Electrophoretic Mobility Shift Analysis Research Articles

Staf50 is a novel p53 target gene conferring reduced clonogenic growth of leukemic U-937 cells.

The tumor suppressor gene p53 is a transcription factor that mediates both cell cycle arrest and apoptosis. Interestingly, p53 also induces differentiation of a number of tissues, including leukemic cells. However, although p53-mediated differentiation of leukemic U-937 cells depends on the transcriptional activity of p53, a p53 target gene mediating differentiation has hitherto not been identified. To screen for novel p53 target genes in leukemic cells, a cDNA microarray analysis was performed with U-937-4/ptsp53 cells, expressing a temperature-sensitive p53 mutant. We report that transcription of the Staf50 (stimulated transacting factor of 50 kDa) gene is upregulated in response to wild-type p53 in U-937-4, K562 and MCF-7 cells. Staf50 was directly activated by p53, as determined by the independence of de novo protein synthesis. Moreover, while the proximal promoter of Staf50 was found not to be p53 responsive, a functional enhancer-like p53-response element in intron 1 of the Staf50 gene was identified that was also transactivated by the p53-family member p73. Direct binding of p53 to the response element was shown by electrophoretic mobility shift analysis. Ectopic expression of Staf50 in U-937 cells resulted in reduced clonogenic growth. Moreover, levels of endogenous Staf50 mRNA correlated to all-trans retinoic acid-induced differentiation of promyelocytic NB-4 and HL60 cells, suggesting that Staf50 could be involved in proliferation and/or differentiation of leukemic cells.

Architecture and assembly of mammalian H/ACA small nucleolar and telomerase ribonucleoproteins

Mammalian H/ACA small nucleolar RNAs and telomerase RNA share common sequence and secondary structure motifs that form ribonucleoprotein particles (RNPs) with the same four core proteins, NAP57 (also dyskerin or in yeast Cbf5p), GAR1, NHP2, and NOP10. The assembly and molecular interactions of the components of H/ACA RNPs are unknown. Using in vitro transcription/translation in combination with immunoprecipitation of core proteins, UV-crosslinking, and electrophoretic mobility shift assays, we demonstrate the following. NOP10 associates with NAP57 as a prerequisite for NHP2 binding. Although NHP2 on its own binds RNA nonspecifically, this NAP57-NOP10-NHP2 core trimer specifically recognizes H/ACA RNAs. GAR1 associates independently with NAP57 near the pseudouridylase core of mature H/ACA RNPs. In contrast to other RNPs whose assembly is initiated by protein-RNA interactions, the four H/ACA core proteins form a protein-only particle that associates with H/ACA RNAs. Nonetheless, functional H/ACA snoRNPs assembled in cytosolic extracts are stable and do not exchange their RNA components, suggesting that new particle formation requires de novo synthesis.

Redox regulation of DNA binding activity of DREF (DNA replication-related element binding factor) in Drosophila

DREF [DRE (DNA replication-related element) binding factor] is an 80 kDa polypeptide homodimer which plays an important role in regulating cell proliferation-related genes. Both DNA binding and dimer formation activities are associated with residues 16-115 of the N-terminal region. However, the mechanisms by which DREF dimerization and DNA binding are regulated remain unknown. Here, we report that the DNA binding activity of DREF is regulated by a redox mechanism, and that the cysteine residues are involved in this regulation. Electrophoretic mobility shift analysis using Drosophila Kc cell extracts or recombinant DREF proteins indicated that the DNA binding domain is sufficient for redox regulation. Site-directed mutagenesis and transient transfection assays showed that Cys59 and/or Cys62 are critical both for DNA binding and for redox regulation, whereas Cys91 is dispensable. In addition, experiments using Kc cells indicated that the DNA binding activity and function of DREF are affected by the intracellular redox state. These findings give insight into the exact nature of DREF function in the regulation of target genes by the intracellular redox state.

Who's Up First?

The tumor suppressor p53, a transcription factor activated in response to various stresses, promotes apoptosis and cell cycle arrest. PML, the protein product of the promyelocytic leukemia gene ( PML ), also acts as a tumor suppressor and is believed to act upstream of p53 to regulate its activity. De Stanchina et al. investigated the role of PML in cellular senescence and discovered that PML functioned as a direct p53 target. The authors used Western analysis to show that, in mouse embryo fibroblasts, the expression of p53 and PML was stimulated by oncogenic Ras. Stimulation of PML expression by Ras or in response to DNA damage depended on the presence of p53, and transfer of p53 into mouse and human cells lacking p53 increased the expression of PML mRNA and protein. Regions of the PML gene that contained putative p53 response elements (REs) enabled p53 to activate a heterologous gene reporter. Mutational analysis indicated that this sensitivity depended on REs. Moreover, electrophoretic mobility-shift analysis indicated that p53 bound to PML REs in vitro, and chromatin immunoprecipitation analysis indicated that p53 bound to the PML gene of cells expressing oncogenic Ras. Cells in which PML was suppressed (with short-hairpin RNA) showed specific defects in p53-mediated cell cycle arrest, senescence, and apoptosis (but not in some other p53 effects). Thus, PML appears to function as a direct target and an effector of p53. The authors discuss scenarios in which these data can be reconciled with previous research suggesting that PML acts upstream of p53. E. de Stanchina, E. Querido, M. Narita, R. V. Davuluri, P. P. Pandolfi, G. Ferbeyre, S. W. Lowe, PML is a direct p53 target that modulates p53 effector functions. Mol. Cell 13 , 523-535 (2004). [Online Journal]

Repression of Gonadotropin-Releasing Hormone (GnRH) Gene Expression by Melatonin May Involve Transcription Factors COUP-TFI and C/EBP Beta Binding at the GnRH Enhancer

Melatonin activates membrane-bound G-protein-coupled receptors mt1 and MT2, but may also bind a family of orphan nuclear receptors, including RORα and RZRβ, representing another potential molecular mechanism of melatonin action. Recently, we demonstrated that melatonin downregulates gonadotropin-releasing hormone (GnRH) gene expression in the GT1–7 cell line, specifically at the level of the neuron-specific GnRH gene enhancer. In this study, we have examined the region located at –1736/–1728 of the GnRH enhancer shown to be involved in the repression of GnRH by melatonin. This region includes hexameric consensus binding sites for orphan nuclear receptors, including ROR/RZR and COUP-TFI, as well as other defined consensus binding sites for AP-1 and C/EBP. Using electrophoretic mobility shift analysis (EMSA), we have demonstrated that GT1–7 nuclear proteins bind specifically to this region of the GnRH enhancer to form 4 complexes. EMSA antibody supershift analysis indicates that the transcription factors COUP-TFI and C/EBP beta bind two specific complexes, but RORα, Oct-1, Pbx-1, c-fos, or c-jun antibodies failed to produce any detectable supershifts. These results provide the first evidence that melatonin may mediate its direct neuroendocrine control of GnRH gene expression through transcription factor binding at specific regions of the GnRH enhancer.

Role of CREB in Transcriptional Regulation of CCAAT/Enhancer-binding Protein β Gene during Adipogenesis

The proximal promoter of the C/EBPbeta gene possesses dual cis regulatory elements (TGA1 and TGA2), both of which contain core CREB binding sites. Comparison of the activities of C/EBPbeta promoter-reporter genes with 5'-truncations or site-directed mutations in the TGA elements showed that both are required for maximal promoter function. Electrophoretic mobility shift and chromatin immunoprecipitation (ChIP) analyses with antibodies specific to CREB and ATF1 showed that these CREB family members associate with the proximal promoter both in vitro and ex vivo. Immunoblotting and ChIP analysis revealed that other CREB family members, CREM and ATF1, are up-regulated and associate with the proximal C/EBPbeta promoter in mouse embryonic fibroblasts (MEFs) from CREB(-/-) mice. ChIP analysis of wild-type MEFs and 3T3-L1 preadipocytes revealed that interaction of phospho-CREB, the active form of CREB, with the C/EBPbeta gene promoter occurs only after induction of differentiation of 3T3-L1 preadipocytes and MEFs. Consistent with the interaction of CREB and ATF1 at the TGA regulatory elements, expression of constitutively active CREB strongly activated C/EBPbeta promoter-reporter genes, induced expression of endogenous C/EBPbeta, and caused adipogenesis in the absence of the hormonal inducers normally required. Conversely, expression of a dominant-negative CREB blocked promoter-reporter activity, expression of C/EBPbeta, and adipogenesis. When subjected to the standard adipocyte differentiation protocol, wild-type MEFs differentiate into adipocytes at high frequency, whereas CREB(-/-) MEFs exhibit greatly reduced expression of C/EBPbeta and differentiation. The low level of expression of C/EBPbeta and differentiation in CREB(-/-) MEFs appears to be due to up-regulation of other CREB protein family members, i.e. ATF1 and CREM.

Tumor Necrosis Factor-α Inhibits Endothelial Nitric-oxide Synthase Gene Promoter Activity in Bovine Aortic Endothelial Cells

Tumor necrosis factor-alpha (TNF-alpha) has been shown to reduce endothelial nitric-oxide synthase (eNOS) gene expression through post-transcriptional regulation of mRNA stability. The current study documented an independent effect of the cytokine on the eNOS gene promoter. TNF-alpha effected a time- and dose-dependent reduction in activity of a transiently transfected human -1197 eNOS-luciferase reporter. This reduction was inhibited by co-transfection of dominant negative IKKbeta as well as a nonphosphorylatable constitutively suppressive mutant of IkappaB implying involvement of the NFkappaB cascade in the inhibitory effect. The locus of the TNF-alpha-dependent inhibition was traced to two Sp1-binding sites positioned between -109 and -95 and -81 and -67 relative to the transcription start site. Electrophoretic mobility shift analysis and immunoperturbation studies showed evidence for Sp1 and Sp3 binding to each element. TNF-alpha treatment had no effect on the binding pattern to the downstream (-81 to -67) site but did suppress association of Sp1 and Sp3 to the upstream (-109 to -95) site. Collectively, these data indicate that TNF-alpha exerts transcriptional, as well as post-transcriptional, effects on eNOS gene expression and suggest a potential mechanism to account for the endothelial dysfunction that accompanies disorders such as diabetes mellitus and heart failure.

A Chromatin Immunoprecipitation Screen Reveals Protein Kinase Cβ as a Direct RUNX1 Target Gene

RUNX1 (also known as AML1) is a DNA-binding transcription factor that functions as a tumor suppressor and developmental determinant in hematopoietic cells. Target promoters have been identified primarily through the use of differential expression strategies and candidate gene approaches but not biochemical screens. Using a chromatin immunoprecipitation screen, we identified protein kinase Cbeta as a direct RUNX1 target gene and demonstrate that endogenous RUNX1 binds the chromatinized protein kinase Cbeta promoter of U937 cells. A phylogenetically conserved RUNX1-binding site within the PKCbeta promoter binds RUNX1 in electrophoretic mobility shift analyses and confers RUNX1 responsiveness on a heterologous promoter. Changes in RUNX1 activity affect endogenous protein kinase Cbeta expression, and a dominant-negative form of RUNX1 protects U937 cells from apoptotic stimuli previously shown to be dependent on protein kinase Cbeta. This protection can be reversed by the ectopic expression of protein kinase Cbeta. Together these findings demonstrate that protein kinase Cbeta is a direct, downstream target of RUNX1 and links RUNX1 to a myeloid apoptotic pathway.

Fibroblast Growth Factor-2 Represses Platelet-derived Growth Factor Receptor-α (PDGFR-α) Transcription via ERK1/2-dependent Sp1 Phosphorylation and an Atypical cis-Acting Element in the Proximal PDGFR-α Promoter

Platelet-derived growth factor (PDGF) is a potent mitogen and chemoattractant for vascular smooth muscle cells (SMCs) whose biological activity is mediated via its high affinity interaction with specific cell surface receptors. The molecular mechanisms governing the expression of PDGF receptor-alpha (PDGFR-alpha) are poorly understood. Here we demonstrate that PDGFR-alpha protein and transcriptional regulation in SMCs is under the positive regulatory influence of the zinc finger nuclear protein, Sp1. Electrophoretic mobility shift, competition, and supershift analysis revealed the existence of an atypical G-rich Sp1-binding element located in the PDGFR-alpha promoter -61 to -52 bp upstream of the transcriptional start site. Mutation of this sequence ablated endogenous Sp1 binding and activation of the PDGFR-alpha promoter. PDGFR-alpha transcription, mRNA, and protein expression were repressed in SMCs exposed to fibroblast growth factor-2 (FGF-2). This inhibition was rescued by the blockade of extracellular signal-regulated kinase-1/2 (ERK1/2). FGF-2 repression of PDGFR-alpha transcription was abrogated upon mutation of this Sp1-response element. FGF-2 stimulated Sp1 phosphorylation in an ERK1/2- but not p38-dependent manner, the growth factor enhancing Sp1 interaction with the PDGFR-alpha promoter. Mutation of residues Thr(453) and Thr(739) in Sp1 (amino acids phosphorylated by ERK) blocked FGF-2 repression of PDGFR-alpha transcription. These findings, taken together, demonstrate that FGF-2 stimulates ERK1/2-dependent Sp1 phosphorylation, thereby repressing PDGFR-alpha transcription via the -61/-52 element in the PDGFR-alpha promoter. Phosphorylation triggered by FGF-2 switches Sp1 from an activator to a repressor of PDGFR-alpha transcription, a finding previously unreported in any Sp1-dependent gene.

A novel action of minocycline: inhibition of human immunodeficiency virus type 1 infection in microglia.

Human immunodeficiency virus type 1 (HIV-1) infection of the brain produces a characteristic disease called acquired immunodeficiency syndrome (AIDS) dementia in which productive infection and inflammatory activation of microglia and macrophages play a central role. In this report, the authors demonstrate that minocycline (MC), a second-generation tetracycline with proven safety and penetration to the central nervous system, potently inhibited viral production from microglia. Inhibition of viral release was sustained through the entire course of infection and even when the drug exposure was limited to the first day of infection. Minocycline was effective even at low viral doses, and against R5- and X4R5-HIV, as well as in single-cycle reporter virus assays. Electrophoretic mobility shift analysis showed that minocycline inhibited nuclear factor (NF)-kappaB activation in microglia. HIV-1 long terminal repeat (LTR)-promoter activity in U38 cells was also inhibited. These results, combined with recently demonstrated in vivo anti-inflammatory effects of MC on microglia, suggest a potential utility for MC as an effective adjunct therapy for AIDS dementia.

AML-1, PU.1, and Sp3 regulate expression of human bactericidal/permeability-increasing protein

Bactericidal/permeability-increasing protein (BPI) is an antimicrobial protein in neutrophils, stored in azurophil granules. Expression of BPI is absent in neutrophils of newborns and patients with secondary granule deficiency (SGD), possibly contributing to dysfunction of neutrophils. We report two alternative transcription start sites at 52 and 22 bp upstream of the translation start. A proximal 222 bp promoter conferring expression in myeloid cells was identified, and critical cis-acting sites for myeloid expression were contained within the 159 bp upstream of translation start. Within this region, direct binding and transactivation by AML-1, PU.1, and Sp3 were demonstrated, as judged by electrophoretic mobility shift analysis. Moreover, transient transfections of C/EBPα or C/EBPε to HeLa cells resulted in increased promoter activity, indicating a direct or indirect role for C/EBP. In conclusion, we provide evidence for AML-1, PU.1, and Sp3 cooperatively and directly mediating BPI-expression during myeloid differentiation.

Metal ions alter lipopolysaccharide‐induced NFκB binding in monocytes

Metals are components of a variety of biomaterials used in orthopedic and dental appliances; however, their biocompatibility with the surrounding tissues is not completely understood. Monocytes are important immune cells that respond to inflammatory stimuli by rapidly producing a variety of inflammatory proteins. Regulation of this response often involves activation of the transcription factor NF kappa B. The current study was designed to determine whether monocyte activation of NF kappa B in response to bacterial lipopolysaccharide (LPS) is affected by pretreatment with metal ions. Concentrations of metal ions that affected cell number after 24 h of exposure were first determined. Then THP-1 human monocytes were cultured for 2 h in media containing metal ions at concentrations below levels that altered cell growth. Parallel cultures were treated with 10 microg/mL Escherichia coli LPS, and all samples were cultured an additional 2 h. Nuclear proteins were extracted and normalized amounts were incubated with [(32)P]-end-labeled NF kappa B consensus oligonucleotide. NF kappa B-DNA complexes were identified and quantified by electrophoretic mobility shift analysis. The extent of NF kappa B-DNA complex formation after metal ion pretreatment with or without LPS induction was compared to no treatment or LPS-only treated controls. Finally, LPS-induced IL1 beta secretion was measured from palladium-treated and control cells. Concentrations were identified for each metal ion (Ag(+), Co(2+), Cu(2+), Hg(2+), Ni(2+), and Pd(2+)) that did not reduce cell number after 24 h of exposure (ranging from 5 microM for Ag(+) and Hg(2+) to 200 microM for Ni(2+)). Exposures of 2 h at these concentrations did not alter cell morphology, staining with trypan blue, or cell number. LPS exposure had no effect on cell number with or without metal ions after 2 h. When metal treatment alone was assessed, none of the metal ions had a significant effect on NF kappa B-DNA binding. However, pretreatment with Co(2+), Ni(2+), Ag(1+), Hg(2+), and Pd(2+) significantly decreased NF kappa B-DNA binding by 40-70% versus LPS alone. Only Cu(2+) had no effect on LPS-induced NF kappa B-DNA complex formation. Pd(2+) lowered, but did not abolish, IL1 beta secretion at concentrations comparable to those that altered NF kappa B-DNA binding. These results suggest that many commonly used metals alter monocyte function at concentrations that are not overtly toxic, and that protein levels controlled in part by NF kappa B also may be altered.

A functional ISRE is required for myeloid transcription of the p47 phox gene

Expression of p47 phox , a component of the phagocytic NADPH oxidase, is both tissue-specific and developmentally regulated. We have investigated transcription from the p47 phox gene promoter by reporter gene analysis of myeloid PLB985 cells stably transfected with a series of p47 phox promoter constructs. Stable transfection with constructs containing up to 3100 bp of proximal promoter sequence demonstrated that as little as 144 bp of proximal promoter sequence was able to direct significant reporter gene activity in myeloid cells, but not in HeLa cells. Mutation of a previously uncharacterised interferon-stimulated response element (ISRE) consensus located at positions −104 to−116, or of an established binding site for the Ets family transcription factor, PU.1 (located at positions −39 to −44), abolished transcription in stably transfected myeloid cells. Electrophoretic mobility shift analysis (EMSA) with myeloid cell nuclear extracts demonstrated that an oligonucleotide containing the p47 phox ISRE consensus was able to compete binding at another bona fide ISRE. Complexes formed on the p47 phox ISRE itself were competed by other ISRE consensus sequences. We conclude that transcription of p47 phox in myeloid cells requires a functional ISRE in addition to the previously identified PU.1 binding site.

The transcription factor, NFI/CTF plays a positive regulatory role in expression of the hSMUG1 gene

SMUG1 is a recently discovered uracil-DNA glycosylase with the ability to remove uracil from single-stranded as well as double-stranded DNA. SMUG1 also has the capacity to excise oxidized pyrimidine bases such as 5-hydroxymethyluracil and 5-formyluracil from DNA. Very little is known about the regulation of this enzyme. Therefore, we undertook this study to begin to elucidate the mechanisms of hSMUG1 gene expression. Northern blot analysis performed on mRNAs derived from different cell lines reveals that the steady-state levels of hSMUG1 transcript are about 10-fold lower relative to UDG. In addition to the 1.6 kb transcript known to encode a functional hSMUG1 protein, an alternate 0.7 kb transcript was uncovered that contains an open reading frame. Interestingly, this alternate transcript is missing a carboxy-terminal domain which is necessary for catalytic activity. Utilizing a luciferase reporter assay system we show that significant promoter activity is associated with a 2000 bp region, located immediately upstream of the first transcribed, non-translated exon. 5′ deletion analysis of this 2000 bp region reveals that there are both negative and positive regulatory elements that control expression of SMUG1. Using electrophoretic mobility shift analysis we show that a number of DNA–protein complexes are formed within the region (−705 to −604) of positive regulation. At least two of these complexes contain the transcription factor NFI/CTF as demonstrated by oligonucleotide competition studies with NFI/CTF consensus sequence containing both protein-binding half-sites. We further demonstrate that purified NFI-C protein will bind to this positive regulatory region within the hSMUG1 gene. DNase I footprint analysis reveals that the 3′ half-site is protected when using crude nuclear extract as a protein source. However, the introduction of mutations into either or both of the half-sites indicates that the individual half-sites contribute to NFI/CTF binding. Overexpression of NFI-C in NIH-3T3 cells results in an increase in SMUG1 enzyme activity. Collectively, these data indicate that the NFI/CTF consensus site may function as a cis-element in the SMUG1 promoter and that this transcription factor contributes to the positive regulation of SMUG1 gene expression.

Human catechol- O-methyltransferase down-regulation by estradiol

Catechol- O-methyltransferase (COMT) is a crucial enzyme in dopamine and levodopa metabolism. Previously we reported that physiological concentrations of 17β-estradiol (E2) down-regulated steady-state 1.3-kb COMT mRNA levels in MCF-7 cells. In this study, we investigated whether similar reductions occurred in a glial cell line (U138MG) and whether COMT protein and activity levels paralleled the reduction in COMT mRNA levels in MCF-7 cells. In addition, we explored the mechanism of E2 action. E2 had no effect on COMT mRNA levels in U138MG cells, but significantly reduced COMT protein and activity in MCF-7 cells (activity by 53% at 10 −7 M of E2, by 45% at 10 −8 M, and by 28% at 10 −9 M relative to non-E2-treated cells). A specific estrogen receptor antagonist (ICI 182780) blocked these estrogenic effects. Estrogen receptor in nuclear extracts of MCF-7 cells, which were pretreated with E2 (10 −9 M) for 48 h, bound to the whole proximal and distal promoter regions, as determined by electrophoretic mobility shift analysis (EMSA). We propose that E2 decreased COMT activity through down-regulation of its gene and protein expression mediated via ER interaction with response elements in the promoter region of the gene. Our findings may explain the lower of COMT activity in women compared to that in men, and, in part, the beneficial effects of E2 therapy in post-menopausal Parkinson’s disease patients.

Multiple mechanisms for oxygen-induced regulation of the Clara cell secretory protein gene.

The Clara cell secretory protein (CCSP) imparts a protective effect to the lung during oxidant injury. However, exposure to supplemental oxygen, a common therapeutic modality for lung disease, represses the expression of CCSP in the adult mouse lung. We investigated the mechanisms of hyperoxia-induced repression of the mouse CCSP promoter. Deletion experiments in vivo and in vitro indicated that the hyperoxia-responsive elements are localized to the proximal -166 bp of the CCSP promoter. Electrophoretic mobility shift and supershift analyses demonstrated increased binding of c-Jun at the activator protein-1 site, increased binding of CCAAT/enhancer binding protein (C/EBP) beta at the C/EBP sites, and decreased binding at the Nkx2.1 sites. Western analyses revealed that hyperoxia exposure induced an increase in the expression of the C/EBPbeta isoform liver-inhibiting protein (LIP) and an increase in cytoplasmic Nkx2.1. Cotransfection of LIP or c-Jun expression plasmids decreased the transcriptional activity of the proximal -166-bp CCSP promoter. These observations suggest that hyperoxia-induced repression of the CCSP gene is mediated, at least in part, at the level of transcription and that multiple mechanisms mediate this repression. Moreover, these novel observations may provide insights for generation of therapeutic interventions for the amelioration of oxidant-induced lung injury.

Trypanosoma cruzi poly-zinc finger protein: a novel DNA/RNA-binding CCHC-zinc finger protein.

A poly-zinc finger protein, designated PZFP1 was identified in Trypanosoma cruzi for the first time. The protein has 191 amino acids, contains seven motifs Cys(X)(2)Cys(X)(4)His(X)(4)Cys. A recombinant PZFP1 was generated in E. coli and the expected 21kDa polypeptide co-purified with two other inducible products of about 42 and 63kDa. Western blot analysis of cell extracts using an anti-PZFP1 antibody recognized a major band of 41kDa. Electrophoretic mobility shift analysis demonstrated that both, recombinant and native PZFP1, specifically interact with single-stranded DNA or RNA oligonucleotides carrying recognition sequences of other CCHC proteins. The protein was localized mainly in the cytoplasm and nucleus as observed by indirect immunofluorescence analysis. PZFP1 interacted specifically with a T. cruzi serine-arginine-rich protein (TcSR) in a yeast two-hybrid assay, suggesting a role in pre-mRNA processing.

Saccharomyces cerevisiae PIP2 Mediating Oleic Acid Induction and Peroxisome Proliferation Is Regulated by Adr1p and Pip2p-Oaf1p

Saccharomyces cerevisiae genes involved in fatty acid degradation contain in their promoters oleate response elements (OREs) and type 1 upstream activation sequences (UAS1s) that bind Pip2p-Oaf1p and Adr1p, respectively. The promoter of the PIP2 gene was found to contain a potential UAS1 that consists of a tandem array of CYCCRR half-sites in an overlapping arrangement with a previously characterized ORE. Electrophoretic mobility shift analysis demonstrated that Adr1p bound to UAS1PIP2, and Northern analysis in combination with a lacZ reporter gene confirmed that Adr1p influenced the transcription of PIP2. Immunoprecipitation showed that, in adr1delta mutant cells grown on oleic acid, Pip2p was less abundant compared with the corresponding wild-type. In addition, the amount of Pip2p-Oaf1p that bound to a target ORE in vitro was reduced in mutant extracts compared with the wild-type. Transcription of the oleic acid-inducible genes SPS19 and CTA1, which rely on both Pip2p-Oaf1p and Adr1p for their regulation, was reduced in adr1delta mutant cells. However, by ectopically restoring levels of Pip2p in adr1delta cells grown on oleic acid medium, transcription of both genes increased 2-fold compared with the control. This partial suppression of the adr1delta mutant phenotype was additionally manifested by moderate utilization of oleic acid. Hence, both the expression as well as the action of the two transcription factors, Adr1p and Pip2p-Oaf1p, are interconnected, which allows for an elaborate control of fatty acid-inducible genes.

The pkr kinase promoter binds both Sp1 and Sp3, but only Sp3 functions as part of the interferon-inducible complex with ISGF-3 proteins

The protein kinase regulated by RNA (PKR) is an important mediator of the antiviral and antiproliferative actions of interferon (IFN). The promoter of the PKR gene contains a novel 15-bp element designated KCS that is required for both basal and IFN-inducible transcription, with KCS function dependent upon both position and orientation relative to the ISRE element. Novel inducible protein complexes (iKIBP1, iKIBP2) that require both the KCS and the ISRE element sequences for their formation have been identified and characterized. Transcription factors Sp1 and Sp3 were found to be KCS-binding proteins by electrophoretic mobility shift analyses (EMSA) and Sepharose bead-KCS oligonucleotide pull-down assays. However, only Sp3 but not Sp1 was a constituent of the inducible iKIBP complexes. EMSA also identified STAT1, STAT2, and IRF-9 as components of the iKIBP complexes, indicating that ISGF-3 participates in iKIBP complex formation. Proteins bound at the KCS element in the absence of ISRE were able to recruit both STAT1 and STAT2 to the KCS element; recruitment was dependent upon IFN-α treatment. Chromatin immunoprecipitation assays revealed that the binding of Sp3, similar to STAT1 and STAT2, at the PKR promoter in vivo was IFN-dependent, but that Sp1 binding was not dependent upon IFN treatment. These results, taken together, strongly suggest a role for Sp1 in basal and Sp3 in inducible transcription of PKR and that a potential function of the KCS element is to facilitate the recruitment of ISGF-3 complex components to the PKR promoter to stimulate transcription.

Role of CRE-binding protein (CREB) in aromatase expression in breast adipose.

Estrogen biosynthesis from C19 steroids is catalyzed by aromatase cytochrome P450. Aromatase is expressed in breast adipose tissue through the use of a distal, cytokine-responsive promoter (promoter I.4). Breast tumors, however, secrete soluble factors that over-stimulate aromatase expression through an alternative proximal cAMP-responsive promoter, promoter II. We have mapped the cAMP-responsive regions of promoter II by transient transfection of 3T3-L1 preadipocytes with aromatase promoter II reporter genes. 5' deletion and mutation analyses identified two cAMP response element (CRE)-like sequences (CRE1 and CRE2) that were essential for cAMP-induced promoter II activity. Electrophoretic mobility shift analysis demonstrated that CRE binding protein (CREB) bound to each element, and that this interaction was enhanced in the presence of cAMP. Quantification of CREB mRNA expression in adipose tissue from normal and tumor bearing breast adipose tissue revealed that CREB expression is approximately five times higher in tumor bearing than in normal breast adipose tissue. Thus, the over expression of aromatase in adipose tissue surrounding breast tumors could arise through increases in both CREB expression and CREB transcriptional activity. Pharmacological inhibition of CREB activity, previously shown to have anti-proliferative effects on cancer cells, might therefore have additional benefits through inhibition of aromatase expression and thus estrogen production in breast adipose.