Binding Site For Nuclear Factor Research Articles

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.

Induction of sodium iodide symporter gene and molecular characterisation of HNF3β/FoxA2, TTF-1 and C/EBPβ in thyroid carcinoma cells

Thyroid carcinoma cells often do not express thyroid-specific genes including sodium iodide symporter (NIS), thyroperoxidase (TPO), thyroglobulin (TG), and thyrotropin-stimulating hormone receptor (TSHR). Treatment of thyroid carcinoma cells (four papillary and two anaplastic cell lines) with histone deacetylase inhibitors (SAHA or VPA) modestly induced the expression of the NIS gene. The promoter regions of the thyroid-specific genes contained binding sites for hepatocyte nuclear factor 3 β (HNF3β)/forkhead box A2 (FoxA2), thyroid transcription factor 1 (TTF-1), and CCAAT/enhancer binding protein β (C/EBPβ). Quantitative reverse transcription-polymerase chain reaction (RT–PCR) showed decreased expression of HNF3β/FoxA2 and TTF-1 mRNA in papillary thyroid carcinoma cell lines, when compared with normal thyroid cells. Forced expression of these genes in papillary thyroid carcinoma cells inhibited their growth. Furthermore, the CpG island in the promoter region of HNF3β/FoxA2 was aberrantly methylated; and treatment with 5-aza-2-deoxycytidine (5-Az) induced its expression. Immunohistochemical staining showed that C/EBPβ was localised in the nucleus in normal thyroid cells but was detected in the cytoplasm in papillary thyroid carcinoma cells. Subcellular fractionation of papillary thyroid carcinoma cell lines also demonstrated high levels of expression of C/EBPβ in the cytoplasm, suggesting that a large proportion of C/EBPβ protein is inappropriately localised in the cytoplasm. In summary, these findings reveal novel abnormalities in thyroid carcinoma cells

Selenoprotein P expression is controlled through interaction of the coactivator PGC-1α with FoxO1a and hepatocyte nuclear factor 4α transcription factors

Selenoprotein P (SeP), the major selenoprotein in plasma, is produced mainly by the liver, although SeP expression is detected in many organs. Recently, we reported stimulation of SeP promoter activity by the forkhead box transcription factor FoxO1a in hepatoma cells and its attenuation by insulin. Here, we demonstrate that this translates into fine-tuning of SeP production and secretion by insulin. Overexpression of peroxisomal proliferator activated receptor-gamma coactivator 1alpha (PGC-1alpha) enhanced the stimulatory effect of FoxO1a on SeP promoter activity. We identified a novel functional binding site for hepatocyte nuclear factor (HNF)-4alpha, termed hepatocyte nuclear factor binding element 1, in the human SeP promoter directly upstream of the FoxO-responsive element daf16-binding element 2 (DBE2). Point mutations in hepatocyte nuclear factor binding element 1 alone or together with DBE2 decreased basal activity and responsiveness of the SeP promoter to PGC-1alpha. Moreover, the PGC-1alpha-inducing glucocorticoid dexamethasone strongly enhanced SeP messenger RNA levels and protein secretion in cultured rat hepatocytes, whereas insulin suppressed the stimulation of both PGC-1alpha and SeP caused by dexamethasone treatment. In a brain-derived neuroblastoma cell line with low basal SeP expression, SeP transcription was stimulated by PGC-1alpha together with FoxO1a, and overexpression of HNF-4alpha potentiated this effect. High-level expression of SeP in liver is ensured by concerted action of the coactivator PGC-1alpha and the transcription factors FoxO1a and HNF-4alpha. Hence, the production of SeP is regulated similarly to that of the gluconeogenic enzyme glucose-6-phosphatase. As hepatic SeP production is crucial for selenium distribution throughout the body, the present study establishes PGC-1alpha as a key regulator of selenium homeostasis.

Transcriptional regulation of the proximal promoter of the murine pyruvate carboxylase gene in liver cells

Pyruvate carboxylase (PC) plays a crucial role in intermediary metabolism i.e. gluconeogenesis, lipogenesis and insulin secretion. Mammalian PC genes are regulated by 2 promoters, the proximal (P1) and the distal (P2) promoters which mediate alternative transcription in tissue‐specific manner. P1 promoter is active in liver and adipocytes while the P2 is active in pancreatic islets. In islets, P2 promoter is regulated by Sp1/Sp3, USF1, 2 and Foxa2 while P1 promoter is regulated by the PPARgamma. P1 is active in both liver and adipocytes, suggesting that this promoter is regulated by the distinct sets of liver and adipocyte transcription factors. To investigate this, we identify the liver‐specific transcription factors that regulate PC expression. We generate a series of 5’‐nested deletion mutants of the 2.3 kb P1 promoter of the mouse PC gene followed by transient transfection in HepG2 cells. This result showed the presence of 3 regulatory regions, located in regions A (−166/−80), B (−166/−256) and C (−335/−603). Region A contains 3 sites for Sp1/Sp3 while the region C contains a putative binding site for the liver‐enrich nuclear factor, HNF4alpha which could potentially bind to its responsive element identical to the PPRE. We are investigating the molecular interaction between HNF4 alpha to this site as well as identifying the nuclear proteins that bind region B of the P1 promoter.

Characterization of Two Hepatitis B Virus Populations in a Single Korean Hepatocellular Carcinoma Patient with an HBeAg-Negative Serostatus: A Novel X-Gene-Deleted Strain with Inverted Duplication Sequences of Upstream Enhancer Site II

Objectives: The aim of the study was to elucidate mutation patterns related to hepatocarcinogenesis in a Korean hepatocellular carcinoma (HCC) patient. Methods: We analyzed full genome sequences of 6 hepatitis B virus (HBV) clones from an HCC patient. Results: This patient harbored 2 HBV populations with genomes of different lengths (3,221 and 2,212 bp). In addition, we found 2 characteristic features not described so far in the full-genome sequence of deleted strains. First, 3 large deletion events (847, 144 and 48 bp) and a premature termination of the 182th codon of the surface antigen could lead to truncated or possibly nonfunctional forms of all HBV proteins. Second, these showed a novel mutation type not reported to date, which is a complex of an inverted duplication of 36-bp sequences containing an upstream enhancer site II (UEII), a remote insertion, and a large deletion event of the X region by homologous recombination. Conclusion: The fact that UEII is a binding site of liver-specific nuclear factor, which is expressed only in highly differentiated liver cells such as cancerous HepG2, strongly suggests a relationship between this novel mutation and hepatocarcinogenesis in this patient.

Thioredoxin1 Upregulates Mitochondrial Proteins Related to Oxidative Phosphorylation and TCA Cycle in the Heart

Thioredoxin1 (Trx1) inhibits hypertrophy and exhibits protective functions in the heart. To elucidate further the cardiac functions of Trx1, we used a DNA microarray analysis, with hearts from transgenic mice with cardiac- specific overexpression of Trx1 (Tg-Trx1, n = 4) and nontransgenic controls (n = 4). Expression of a large number of genes is regulated in Tg-Trx1, with a greater number of genes downregulated, versus upregulated, at high-fold changes. The peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1gamma) gene was among the top 50 significantly upregulated genes. By pathway analyses, we found that genes involved in both mitochondrial oxidative phosphorylation and the TCA cycle were upregulated in Tg-Trx1. We confirmed upregulation of cytochrome c oxidase (COX) components and mitochondrial transcription factor A in Tg-Trx1. The activity of citrate synthase and COX and the cardiac ATP content were significantly higher in Tg-Trx1. A transcription factor binding-site analysis showed that upregulated genes frequently contained binding sites for nuclear respiratory factor 1 (NRF1). Expression of NRF1 and PGC-1gamma was upregulated in Tg-Trx1, and Trx1 stimulated the transcriptional activity of NRF1 and NRF2 in cardiac myocytes. These results suggest that, in cardiac myocytes, Trx1 upregulates mitochondrial proteins and enhances mitochondrial functions, possibly through PGC-1alpha and NRFs.

Cis-Regulatory sequences of the genes involved in apoptosis, cell growth, and proliferation may provide a target for some of the effects of acute ethanol exposure

The physiological effects of alcohol are known to include drunkenness, toxicity, and addiction leading to alcohol-related health and societal problems. Some of these effects are mediated by regulation of expression of many genes involved in alcohol response pathways. Analysis of the regulatory elements and biological interaction of the genes that show coexpression in response to alcohol may give an insight into how they are regulated. Fifty-two ethanol-responsive (ER) genes displaying differential expression in mouse brain in response to acute ethanol exposure were subjected to bioinformatics analysis to identify known or putative transcription factor binding sites and cis-regulatory modules in the promoter regions that may be involved in their responsiveness to alcohol. Functional interactions of these genes were also examined to assess their cumulative contribution to metabolomic pathways. Clustering and promoter sequence analysis of the ER genes revealed the DNA binding site for nuclear transcription factor Y (NFY) as the most significant. NFY also take part in the proposed biological association network of a number of ER genes, where these genes interact with themselves and other cellular components, and may generate a major cumulative effect on apoptosis, cell survival, and proliferation in response to alcohol. NFY has the potential to play a critical role in mediating the expression of a set of ER genes whose interactions contribute to apoptosis, cell survival, and proliferation, which in turn may affect alcohol-related behaviors.

The 5′‐untranslated sequence of the amyloid precursor protein transcript exerts post‐transcriptional control of gene expression by variable mechanisms

The promoter of the human amyloid precursor protein (APP) gene directs cell-type specific expression with about 121 base pairs 5′ to the main transcriptional start site (+1). Transcription from this region is activated by transcription factors such as CTCF and USF. Downstream from the transcriptional start site the APP transcriptional unit contains an untranslated region (UTR) of 147 base pairs. We have here investigated the role of the APP-UTR in regulating expression from the APP promoter. For this purpose, deletions and transverse block mutations were introduced within the UTR. The constructs were analyzed by transient transfection, in vitro transcription, in vitro translation, DNase I footprinting, and mobility shift electrophoresis. The results showed that mutating sequences close to the transcriptional start site (position +1 to +25) interfered with transcriptional activity. In contrast, mutations between position +61 and +105 reduced expression in vivo in a transcriptionally independent manner. Within this domain two subdomains were identified. Mutations within one domain reduced translational activity in vitro. In contrast, mutations within a second domain eliminated a nuclear factor binding site while having no effect on translational activity. The protein that binds to the APP-UTR has not been identified but it is distinct from the thyroxin response element. In summary, two domains within the APP-UTR (position +61 to +105) affect expression from the APP promoter by independent post-transcriptional mechanisms. [Supported by NS30994].

Characteristics and clustering of human ribosomal protein genes

BackgroundThe ribosome is a central player in the translation system, which in mammals consists of four RNA species and 79 ribosomal proteins (RPs). The control mechanisms of gene expression and the functions of RPs are believed to be identical. Most RP genes have common promoters and were therefore assumed to have a unified gene expression control mechanism.ResultsWe systematically analyzed the homogeneity and heterogeneity of RP genes on the basis of their expression profiles, promoter structures, encoded amino acid compositions, and codon compositions. The results revealed that (1) most RP genes are coordinately expressed at the mRNA level, with higher signals in the spleen, lymph node dissection (LND), and fetal brain. However, 17 genes, including the P protein genes (RPLP0, RPLP1, RPLP2), are expressed in a tissue-specific manner. (2) Most promoters have GC boxes and possible binding sites for nuclear respiratory factor 2, Yin and Yang 1, and/or activator protein 1. However, they do not have canonical TATA boxes. (3) Analysis of the amino acid composition of the encoded proteins indicated a high lysine and arginine content. (4) The major RP genes exhibit a characteristic synonymous codon composition with high rates of G or C in the third-codon position and a high content of AAG, CAG, ATC, GAG, CAC, and CTG.ConclusionEleven of the RP genes are still identified as being unique and did not exhibit at least some of the above characteristics, indicating that they may have unknown functions not present in other RP genes. Furthermore, we found sequences conserved between human and mouse genes around the transcription start sites and in the intronic regions. This study suggests certain overall trends and characteristic features of human RP genes.

Activin A-induced expression of PAX4 in AR42J-B13 cells involves the increase in transactivation of E47/E12

Pax4 is a paired-homeodomain containing transcriptional factor that controls the differentiation of pancreatic β cells. The aim of this study was to investigate the mechanism of PAX4 expression by activin A. By reporter gene analysis using AR42J-B13 cells, in which treatment with activin A induced PAX4 mRNA expression, we identified that a short sequence located ∼1930 bp upstream of the transcriptional start site is essential for activin A induced PAX4 promoter activation. This region contains an E box and binding sites for hepatocyte nuclear factor (HNF)-1α. Mutation introduced in each binding site markedly reduced activin A responsiveness. It has been reported that HNF-1α synergizes with basic helix–loop–helix (bHLH) proteins in activating the PAX4 promoter, and we demonstrated that activin A strongly enhanced the functional activity of E47/E12 without the increase in its binding ability. In addition, suppression of E47/E12 expression in AR42J-B13 cells using siRNA oligonucleotides results in the significant decrease in the intrinsic activin A-induced PAX4 expression. Our results suggest that activin A enhances PAX4 expression by enhanced transactivation of E47/E12 proteins and might result in a cumulative transactivation of the promoter.

Regulation of bile acid biosynthesis by hepatocyte nuclear factor 4α

Hepatocyte nuclear factor 4alpha (HNF4alpha) regulates many genes that are preferentially expressed in liver. Mice lacking hepatic expression of HNF4alpha (HNF4alphaDeltaL) exhibited markedly increased levels of serum bile acids (BAs) compared with HNF4alpha-floxed (HNF4alphaF/F) mice. The expression of genes involved in the hydroxylation and side chain beta-oxidation of cholesterol, including oxysterol 7alpha-hydroxylase, sterol 12alpha-hydroxylase (CYP8B1), and sterol carrier protein x, was markedly decreased in HNF4alphaDeltaL mice. Cholesterol 7alpha-hydroxylase mRNA and protein were diminished only during the dark cycle in HNF4alphaDeltaL mice, whereas expression in the light cycle was not different between HNF4alphaDeltaL and HNF4alphaF/F mice. Because CYP8B1 expression was reduced in HNF4alphaDeltaL mice, it was studied in more detail. In agreement with the mRNA levels, CYP8B1 enzyme activity was absent in HNF4alphaDeltaL mice. An HNF4alpha binding site was found in the mouse Cyp8b1 promoter that was able to direct HNF4alpha-dependent transcription. Surprisingly, cholic acid-derived BAs, produced as a result of CYP8B1 activity, were still observed in the serum and gallbladder of these mice. These studies reveal that HNF4alpha plays a central role in BA homeostasis by regulation of genes involved in BA biosynthesis, including hydroxylation and side chain beta-oxidation of cholesterol in vivo.

Proximal HNF1 element is essential for the induction of human UDP-glucuronosyltransferase 1A1 by glucocorticoid receptor

Previous study showed noinduction of the reporter gene (−3174/+14) of UGT1A1 in HepG2 by bilirubin, but induction by dexamethasone (DEX). This induction was enhanced seven-fold by the co-expression of human glucocorticoid receptor (GR) and was inhibited by a GR antagonist, RU486, indicating stimulation by DEX-GR. Meanwhile, we could not detect stimulation by β-estradiol, phenobarbital or rifampicin (RIF) in the presence of GR. We investigated the position playing a role in this induction by GR in the promoter region of UGT1A1 using deletion mutants, and clarified the essential sequence (−75/−63) for the binding site of hepatocyte nuclear factor 1 (HNF1). However, GR did not bind directly to this sequence, because UGT-PE2 did not compete for binding to a glucocorticoid responsive element (GRE) probe in an electrophoretic mobility shift assay (EMSA) method. Labeled [ 32P]DNA probe of HNF1 binds with nuclear extracts as shown by the EMSA. This shift of the complex of probe–protein was not inhibited by unlabeled GRE but was inhibited by unlabeled HNF1 element. This shift was not influenced by the addition of anti-GR, but was super-shifted by the addition of anti-HNF1. GR did not stimulate the induction of HNF1, because we detected no-elevation of the mRNA level of HNF1 by reverse transcription-polymerase chain reaction (RT-PCR). Therefore, the induction of UGT1A1 by DEX-GR did not depend on the elevation of HNF1 but on the interaction of GR with HNF1 or the activation of HNF1 through the transcription of other proteins. Also given the lack of evidence of binding of DEX-GR to HNF1 in the EMSA, the data suggest that the mechanism of DEX-GRE effect on HNF1 is indirect by whatever mechanisms.

T1764G1766 core promoter double mutants are restricted to Hepatitis B virus strains with an A1757 and are common in genotype D

To investigate the role of pre-core and basal core promoter (BCP) mutants in hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (e-CHB) in Iran, Hepatitis B virus strains from 30 patients and 42 anti-HBe-positive asymptomatic carriers (ASCs) were characterized. G1896A pre-core stop mutants, detected in 77 % of e-CHB patients and 85 % of ASCs, showed no association with virus load or aminotransferase levels. Twenty per cent of e-CHB patients and 31 % of ASCs harboured T1762A1764 mutants. When this double mutation was associated with G1757, it was linked to a higher virus load in patients than when it was associated with A1757 (10(5.2+/-1.8) vs 10(3.2+/-0.8) copies ml(-1); P=0.004). Interestingly, the most common BCP mutations were T1764 and G1766, which were present in 33 % of e-CHB patients and 29 % of ASCs. These were associated with higher virus load and aminotransferase levels compared with patients lacking core promoter mutations, although this was not significant. The T1764G1766 double mutation was only present in strains with A1757 (P<0.001), which is more frequent in strains of genotype D than in those belonging to other genotypes. On the other hand, the T1762A1764 double mutation was found more frequently in association with G1757 than with A1757. The T1762A1764 double mutation forms a binding site for hepatocyte nuclear factor 1 (HNF1), which is constrained by A1757. However, the T1764G1766 double mutant may form a binding site for HNF3. Thus, position 1757 affects the emergence of promoter double mutants and would predict a relative genotypic restriction of both the T1762A1764 and the T1764G1766 double mutants.

Control of ACAT2 liver expression by HNF1

ACAT catalyzes the formation of cholesteryl esters from cholesterol and long-chain fatty acids. There are two known genes encoding the two ACAT enzymes, ACAT1 and ACAT2 (also known as Soat1 and Soat2). In adult humans, ACAT1 is present in most tissues, whereas ACAT2 is localized to enterocytes and hepatocytes. In this report, we elucidate the mechanisms that control the liver-specific expression of the human ACAT2 gene. We identified hepatic nuclear factor 1 (HNF1) as an important liver-specific trans-acting element for the human ACAT2 gene using the human hepatocellular carcinoma cell lines HuH7 and HepG2. Targeted deletion of the HNF1 binding site in the DNA sequence abolished not only the basal promoter function in HepG2 and HuH7 cells but also the induction of the ACAT2 promoter by HNF1. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that the transcription factors HNF1alpha and HNF1beta interact with this region in the human ACAT2 gene in vitro and in vivo. These data indicate that a) the identified HNF1 binding site serves as a positive regulator sequence, b) the binding site is functionally active both in vivo and in vitro, and c) the transcription factors HNF1alpha and HNF1beta, which bind to this site, play an important part in the regulation of the human ACAT2 promoter.

Cloning and functional analysis of promoters of three GnRH genes in a cichlid

Mechanisms regulating gonadotropin-releasing hormone (GnRH) types, a key molecule for reproductive physiology, remain unclear. In the present study, we cloned the promoters of GnRH1, GnRH2, and GnRH3 genes in the tilapia, Oreochromis niloticus; and found putative binding sites for glucocorticoid receptors, Sp1, C/EBP, GATA, and Oct-1, but not for androgen receptors in all three GnRH promoters using computer analysis. The presence of binding sites for progesterone receptors in GnRH1, estrogen receptors in GnRH1 and GnRH2, and thyroid hormone receptors in GnRH1 and GnRH3 suggests direct action of steroid hormones on GnRH types. Our observation of SOX and LINE-like sequences exclusively in GnRH1, COUP in GnRH2, and retinoid X receptors in GnRH3 suggests their role in sexual differentiation, midbrain segmentation, and visual cue integration, respectively. Thus, the characteristic binding sites for nuclear receptors and transcription factors support the notion that each GnRH type is regulated differently and has distinct physiological roles.

Fulminant Hepatitis B Associated with a Specific Insertion in the Basal Core Promoter Region of Hepatitis B Virus DNA after Immunosuppressive Treatment

We report a case of hepatitis B virus reactivation that occurred after immunosuppressive treatment in a hepatitis B surface antigen-negative patient with detectable antibodies to hepatitis B core antigen and hepatitis B surface antigen. Sequence analysis revealed an 11-bp insertion in the core promoter region of hepatitis virus DNA, creating a novel hepatocyte nuclear factor-1 binding site. This led to enhanced viral replication and fulminant hepatitis.

Nuclear factor 1 regulates adipose tissue-specific expression in the mouse GLUT4 gene

Previous studies demonstrated that an adipose tissue-specific element(s) (ASE) of the murine GLUT4 gene is located between −551 and −506 in the 5′-flanking sequence and that a high-fat responsive element(s) for down-regulation of the GLUT4 gene is located between bases −701 and −552. A binding site for nuclear factor 1 (NF1), that mediates insulin and cAMP-induced repression of GLUT4 in 3T3-L1 adipocytes is located between bases −700 and −688. To examine the role of NF1 in the regulation of GLUT4 gene expression in white adipose tissues (WAT) in vivo, we created two types of transgenic mice harboring mutated either 5′ or 3′ half-site of NF1-binding sites in GLUT4 minigene constructs. In both cases, the GLUT4 minigene was not expressed in WAT, while expression was maintained in brown adipose tissue, skeletal muscle, and heart. This was an unexpected finding, since a −551 GLUT4 minigene that did not have the NF1-binding site was expressed in WAT. We propose a model that explains the requirement for both the ASE and the NF1-binding site for expression of GLUT4 in WAT.

A signal transducer and activator of transcription 6 haplotype influences the regulation of serum IgE levels

Because of its central role in the IL-4/IL-13 pathway, the intracellular signaling molecule signal transducer and activator of transcription 6 ( STAT6 ) may be crucial for IgE production in asthma and allergy. We analyzed the association between polymorphisms in the STAT6 gene and the regulation of serum IgE levels. Methods In a population of 1120 German schoolchildren (age 9-11 years), we genotyped 6 previously identified polymorphisms spanning the STAT6 gene by using the matrix-assisted laser desorption ionization-time of flight mass spectrometry method. Haplotypes were estimated and population-derived IgE percentiles (50% IgE > 60 IU/mL, 66% IgE > 115 IU/mL, and 90% IgE > 457 IU/mL) were modeled as outcome variables in haplotype-trend regression analysis. Polymorphisms located in intron 2 (C2892T) and the 3' untranslated region (T12888C) significantly and consistently contributed to elevated total serum IgE levels. One STAT6 haplotype showed increased odds ratios of 1.58 (95% CI, 1.08-2.32; P = .020), 1.82 (95% CI, 1.19-2.77; P = .006), and 3.92 (95% CI, 1.93-7.96; P = .0002) for elevated IgE levels at percentiles 50%, 66%, and 90%, respectively. Because C2892T is located within a nuclear factor kappaB transcription factor binding site, a functional role of this polymorphism is very likely. The data indicate that within the IL-4/IL-13 pathway, genetic variants in the STAT6 gene significantly contribute to the regulation of serum IgE levels.

Identification of a functional CBF-binding CCAAT-like motif in the core promoter of the mouse pro-alpha 1(V) collagen gene (Col5a1).

We used structure-function analysis of the core promoter region to elucidate the transcriptional features of the mouse alpha 1(V) collagen gene (Col5a1). The core promoter, which lacks a typical TATA motif and has a high GC content, was defined within the 231 bp immediately upstream from the major transcription start site by transient transfection experiments. In this region, we identified three nuclear-factor binding sites by electrophoretic mobility shift assay: BS1 (-195 to -167), BS2 (-134 to -106), and BS3 (-110 to -80). Oligonucleotide competition and supershift assays revealed that Sp1, CBF, and Sp1-related protein specifically bind to BS1, BS2, and BS3, respectively. The CCAAT-like motif, CAAAT, and flanking sequences are conserved between the mouse and human gene. CBF, which recognizes this motif, activated the Col5a1 promoter, as previously reported for Col1a1 and Col1a2. Furthermore, overexpression of a wild-type and mutant forms of CBF-B subunit altered this activity. These results suggest that CBF is a key factor in the coordinated expression of type I and V collagen genes.

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.