are required for the biosynthesis of cortisol and sex steroids. Human P450c17 is expressed in a cAMP-responsive, cell-specific, developmentally programmed fashion, but little is known about its transcriptional regulation. Expression of deletion mutants of up to 2,500 bp of human 5'-flanking DNA in human adrenal NCI-H295A cells indicated that most regulatory activity was confined to the first 227 bp. Deoxyribonuclease I footprinting of the proximal promoter identified the TATA box, an steroidogenic factor-1 site, and three previously uncharacterized sites at -107/85, at -178/-152, and at -220/-185. EMSAs and methylation interference assays suggested that the -107/-85 site and the -178/-152 site bind members of the NF-1 (nuclear factor-1) family of transcription factors. An NF-1 consensus sequence generated similar DNA/protein complexes, and antibodies against NF-1C2/CTF2 supershifted the complexes formed by the -107/-85 site, the -178/-152 site, and the NF-1 consensus site. Western blots of nuclear extracts from NCI-H295A cells probed with this NF-1 antiserum identified two NF-1 isoforms between 50 and 55 kDa. The presence of NF-1C2 (CTF2) and CTF5 in NCI-H295A cells was demonstrated by RT-PCR and sequencing. Mutation of both the -107/-85 and the -178/-152 NF-1 sites reduced basal transcription by half. Supershift assays showed that the ubiquitous proteins Sp1 and Sp3 both bind to the -227/-184 region, and that mutation of their binding sites reduced transcription by 75%. Mutation of the Sp1/Sp3 site plus the two NF-1 sites eliminated almost all detectable transcription. Thus, Sp1 and Sp3 binding to the -227/-184 site and NF-1C proteins binding to the -107/-85 and the -178/-152 sites are crucial for adrenal transcription of the human gene for P450c17.

Chromatin Assembly Enhances Binding to the CYP2B1Phenobarbital-responsive Unit (PBRU) of Nuclear Factor-1, Which Binds Simultaneously with Constitutive Androstane Receptor (CAR)/Retinoid X Receptor (RXR) and Enhances CAR/RXR-mediated Activation of the PBRU

Phenobarbital induction of CYP2B genes is mediated by a complex phenobarbital-responsive enhancer (PBRU), which contains a binding site for nuclear factor-1 (NF-1) flanked by two DR-4 nuclear receptor (NR) binding sites for a heterodimer of constitutive androstane receptor (CAR) and retinoid X receptor (RXR). To examine potential interactions between NF-1 and CAR/RXR, binding of purified recombinant proteins to DNA, or to chromatin assembled using Drosophila embryo extract, was examined. NF-1 and CAR/RXR bound simultaneously and independently to the overlapping NF-1 and NR-1 sites; binding of CAR/RXR to the NR-2 site was modestly increased by NF-1 binding; and CAR/RXR bound to a new site in the PBRU region, designated NR-3. Assembly of plasmid DNA into chromatin using Drosophila extract resulted in linearly phased nucleosomes in the PBRU region. The apparent binding affinity of NF-1 was increased by about 10-fold in assembled chromatin compared with DNA, whereas CAR/RXR binding was decreased. As observed for DNA, however, simultaneous, largely independent, binding to the NF-1 and NR sites was observed. CAR-mediated transactivation of the PBRU in cultured cells of hepatic origin was inhibited by mutations in the NF-1 site, and overexpression of NF-1 increased CAR transactivation in HepG2 cells. These studies demonstrate that NF-1 and CAR/RXR can both bind to the PBRU at the same time and that chromatin assembly increases NF-1 binding, which is consistent with previous in vivo footprinting studies in which the NF-1 site was occupied in untreated animals and the NF-1 and flanking NR sites were occupied after phenobarbital treatment. CAR-mediated trans-activation of the PBRU was increased by NF-1, analogous to NF-1 effects on phenobarbital induction in previous transient transfection studies and consistent with mediation of phenobarbital induction by CAR.

SF-1 (steroidogenic factor-1), C/EBPbeta (CCAAT/enhancer binding protein), and ubiquitous transcription factors NF1 (nuclear factor 1) and Sp1 (selective promoter factor 1) are required for regulation of the mouse aldose reductase-like gene (AKR1B7) expression in adrenocortical cells.

The MVDP (mouse vas deferens protein) gene encodes an aldose reductase-like protein (AKR1B7) that is responsible for detoxifying isocaproaldehyde generated by steroidogenesis. In adrenocortical cell cultures, hormonal regulation of MVDP gene occurs through the cAMP pathway. We show that in adrenals, the pituitary hormone ACTH regulates MVDP gene expression in a coordinate fashion with steroidogenic genes. Cell transfection and DNA-binding studies were used to investigate the molecular mechanisms underlying MVDP gene regulation in Y1 adrenocortical cells. Progressive deletions of upstream regulatory regions identified a -121/+41 fragment that was sufficient for basal and cAMP-mediated transcriptional activities. Gel shift assays showed that CTF1/nuclear factor 1 (NF1), CCAAT enhancer binding protein-ss (C/EBPss), and selective promoter factor 1 (Sp1) factors bound to cis-acting elements at positions -76, -61, and -52, respectively. We report that the cell-specific steroidogenic factor-1 (SF-1) interacts specifically with a novel regulatory element located in the downstream half-site of the proximal androgen response element (AREp) at position -102. Functional analysis of SF-1 and NF1 sites in the -121/+41 promoter showed that mutation of one of them decreases both constitutive and forskolin-stimulated promoter activity without affecting the fold induction (forskolin stimulated/basal). Individual mutations of C/EBP and Sp1 sites resulted in a loss of more than 50% of the cAMP-dependent induction. When both sites were mutated simultaneously, cAMP responsiveness was nearly abolished. Thus, in adrenocortical cells, both SF-1 and NF1 are required for high expression of the MVDP promoter while Sp1 and C/EBPss functionally interact in an additive manner to mediate cAMP-dependent regulation. Furthermore, we report that MVDP gene regulation is impaired in stably transfected Y1 clones expressing DAX-1. Taken together, our findings suggest that detoxifying enzymes of the aldose reductase family may constitute new potential targets for regulators of adrenal and gonadal differentiation and function, e.g. SF-1 and DAX-1.

A Conserved Nuclear Receptor Consensus Sequence (DR-4) Mediates Transcriptional Activation of the Chicken CYP2H1 Gene by Phenobarbital in a Hepatoma Cell Line

Phenobarbital-responsive DNA elements were identified in the 5'-flanking region of the chicken CYP2H1 gene by in reporter gene assays in a chicken hepatoma cell line (leghorn male hepatoma (LMH)). A 264-base pair (bp) enhancer sequence (phenobarbital-responsive unit (PBRU)) responded to phenobarbital and a variety of phenobarbital-type inducers. Analysis of putative transcription factor binding sites within the 264-bp element revealed a nuclear receptor half-site repeat (DR-4) neighboring a putative nuclear factor-1 site. This motif resembles phenobarbital response elements in the flanking regions of three phenobarbital-inducible genes, rat CYP2B2, mouse Cyp2b10, and human CYP2B6. Activation of the 264-bp element was eliminated after site-directed mutagenesis of the DR-4 hexamer half-sites. Evidence for evolutionary conservation of this recognition site was indicated by activation in LMH cells of a mouse Cyp2b10 phenobarbital-responsive enhancer by the same spectrum of inducers that activate the CYP2H1 264-bp PBRU. Inhibition of this activation by okadaic acid may explain the reported inhibitory effects on induction of CYP2B1/2 and Cyp2b10 by this phosphatase inhibitor. We show that this inhibition occurs directly on the 264-bp PBRU, whereas the proximal promoter of CYP2H1 is induced by okadaic acid in reporter gene assays. These experiments exploit the unique phenobarbital inducibility of the hepatoma-derived cell line LMH.

Hormone activation induces nucleosome positioning in vivo.

The mouse mammary tumor virus (MMTV) promoter is induced by glucocorticoid hormone. A robust hormone- and receptor-dependent activation could be reproduced in Xenopus laevis oocytes. The homogeneous response in this system allowed a detailed analysis of the transition in chromatin structure following hormone activation. This revealed two novel findings: hormone activation led to the establishment of specific translational positioning of nucleosomes despite the lack of significant positioning in the inactive state; and, in the active promoter, a subnucleosomal particle encompassing the glucocorticoid receptor (GR)-binding region was detected. The presence of only a single GR-binding site was sufficient for the structural transition to occur. Both basal promoter elements and ongoing transcription were dispensable. These data reveal a stepwise process in the transcriptional activation by glucocorticoid hormone.

BAG-1, a novel Bcl-2-interacting protein, activates expression of human JC virus.

Transcription of the human polyomavirus JC virus (JCV) genome is regulated by cellular proteins and the large tumour (T) antigen. Earlier studies led to the identification of nuclear factor-1 (NF-1)-binding sites in the JCV enhancer by DNase I protection assays of extracts from retinoic acid (RA)-differentiated P19 embryonal carcinoma (EC) cells. In this study, a cDNA clone that encodes a protein capable of binding to the JCV NF-1 sites was isolated from an RA-differentiated EC cell cDNA library. Sequence analysis revealed that the cDNA isolated was identical to the previously described Bcl-2-interacting protein BAG-1 (Bcl-2-associated athano gene-1). Results from RNA studies indicated that BAG-1 is expressed in several cell types. Co-transfection of a recombinant BAG-1 expression plasmid with JCV promoters indicated that BAG-1 stimulates transcription of the JCV(E) promoter and to a lesser extent the JCV(L) promoter. Mutations in the NF-1 sites in the JCV(E) promoter eliminated the activation by BAG-1. Thus, BAG-1 is a novel transcription factor that may play a role in JCV expression.

The role of Sp family members, basic Kruppel-like factor, and E box factors in the basal and IFN-gamma regulated expression of the human complement C4 promoter.

The fourth component of human complement (C4) is a serum protein that is expressed in the liver and other organs. The promoter region of the C4 gene has been analyzed in reporter gene assays in two cell lines that represent hepatic (HepG2) and monocytic (U937) lineages. Analysis indicated that regions important for basal transcription in HepG2 cells included Sp1 and E box sites within the first 100 bp upstream of the transcription initiation site but not the nuclear factor-1 site important in the control of the mouse C4 gene. Also, a region encompassing -468 to -310 was able to repress activity 2-fold. However, when a CACCC or GT box sequence at -140 was mutated the repressive activity of the upstream region resulted in almost no activity. The -140 region consists of a series of four closely positioned GT boxes that were shown to bind Sp1, Sp3, and basic Krupple-like factor in EMSA. This novel two-part regulatory element may be involved in the regulated expression of C4. However, IFN-gamma a major activator of C4 expression did not signal through this two-part regulatory element. We were able to map the position of an IFN-gamma responsive element in U937. IFN-gamma was able to increase transcription by up to 20-fold with mutations in the E box sequence at -78 to -73, thus completely abolishing induction. We conclude that the E box binding factors, which appear to be distinct from upstream stimulatory factors 1 and 2, are totally responsible for IFN-gamma induction of C4.