Genomic structure and transcriptional regulation of the human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase gene

The NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) is a catabolic enzyme that controls the biological activities of prostaglandins by converting them into inactive keto-metabolites. Here we report the genomic organisation of the complete human PGDH gene and characterise its transcriptional regulation. The PGDH gene spans about 31 kb on chromosome 4 and contains 7 exons. Within 2.4 kb of the 5'-flanking sequence we identified two regions with clustered putative transcription factor binding sites. The distal promoter element PGDH-DE (positions-2152/-1944 relative to the start codon) contains binding sites for Ets and activating protein-1 (AP-1) flanked by two cAMP-responsive element-binding protein binding sites (CREB1, CREB2), whereas the proximal element PGDH-PE (-235/-153) includes an Ets and an AP-1 binding sequence. By electrophoretic mobility shift assay, no high affinity binding of Ets or AP-1 factors was observed with PGDH-PE, whereas we confirmed interaction of members of the Ets, AP-1 and CREB families of transcription factors with PGDH-DE. Transcriptional control of the PGDH promoter was assessed by transiently transfecting JEG-3 choriocarcinoma cells. A luciferase reporter gene construct containing the PGDH-PE was not induced by c-jun/c-fos in the absence or presence of co-expressed Ets-1. A construct carrying the PGDH-DE in front of the minimal homologous promoter was activated by co-transfection of expression vectors for AP-1 proteins. Mutation of the AP-1 or CREB2 site reduced the response to c-jun/c-fos, whereas mutation of the Ets site of the distal element reduced basal promoter activity. CREB activated the PGDH-DE construct through the CREB1 site. These results defined the distal element as an integrator of transcriptional regulation by AP-1, Ets and CREB proteins.

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Krüppel-like factor 4 represses transcription of the survivin gene in esophageal cancer cell lines

Biological Chemistry ◽

10.1515/bc.2009.060 ◽

2009 ◽

Vol 390 (5/6) ◽

Cited By ~ 14

Author(s):

Guo Zhang ◽

Hongxia Zhu ◽

Yihua Wang ◽

Shangbin Yang ◽

Mei Liu ◽

...

Keyword(s):

Cancer Cells ◽

Binding Sites ◽

Ectopic Expression ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Mobility Shift ◽

Aberrant Expression ◽

Survivin Gene ◽

Survivin Promoter

Abstract Aberrant expression of survivin has been shown to be regulated at the transcription level in cancer cells. In this study, we demonstrate that there are six putative binding sites of Krüppel-like factor 4 (KLF4) within the 2000-bp region upstream of the transcription start site of the human survivin gene. Luciferase reporter gene assays revealed that survivin promoter activity is repressed upon overexpression of KLF4 in EC9706 cells. A chromatin immunoprecipitation assay indicated that KLF4 indeed binds the survivin promoter in vivo. It specifically binds the site located at position -40 among the six binding sites as determined by electrophoretic mobility shift assay. Ectopic expression of KLF4 decreases the mRNA and protein levels of survivin. Furthermore, overexpression of survivin partially reverses KLF4-induced cell apoptosis. These results indicate that KLF4 is a transcriptional repressor of the human survivin gene in esophageal squamous cancer cells.

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Transcriptional Regulation of Human 11β-Hydroxylase (hCYP11B1)

Endocrinology ◽

10.1210/endo.141.10.7689 ◽

2000 ◽

Vol 141 (10) ◽

pp. 3587-3594 ◽

Cited By ~ 38

Author(s):

Xiao-Li Wang ◽

Mary Bassett ◽

Yin Zhang ◽

Su Yin ◽

Colin Clyne ◽

...

Keyword(s):

Adrenal Cortex ◽

Electrophoretic Mobility ◽

Binding Protein ◽

Regulatory Elements ◽

Expression Vectors ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Mobility Shift ◽

Maximal Induction ◽

Electrophoretic Mobility Shift Assays

Abstract Steroid 11β-hydroxylase is a mitochondrial enzyme that catalyzes the conversion of deoxycortisol to cortisol. The gene encoding human 11β-hydroxylase (hCYP11B1) is expressed in the adrenal cortex under the control of circulating levels of ACTH. The current study was undertaken to define the cis-regulatory elements and transacting factors that regulate hCYP11B1 transcription. The hCYP11B1 5′-flanking DNA was studied using transient transfection of luciferase reporter constructs in NCI-H295R human adrenocortical cells. A cAMP analogue ((Bu)2cAMP) increased expression of a construct containing −1102 bp of hCYP11B1 5′-flanking DNA (pB1–1102). An element at position −71/−64 (TGACGTGA, previously termed Ad1) resembling a consensus cAMP response element (CRE) was required for maximal induction by cAMP. The Ad1 element bound several transcriptional factors in electrophoretic mobility shift assays, including CRE-binding protein, activating transcription factor-1 (ATF-1), and ATF-2, but only the ATF-2 complex migrated similarly to a complex seen using H295R nuclear extract. In addition, Western analysis of H295R and adrenal lysates demonstrated expression of high levels of ATF-2 and ATF-1. CRE-binding protein levels varied among the strains of H295R cells tested. Transcription of CYP11B1 also appeared to be regulated by steroidogenic factor-1 (SF-1). Luciferase reporter gene activity was increased after cotransfection with expression vectors containing SF-1. An element in hCYP11B1 at positions −242/−234 (CCAAGGCTC), previously termed Ad4, was required for maximal induction by SF-1 and was found to bind SF-1 in electrophoretic mobility shift assays. The key role for SF-1 in hCYP11B1 transcription is in contrast to its lack of an effect on expression of the hCYP11B2 (aldosterone synthase) isozyme. The differential effects of SF-1 on transcription of hCYP11B1 and hCYP11B2 may be one of the mechanisms controlling differential expression of these isozymes within the zonae fasciculata and glomerulosa of the human adrenal cortex.

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Characterization of an interleukin 4 (IL-4) responsive region in the immunoglobulin heavy chain germline epsilon promoter: regulation by NF-IL-4, a C/EBP family member and NF-kappa B/p50.

Journal of Experimental Medicine ◽

10.1084/jem.181.1.181 ◽

1995 ◽

Vol 181 (1) ◽

pp. 181-192 ◽

Cited By ~ 131

Author(s):

S Delphin ◽

J Stavnezer

Keyword(s):

Transcription Factors ◽

Binding Sites ◽

Immunoglobulin E ◽

Large Body ◽

Interleukin 4 ◽

Luciferase Reporter ◽

Class Switching ◽

Luciferase Reporter Gene ◽

Nf Kappa B ◽

Mobility Shift

A large body of data indicate that antibody class switching is directed by cytokines by inducing or repressing transcription from unrearranged, or germline, CH genes. Interleukin 4 (IL-4) induces transcription of the germline C epsilon genes in activated B cells and subsequently, cells in this population will undergo switch recombination to immunoglobulin E. Furthermore, the data suggest that transcription of germline C epsilon genes is required for class switching. In this paper we define DNA elements required for induction of transcription of the germline C epsilon genes by IL-4. To do this, segments of DNA from the 5' flank of the initiation sites for germline epsilon RNA were ligated to a luciferase reporter gene and transfected into two mouse B cell lines, one of which can be induced to switch to IgE. By analysis of a series of 5' deletion constructs and linker-scanning mutations, we demonstrate that a 46-bp segment (residing at -126/-79 relative to the first RNA initiation site) contains an IL-4 responsive region. By electrophoretic mobility shift assays, we find that this segment binds three transcription factors: the recently described NF-IL4, one or more members of the C/EBP family of transcription factors, and NF-kappa B/p50. Mutation of any of the binding sites for these three factors abolishes or reduces IL-4 inducibility of the epsilon promoter. A 27-bp segment within this IL-4 response region containing binding sites for NF-IL4 and a C/EBP factor is sufficient to transfer IL-4 inducibility to a minimal c-fos promoter.

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Transcriptional Regulation of PEN-2, a Key Component of the γ-Secretase Complex, by CREB

Molecular and Cellular Biology ◽

10.1128/mcb.26.4.1347-1354.2006 ◽

2006 ◽

Vol 26 (4) ◽

pp. 1347-1354 ◽

Cited By ~ 30

Author(s):

Ruishan Wang ◽

Yun-wu Zhang ◽

Ping Sun ◽

Runzhong Liu ◽

Xian Zhang ◽

...

Keyword(s):

Transcriptional Regulation ◽

Transcriptional Activity ◽

Start Codon ◽

Gamma Secretase ◽

Mobility Shift ◽

Endoproteolytic Cleavage ◽

Notch Cleavage ◽

Translational Start

ABSTRACT Gamma-secretase, which is responsible for the intramembranous cleavage of Alzheimer's β-amyloid precursor protein (APP), the signaling receptor Notch, and many other substrates, is a multiprotein complex consisting of at least four components: presenilin (PS), nicastrin, APH-1, and PEN-2. Despite the fact that PEN-2 is known to mediate endoproteolytic cleavage of full-length PS and APH-1 and nicastrin are required for maintaining the stability of the complex, the detailed physiological function of each component remain elusive. Unlike that of PS, the transcriptional regulation of PEN-2, APH-1, and nicastrin has not been investigated. Here, we characterized the upstream regions of the human PEN-2 gene and identified a 238-bp fragment located 353 bp upstream of the translational start codon as the key region necessary for the promoter activity. Further analysis revealed a CREB binding site located in the 238-bp region that is essential for the transcriptional activity of the PEN-2 promoter. Mutation of the CREB site abolished the transcriptional activity of the PEN-2 promoter. Electrophoretic mobility shift assays and chromatin immunoprecipitation analysis showed the binding of CREB to the PEN-2 promoter region both in vitro and in vivo. Activation of the CREB transcriptional factor by forskolin dramatically promoted the expression of PEN-2 mRNA and protein, whereas the other components of the γ-secretase complex remained unaffected. Forskolin treatment slightly increases the secretion of soluble APPα and Aβ without affecting Notch cleavage. These results demonstrate that expression of PEN-2 is regulated by CREB and suggest that the specific control of PEN-2 expression may imply additional physiological functions uniquely assigned to PEN-2.

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Lipid deprivation increases surfactant phosphatidylcholine synthesis via a sterol-sensitive regulatory element within the CTP:phosphocholine cytidylyltransferase promoter

Biochemical Journal ◽

10.1042/bj3620081 ◽

2002 ◽

Vol 362 (1) ◽

pp. 81-88 ◽

Cited By ~ 8

Author(s):

Rama K. MALLAMPALLI ◽

Alan J. RYAN ◽

James L. CARROLL ◽

Timothy F. OSBORNE ◽

Christie P. THOMAS

Keyword(s):

Specific Binding ◽

Core Promoter ◽

Regulatory Element ◽

Simian Virus 40 ◽

Simian Virus ◽

Luciferase Reporter ◽

Standard Diet ◽

Flanking Sequence ◽

Mobility Shift ◽

Ctp:Phosphocholine Cytidylyltransferase

Lipid-deprived mice increase alveolar surfactant disaturated phosphatidylcholine (DSPtdCho) synthesis compared with mice fed a standard diet by increasing expression of CTP:phosphocholine cytidylyltransferase (CCT), the rate-limiting enzyme for DSPtdCho synthesis. We previously observed that lipid deprivation increases mRNA synthesis for CCT [Ryan, McCoy, Mathur, Field and Mallampalli (2000) J. Lipid Res. 41, 1268–1277]. To evaluate regulatory mechanisms for this gene, we cloned the proximal ∼ 1900bp of the 5′ flanking sequence of the murine CCT gene, coupled this to a luciferase reporter, and examined transcriptional regulation in a murine alveolar epithelial type II cell line (MLE-12). The core promoter was localized to a region between −169 and +71bp, which exhibited strong basal activity comparable with the simian virus 40 promoter. The full-length construct, from −1867 to +71, was induced 2–3-fold when cells were cultured in lipoprotein-deficient serum (LPDS), similar to the level of induction of the endogenous CCT gene. By deletional analysis the sterol regulatory element (SRE) was localized within a 240bp region. LPDS activation of the CCT promoter was abolished by mutation of this SRE, and gel mobility-shift assays demonstrated specific binding of recombinant SRE-binding protein to this element within the CCT promoter. These observations indicate that sterol-regulated expression of CCT is mediated by an SRE within its 5′ flanking region.

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CD14 Protein Acts as an Adaptor Molecule for the Immune Recognition of Salmonella Curli Fibers

Journal of Biological Chemistry ◽

10.1074/jbc.m112.447060 ◽

2013 ◽

Vol 288 (20) ◽

pp. 14178-14188 ◽

Cited By ~ 31

Author(s):

Glenn J. Rapsinski ◽

Tiffanny N. Newman ◽

Gertrude O. Oppong ◽

Jos P. M. van Putten ◽

Çagla Tükel

Keyword(s):

Bone Marrow ◽

Cancer Cell Line ◽

Enteric Bacteria ◽

Fiber Formation ◽

Expression Vectors ◽

Luciferase Reporter ◽

Cervical Cancer Cell Line ◽

Immune Recognition ◽

Luciferase Reporter Gene ◽

Membrane Bound

Amyloids, protein aggregates with a cross β-sheet structure, contribute to inflammation in debilitating disorders, including Alzheimer's disease. Enteric bacteria also produce amyloids, termed curli, contributing to inflammation during infection. It has been demonstrated that curli and β-amyloid are recognized by the immune system via the Toll-like receptor (TLR) 2/TLR1 complex. Here we investigated the role of CD14 in the immune recognition of bacterial amyloids. We used HeLa 57A cells, a human cervical cancer cell line containing a luciferase reporter gene under the control of an NF-κB promoter. When HeLa 57A cells were transiently transfected with combinations of human expression vectors containing genes for TLR2, TLR1, and CD14, membrane-bound CD14 enhanced NF-κB activation through the TLR2/TLR1 complex stimulated with curli fibers or recombinant CsgA, the curli major subunit. Similarly, soluble CD14 augmented the TLR2/TLR1 response to curli fibers in the absence of membrane-bound CD14. We further revealed that IL-6 and nitric oxide production were significantly higher by wild-type (C57BL/6) bone marrow-derived macrophages compared with TLR2-deficient or CD14-deficient bone marrow-derived macrophages when stimulated with curli fibers, recombinant CsgA, or synthetic CsgA peptide, CsgA-R4–5. Binding assays demonstrated that recombinant TLR2, TLR1, and CD14 bound purified curli fibers. Interestingly, CD14-curli interaction was specific to the fibrillar form of the amyloid, as demonstrated by using synthetic CsgA peptides proficient and deficient in fiber formation, respectively. Activation of the TLR2/TLR1/CD14 trimolecular complex by amyloids provides novel insights for innate immunity with implications for amyloid-associated diseases.

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Xiao Yao San against Corticosterone-Induced Stress Injury via Upregulating Glucocorticoid Receptor Reaction Element Transcriptional Activity

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2016/5850739 ◽

2016 ◽

Vol 2016 ◽

pp. 1-14 ◽

Cited By ~ 2

Author(s):

Guoping Cao ◽

Shenglan Gong ◽

Fengxue Zhang ◽

Wenjun Fu

Keyword(s):

Hippocampal Neurons ◽

Transcriptional Activity ◽

Nuclear Translocation ◽

Luciferase Reporter ◽

Potential Candidate ◽

Luciferase Reporter Gene ◽

Mobility Shift ◽

Stress Injury ◽

Induced Stress

Previous studies have revealed that uncontrollable stress can impair the synaptic plasticity and firing property of hippocampal neurons, which influenced various hippocampal-dependent tasks including memory, cognition, behavior, and mood. In this work, we had investigated the effects and mechanisms of the Chinese herbal medicine Xiao Yao San (XYS) against corticosterone-induced stress injury in primary hippocampal neurons (PHN) cells. We found that XYS and RU38486 could increase cell viabilities and decrease cell apoptosis by MTT, immunofluorescence, and flow cytometry assays. In addition, we observed that XYS notably inhibited the nuclear translocation of GR and upregulated the mRNA and protein expressions levels of Caveolin-1, GR, BDNF, TrkB, and FKBP4. However, XYS downregulated the FKBP51 expressions. Furthermore, the results of the electrophoretic mobility shift assay (EMSA) and double luciferase reporter gene detection indicated that FKBP4 promotes the transcriptional activity of GR reaction element (GRE) by binding with GR, and FKBP51 processed the opposite action. Thein vivoexperiment also proved the functions of XYS. These results suggested that XYS showed an efficient neuroprotection against corticosterone-induced stress injury in PHN cells by upregulating GRE transcriptional activity, which should be developed as a potential candidate for treating stress injury in the future.

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A distal activation domain is critical in the regulation of the rat androgen receptor gene promoter

Biochemical Journal ◽

10.1042/bj2940779 ◽

1993 ◽

Vol 294 (3) ◽

pp. 779-784 ◽

Cited By ~ 19

Author(s):

C S Song ◽

S Her ◽

M Slomczynska ◽

S J Choi ◽

M H Jung ◽

...

Keyword(s):

Androgen Receptor ◽

Nucleotide Sequence ◽

Receptor Gene ◽

Critical Role ◽

Androgen Receptor Gene ◽

Luciferase Reporter ◽

Upstream Region ◽

Luciferase Reporter Gene ◽

Mobility Shift ◽

Cis Element

The far upstream region of the rat androgen receptor (AR) gene has been cloned, and the nucleotide sequence up to -2656 bp established. Nested deletion mutants of rat AR 5′ flanking sequences were ligated to the luciferase reporter gene, and their promoter activities were examined in transfected COS1 cells. Results show a critical cis-acting domain located between positions -960 and -940. Deletion of this cis element resulted in a greater than 90% decrease in the promoter activity. A nuclear protein that specifically binds to this 21-nucleotide sequence was identified by gel mobility shift analysis. The -960/-940 cis element has no identify to the binding sequence of any known transcription factor. Furthermore, the cognate binding protein is present in both rat and human (HeLa) cell nuclear extracts. We conclude that a novel trans-activator interacting at the -960/-940 region plays a critical role in the regulation of AR gene expression.

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Detection of a Complex That Associates With the Bβ Fibrinogen G−455-A Polymorphism

Blood ◽

10.1182/blood.v92.9.3286.421k20_3286_3293 ◽

1998 ◽

Vol 92 (9) ◽

pp. 3286-3293

Author(s):

Erika T. Brown ◽

Gerald M. Fuller

Keyword(s):

Luciferase Reporter ◽

Molecular Evidence ◽

Luciferase Reporter Gene ◽

Nucleotide Substitutions ◽

Mobility Shift ◽

Binding Characteristics ◽

Base Sequences ◽

American Society ◽

Electrophoretic Mobility Shift Assays

The promoter region of the Bβ fibrinogen gene containing the polymorphic site (G−455-A) shows an increase in fibrinogen levels for individuals containing an adenine rather than a guanine. Two methods were used to explore the possible functional role of this region. Electrophoretic mobility shift assays (EMSAs) were performed using specific DNA probes containing base sequences pertinent to the allelic site. Specific DNA binding proteins were detected and their binding characteristics were determined. Secondly, we placed DNA fragments containing different −455 nucleotide substitutions of the Bβ promoter upstream of a luciferase reporter gene and transfected them into HepG2 cells to determine their effect on transactivation. An adenine at position −455 resulted in greater luciferase activity than when a guanine was present. UV cross-linking bound protein to the DNA demonstrated a 47-kD protein binding preferentially to the site when a guanine rather than an adenine was present at −455. We hypothesize that a transactivation protein complex associates with the site, but its association is stronger when guanine is present, thereby slowing downstream Bβ gene transcription. These data provide the first molecular evidence that accounts for the increase in fibrinogen in individuals carrying this allele.© 1998 by The American Society of Hematology.

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Transcriptional regulation of human and murine 17β-hydroxysteroid dehydrogenase type-7 confers its participation in cholesterol biosynthesis

Journal of Molecular Endocrinology ◽

10.1677/jme.1.02043 ◽

2006 ◽

Vol 37 (1) ◽

pp. 185-197 ◽

Cited By ~ 15

Author(s):

Thomas Ohnesorg ◽

Brigitte Keller ◽

Martin Hrabé de Angelis ◽

Jerzy Adamski

Keyword(s):

Transcriptional Regulation ◽

Binding Sites ◽

Sequence Similarity ◽

Regulatory Element ◽

Cholesterol Biosynthesis ◽

Hydroxysteroid Dehydrogenase ◽

Site Directed Mutagenesis ◽

Mobility Shift ◽

Mobility Shift Assay

In both humans and mice, 17β-hydroxysteroid dehydrogenase type-7 (HSD17B7) was described as possessing dual enzymatic functionality. The enzyme was first shown to be able to convert estrone to estradiol in vitro. Later involvement of this enzyme in postsqualene cholesterol biosynthesis was postulated (conversion of zymosterone to zymosterol) and could be proven in vitro. In this work, we performed a detailed analysis of the transcriptional regulation of both the human and murine genes. Despite relatively low sequence similarity, both promoters contain similar contexts of transcription factor-binding sites. The participation of these sites in transcriptional regulation of HSD17B7 was proven by electro-mobility shift assay and site-directed mutagenesis of the corresponding binding sites. We describe novel involvement of vitamin D receptor/retinoid X receptor and provide new information on the regulation of HSD17B7 expression by sterol regulatory element-binding protein and hepatocyte nuclear factor 4, the latter known from other genes of cholesterogenic enzymes. The results of our study provide unequivocal evidence for a role of HSD17B7 in cholesterol biosynthesis.

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