Upstream Stimulatory Factor Binding Site Research Articles

Fat Mass and Obesity Associated Gene (FTO) Expression Is Regulated Negatively by the Transcription Factor Foxa2

Fat mass and obesity associated gene (FTO) is the first gene associated with body mass index (BMI) and risk for diabetes. FTO is highly expressed in the brain and pancreas, and is involved in regulating dietary intake and energy expenditure. To investigate the transcriptional regulation of FTO expression, we created 5′-deletion constructs of the FTO promoter to determine which transcription factors are most relevant to FTO expression. The presence of an activation region at −201/+34 was confirmed by luciferase activity analysis. A potential Foxa2 (called HNF-3β) binding site and an upstream stimulatory factor (USF)-binding site was identified in the −100 bp fragment upstream of the transcription start site (TSS). Furthermore, using mutagenesis, we identified the Foxa2 binding sequence (−26/−14) as a negative regulatory element to the activity of the human FTO promoter. The USF binding site did not affect the FTO promoter activity. Chromatin immunoprecipitation (ChIP) assays were performed to confirm Foxa2 binding to the FTO promoter. Overexpression of Foxa2 in HEK 293 cells significantly down-regulated FTO promoter activity and expression. Conversely, knockdown of Foxa2 by siRNA significantly up-regulated FTO expression. These findings suggest that Foxa2 negatively regulates the basal transcription and expression of the human FTO gene.

Upstream Stimulatory Factor Is Required for Human Angiotensinogen Expression and Differential Regulation by the A−20C Polymorphism

Among naturally occurring polymorphisms in the 5' flanking region of the human angiotensinogen (AGT) gene, the -20 and -217 polymorphisms have the strongest effects on AGT regulation in AGT-expressing cells derived from liver, kidney, brain, and fat. These polymorphisms may affect allele-specific transcription factor binding, and the high-expressing alleles are both relatively common. We show herein that the -20C allele has higher transcriptional activity than -20A, and the -20A allele confers no additional transactivation potential beyond that of a mutated vector. Gel-shift assays show that upstream stimulatory factor (USF)1 and USF2 preferentially bind the -20C allele, whereas the -20A allele retains a low affinity USF binding site. Plasmid immunoprecipitation assays confirmed preferential association of USF1 with the -20C allele in transfected HepG2 cells. Chromatin immunoprecipitation confirmed that USF1 binds to the endogenous AGT -20C allele in CCF cells, the only cell line tested that carries the -20C allele, and to the human AGT promoter in liver and adipose tissue from transgenic mice. Transduction of AGT-expressing cells with short hairpin RNAs specifically targeting USF1 or USF2, resulted in cell- and allele-specific attenuation of AGT promoter activity. In vivo, knockdown of USF expression in the liver of transgenic mice expressing the -20C allele of AGT resulted in lower AGT expression, a decrease in circulating human AGT protein but no change in expression of GAPDH or hepatocyte nuclear factor-4alpha. We conclude that USF1 functionally and differentially regulates AGT expression via the -20 polymorphism and that the differential expression exhibited by -20 can be accounted for by differential association with USF1.

Control of the Calcitonin Gene-related Peptide Enhancer by Upstream Stimulatory Factor in Trigeminal Ganglion Neurons

The neuropeptide calcitonin gene-related peptide (CGRP) is a key player in migraine. However, the transcription factors controlling CGRP expression in the migraine-relevant trigeminal ganglion neurons are unknown. Previous in vitro studies demonstrated that upstream stimulatory factor (USF) 1 and USF2 bind to the CGRP neuroendocrine-specific 18-bp enhancer, yet discrepant overexpression results in cell lines, and the ubiquitous nature of the USF cast doubts about its role. To test the functional role of USF, we first demonstrated that small interfering RNAs directed against USF1 and USF2 reduced endogenous CGRP RNA and preferentially targeted the USF binding site at the 18-bp enhancer in the neuronal-like CA77 cell line. In cultured rat trigeminal ganglion neurons, knockdown of either USF1 or USF2 reduced CGRP promoter activity. Conversely, overexpression of USF1 or USF2 increased promoter activity. The activation was even greater upon cotransfection with an upstream activator of mitogen-activated protein kinases and was synergistic in a heterologous cell line. To begin to address the paradox of how ubiquitous USF proteins might direct neuronal-specific activity, we examined USF expression and used a series of adenoviral reporters in the cultured ganglia. Unexpectedly, there was more intense USF immunostaining in neurons than nonneuronal cells. Importantly, the 18-bp USF enhancer driving a minimal promoter was sufficient for neuronal specificity, although it was not the only site that directed neuronal expression. These results demonstrate that USF1 and USF2 are important contributors to neuronal-specific and mitogen-activated protein kinase regulation of the CGRP gene in trigeminal ganglion neurons.

Characterization of the 5'‐regulatory regions of the rat and human apelin genes and regulation of breast apelin by USF

Apelin, a peptide widely expressed in the body, is the endogenous ligand for the APJ receptor. To investigate how the apelin gene is regulated transcriptionally, we cloned and characterized approximately 3000 and approximately 4000 bp 5'-upstream fragments of the rat and human apelin genes. Putative CAAT-like box, but not TATA-box sites were identified. The rat (-207/-1 bp) and human (-100/+74 bp) core promoter sequences contain putative binding sites for upstream stimulatory factor (USF)-1/-2. Mutagenesis and overexpression assays showed that USF up-regulates basal and inducible apelin transcription. EMSA and supershift experiments indicated binding of USF-1/-2 to the rat (-114/-109 bp) and human (-84/-79 bp) apelin promoters. ChIP experiments show that USF is recruited to the putative USF binding site in the human apelin promoter in cultured breast cells. In concert with increased breast apelin expression during pregnancy and lactation in rats, EMSAs demonstrate an elevated binding of pregnant and lactating rat breast nuclear proteins to a consensus USF oligonucleotide. In vivo ChIP assays verified increased USF binding to the apelin promoter in breast of lactating rats. Together, our findings show that USF exerts a stimulatory role in regulation of breast apelin expression during pregnancy and lactation.

Induction of the BRCA2 Promoter by Nuclear Factor-κB

BRCA2 is a tumor suppressor gene that has been implicated in response to DNA damage, cell cycle control, and transcription. BRCA2 has been found to be overexpressed in many breast tumors, suggesting that altered expression of the BRCA2 gene may contribute to breast tumorigenesis. To determine how BRCA2 is overexpressed in tumors, we investigated the transcriptional regulation of the BRCA2 promoter. Deletion mapping of the BRCA2 promoter identified three regions associated with 3-fold activation or repression and one upstream stimulatory factor binding site associated with 20-fold activation. Gel shift and cotransfection studies verified the role of USF in regulation of BRCA2 transcription. Analysis of the -144 to -59 region associated with 3-fold activation identified a putative NFkappaB binding site. Cotransfection of the p65 and p50 subunits of NFkappaB up-regulated the BRCA2 promoter 16-fold in a luciferase reporter assay, whereas mutations in the binding site ablated the effect. Gel shift and supershift assays with anti-p65 and -p50 antibodies demonstrated that NFkappaB binds specifically to the NFkappaB site. In addition, ectopic expression of NFkappaB resulted in increased levels of endogeneous BRCA2 expression. Thus, NFkappaB and USF regulate BRCA2 expression through the BRCA2 promoter.

The Orphan Receptor COUP-TF Binds to a Third Glucocorticoid Accessory Factor Element within the Phosphoenolpyruvate Carboxykinase Gene Promoter

The phosphoenolpyruvate carboxykinase (PEPCK) gene promoter contains a glucocorticoid response unit (GRU) that includes, as a linear array, two accessory factor binding sites (AF1 and AF2) and two glucocorticoid receptor binding sites. All of these elements are required for a complete glucocorticoid response. AF1 and AF2 also partially account for the response of the PEPCK gene to retinoic acid and insulin, respectively. A second retinoic acid response element was recently located just downstream of the GRU. In this study we show that mutation of the 3' half-site of this element results in a 60% reduction of the glucocorticoid response of PEPCK promoter-chloramphenicol acetyltransferase (CAT) fusion constructs in transient transfection assays, thus the half-site is now termed AF3. A variety of assays were used to show that chicken ovalbumin upstream promoter transcription factor (COUP-TF) binds specifically to AF3 and that upstream stimulatory factor (USF) binds to an E-box motif located 2 base pairs downstream of AF3. Mutations of AF3 that diminish binding of COUP-TF reduce the glucocorticoid response, but mutation of the USF binding site has no effect. The functional roles of AF1, AF2, and AF3 in the glucocorticoid response were explored using constructs that contained combinations of mutations in all three elements. All three elements are required for a maximal glucocorticoid response, and mutation of any two abolish the response.

Two distinct forms of USF in the Lytechinus sea urchin embryo do not play a role in LpS1 gene inactivation upon disruption of the extracellular matrix.

Recent studies in our laboratory indicated that the upstream stimulatory factor (USF) in the sea urchin embryo of Lytechinus acts as a transcriptional repressor for the aboral ectoderm-specific expression of the LpS1 genes. Disruption of the extracellular matrix (ECM) arrests development prior to gastrulation and inactivates the LpS1 genes. We wanted to determine whether the inactivation of the LpS1 genes by ECM disruption may be due to an increase in USF expression. In the course of the investigation, a second L, variegatus USF cDNA clone (LvUSF2) was isolated and sequenced. The deduced amino acid sequence of LvUSF2 is nearly identical to LvUSF1 except at the amino end, where they are sharply divergent. Like LvUSF1, LvUSF2 has a USF-specific, a basic/hefixloop-helix, and a leucine zipper domain. Genomic DNA blots indicated that the two cDNA clones are derived from one gene, which suggested that the Lytechinus USF1 and USF2 mRNAs, of approximately 6.0 and 4.0 kb, respectively, are the result of differential RNA splicing. ECM disruption in Lytechinus embryos caused a relative drop in USF RNA accumulation levels to approximately 60% of control embryos, while LpS1 RNA accumulation levels dropped to less than 5%. USF protein levels and DNA binding activities in ECM-disrupted embryos also dropped to approximately 60% to that of control embryos. A mutation at the USF binding site in an LpS1 promoter-chloramphenicol acetyl transferase (CAT) fusion DNA construct did not cause a relative increase in CAT activity in ECM disrupted embryos. These results suggest that the induced drop in LpS1 gene expression by ECM disruption is not due to an increase in the repressive activity of USF.

Participation of altered upstream stimulatory factor in the induction of rat heme oxygenase-1 by cadmium.

We have reported that an upstream stimulatory factor (USF) binding site is functional in transcription of the heme oxygenase-1 gene. In this study, we examined the role of USF in the induced state. By transient expression analyses with the chloramphenicol acetyl-transferase gene, we found that the USF binding site plays an important role in the induction of rat heme oxygenase-1 by cadmium, but not by hemin. To elucidate the role of USF, we prepared USF-rich nuclear extracts from control and cadmium-treated rat liver. On electrophoretic mobility shift assay using control nuclear proteins, one slowly migrating band was detected, whereas using nuclear proteins of cadmium-treated rat liver, two fast migrating bands were detected. The molecular masses of the two subunits of USF prepared from cadmium-treated rat liver were approximately 34 kDa as determined by UV cross-linking and subsequent SDS-PAGE, while the two subunits of native USF were 43 kDa and 44 kDa. DNase I footprinting analysis revealed that both the nuclear proteins bound to the same region including the USF binding site. We therefore suppose that cadmium causes some structural changes in the two proteins of USF and that the altered USF participates in the effective initiation of transcription of the rat heme oxygenase-1 gene.

Excitotoxic Brain Lesion Modifies Binding to a USF Binding Site Acting as a Negative Regulatory Element in theProtease Nexin-1Promoter

The expression of the serine protease inhibitorProtease nexin-1 (PN-1)is upregulated in glial cells following different types of lesion in the nervous system. A strong negative regulatory element has been shown by the missing nucleoside technique to be a CACGTG site (E-box) in the proximal part of thePN-1promoter. The factor binding to this site is specifically recognized by antibodies directed against the human upstream stimulatory factor (USF). Point mutations in the E-box binding site which abolish USF bindingin vitroincrease the transcriptional activity of thePN-1promoter. Cotransfection of aPN-1promoter/reporter construct together with an expression vector for humanUSF1confirms the negative regulatory function of this site. Finally, we show that the binding to this USF site changed after ibotenic acid-induced lesion of the caudate putamen in the rat brain.

Ultraviolet A (320-380 nm) radiation causes an alteration in the binding of a specific protein/protein complex to a short region of the promoter of the human heme oxygenase 1 gene.

Ultraviolet A (320-380 nm) radiation strongly stimulates expression of the human heme oxygenase 1 gene as a consequence of an enhancement in transcription rate (1). We have used a 147 bp fragment of the promoter of this gene as a probe for DNA binding activity in nuclear extracts prepared from untreated and UVA treated populations of cultured human skin fibroblasts. Analysis using gel electrophoresis mobility shift assays clearly demonstrates the appearance of a strong binding activity unique to UVA-treated extracts that is formed in the absence of de novo protein synthesis. Footprint analysis defines a binding region from -41 to -50 bp that partially overlaps with a region known to constitutively bind upstream stimulatory factor (USF). Further analysis using synthetic oligonucleotides and gel retardation has confirmed that the crucial sequence for binding the protein present in both control and UVA-treated extracts lies within a 26 bp sequence that includes the core USF binding site. UVA radiation appears to lead to a modification of the USF complex (or closely related proteins(s)) to give a structurally modified protein/protein complex which protects only the upstream half of a region originally defined by foot-printing of the promoter with USF.

Characterization of an alternative promoter in the human growth hormone gene.

Transcription of the human growth hormone (hGH) gene depends on cis-acting elements contained within 300 base pairs of its 5'-flanking sequence. An earlier in vitro study of the transcriptional activity of this 5'-flanking region suggested that transcription can start upstream from position +1. We have investigated this phenomenon by cell-free transcription and transient transfection of chimeric constructs in cultured pituitary cells and in HeLa cells and by analysis of RNA from human pituitary glands and HeLa cells. Transcription initiation sites were identified at positions -54 and -197 by cell-free transcription assays and by RNAse mapping of human pituitary RNA. In transfection assays, the hGH gene 5'-flanking sequence upstream from position -197 displayed transcriptional activity, which critically depended on the upstream stimulatory factor-binding site located between positions -253 and -266. Transcripts initiated upstream from position +1 were detected in human pituitary RNA by polymerase chain reaction amplification and Northern blotting. These transcripts were longer than the mRNA encoding hGH. They might control initiation at position +1 or code for a novel peptide.