Regulation of DHP receptor expression by elements in the 5′-flanking sequence

2000 ◽  
Vol 278 (4) ◽  
pp. H1153-H1162 ◽  
Author(s):  
Lei Liu ◽  
Q. Ivy Fan ◽  
Mohamad R. El-Zaru ◽  
Kathleen Vanderpool ◽  
Ronald N. Hines ◽  
...  
Keyword(s):  
Fusion Gene ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Receptor Expression ◽  
Neonatal Rat ◽  
Luciferase Reporter ◽  
Subunit Gene ◽  
Luciferase Reporter Gene ◽  
Response Element ◽  
Flanking Region

The α1-subunit of the cardiac/vascular Ca2+channel, which is the dihydropyridine (DHP)-binding site (the DHP receptor), provides the pore structure for Ca2+ entry. It contains the binding sites for multiple classes of drugs collectively known as Ca2+ antagonists. As an initial step toward understanding the mechanisms controlling transcription of the rat cardiac α1C-subunit gene, we have cloned a 2.3-kb fragment containing the 5′-flanking sequences and identified the α1C-subunit gene transcription start site. The rat α1C-subunit gene promoter belongs to the TATA-less class of such basal elements. Using deletion analysis of α1C-subunit promoter-luciferase reporter gene constructs, we have characterized the transcriptional modulating activity of the 5′-flanking region and conducted transient transfections in cultured neonatal rat cardiac ventricular myocytes and vascular smooth muscle cells. Sequence scanning identified several potential regulatory elements, including five consensus sequences for the cardiac-specific transcription factor Nkx2.5, an AP-1 site, a cAMP response element, and a hormone response element. Transient transfection experiments with the promoter-luciferase reporter fusion gene demonstrate that the 2-kb 5′-flanking region confers tissue specificity and hormone responsiveness to expression of the Ca2+ channel α1C-subunit gene. Electrophoretic mobility shift assays identified a region of the α1C-subunit gene promoter that can bind transcription factors and appears to be important for gene expression.

scholarly journals Identification of a cAMP response element within the glucose- 6-phosphatase hydrolytic subunit gene promoter which is involved in the transcriptional regulation by cAMP and glucocorticoids in H4IIE hepatoma cells

1999 ◽  
Vol 338 (2) ◽  
pp. 457-463 ◽  
Author(s):  
Dieter SCHMOLL ◽  
Christina WASNER ◽  
Carolyn J. HINDS ◽  
Bernard B. ALLAN ◽  
Reinhard WALTHER ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Consensus Sequence ◽  
Gene Promoter ◽  
Hepatoma Cells ◽  
Luciferase Reporter ◽  
Regulation Of Transcription ◽  
Dibutyryl Camp ◽  
Luciferase Reporter Gene ◽  
Camp Response Element ◽  
Response Element

The expression of a luciferase reporter gene under the control of the human glucose 6-phosphatase gene promoter was stimulated by both dexamethasone and dibutyryl cAMP in H4IIE hepatoma cells. A cis-active element located between nucleotides -161 and -152 in the glucose 6-phosphatase gene promoter was identified and found to be necessary for both basal reporter-gene expression and induction of expression by both dibutyryl cAMP and dexamethasone. Nucleotides -161 to -152 were functionally replaced by the consensus sequence for a cAMP response element. An antibody against the cAMP response element-binding protein caused a supershift in gel-electrophoretic-mobility-shift assays using an oligonucleotide probe representing the glucose 6-phosphatase gene promoter from nucleotides -161 to -152. These results strongly indicate that in H4IIE cells the glucose 6-phosphatase gene-promoter sequence from -161 to -152 is a cAMP response element which is important for the regulation of transcription of the glucose 6-phosphatase gene by both cAMP and glucocorticoids.

scholarly journals Cloning and Functional Analysis of Rat Tweety-Homolog 1 Gene Promoter

2021 ◽  
Author(s):  
Malgorzata Gorniak-Walas ◽  
Karolina Nizinska ◽  
Katarzyna Lukasiuk
Keyword(s):  
Transcriptional Regulation ◽  
Reporter Gene ◽  
Hippocampal Neurons ◽  
Gene Promoter ◽  
Promoter Sequence ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay

AbstractTweety-homolog 1 protein (Ttyh1) is abundantly expressed in neurons in the healthy brain, and its expression is induced under pathological conditions. In hippocampal neurons in vitro, Ttyh1 was implicated in the regulation of primary neuron morphology. However, the mechanisms that underlie transcriptional regulation of the Ttyh1 gene in neurons remain elusive. The present study sought to identify the promoter of the Ttyh1 gene and functionally characterize cis-regulatory elements that are potentially involved in the transcriptional regulation of Ttyh1 expression in rat dissociated hippocampal neurons in vitro. We cloned a 592 bp rat Ttyh1 promoter sequence and designed deletion constructs of the transcription factors specificity protein 1 (Sp1), E2F transcription factor 3 (E2f3), and achaete-scute homolog 1 (Ascl1) that were fused upstream of a luciferase reporter gene in pGL4.10[luc2]. The luciferase reporter gene assay showed the possible involvement of Ascl1, Sp1, and responsive cis-regulatory elements in Ttyh1 expression. These findings provide novel information about Ttyh1 gene regulation in neurons.

Functional overload increases β-MHC promoter activity in rodent fast muscle via the proximal MCAT (βe3) site

2002 ◽  
Vol 282 (3) ◽  
pp. C518-C527 ◽  
Author(s):  
Julia M. Giger ◽  
Fadia Haddad ◽  
Anqi X. Qin ◽  
Kenneth M. Baldwin
Keyword(s):  
Gene Promoter ◽  
Firefly Luciferase ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Type I ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Slow Type ◽  
Responsive Element ◽  
Gel Mobility Shift

Functional overload (OL) of the rat plantaris muscle by the removal of synergistic muscles induces a shift in the myosin heavy chain (MHC) isoform expression profile from the fast isoforms toward the slow type I, or, β-MHC isoform. Different length rat β-MHC promoters were linked to a firefly luciferase reporter gene and injected in control and OL plantaris muscles. Reporter activities of −3,500, −914, −408, and −215 bp promoters increased in response to 1 wk of OL. The smallest −171 bp promoter was not responsive to OL. Mutation analyses of putative regulatory elements within the −171 and −408 bp region were performed. The −408 bp promoters containing mutations of the βe1, distal muscle CAT (MCAT; βe2), CACC, or A/T-rich (GATA), were still responsive to OL. Only the proximal MCAT (βe3) mutation abolished the OL response. Gel mobility shift assays revealed a significantly higher level of complex formation of the βe3 probe with nuclear protein from OL plantaris compared with control plantaris. These results suggest that the βe3 site functions as a putative OL-responsive element in the rat β-MHC gene promoter.

scholarly journals Gamma-Globin Gene Promoter Elements Required for Interaction With Globin Enhancers

Blood ◽  
1998 ◽  
Vol 91 (1) ◽  
pp. 309-318 ◽  
Author(s):  
Scott D. Langdon ◽  
Russel E. Kaufman
Keyword(s):  
Binding Sites ◽  
Globin Gene ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Promoter Strength ◽  
Gene Promoters ◽  
Wild Type ◽  
Luciferase Reporter Gene

Abstract Normal expression of the human β-globin domain genes is dependent on at least three types of regulatory elements located within the β-globin domain: the locus control region (LCR), globin enhancer elements (3′β and 3′Aγ), and the individual globin gene promoter and upstream regions. It has been postulated that regulation occurs through physical interactions between factors bound to these elements, which are located at considerable distances from each other. To identify the elements required for promoter-enhancer interactions from a distance, we have investigated the expression of the wild-type, truncated, and mutated γ-globin promoters linked to the 5′HS2 enhancer. We show that in K562 cells, 5′HS2 increases activity approximately 20-fold from both a wild-type and truncated (-135 → +25) γ promoter and that truncation or site-directed mutagenesis of the tandem CCAAT boxes eliminated the enhancement by 5′HS2. Mutation of the γ-globin gene promoter GATA-1 binding sites did not decrease either promoter strength or enhancement of activity by 5′HS2. To determine if enhanced expression of γ-globin gene promoters carrying mutations associated with hereditary persistence of fetal hemoglobin (HPFH) was due to greater interactions with enhancers, we linked these HPFH γ-globin gene promoters to 5′HS2 and demonstrated a twofold to threefold higher expression than the corresponding wild-type promoter plus enhancer in MEL cells. Addition of the Aγ-globin gene 3′ enhancer to a plasmid containing the γ-globin gene promoter and 5′HS2 did not further enhance promoter strength. Furthermore, we have demonstrated that the previously identified core 5′HS2 enhancer (46-bp tandem AP-1/NF-E2 sites) increased expression only when located 5′, but not 3′, to the γ-globin-luciferase reporter gene, suggesting that its enhancer effect is not by DNA looping. Our results suggest that CCAAT boxes, but not GATA or CACCC binding sites, are required for interaction between the γ-globin promoter and the LCR/5′HS2 and that regulatory elements in addition to the core enhancer may be required for the enhancer to act from a distance.

Regulation of the S100B gene by α1-adrenergic stimulation in cardiac myocytes

2003 ◽  
Vol 284 (1) ◽  
pp. H193-H203 ◽  
Author(s):  
James N. Tsoporis ◽  
Alexander Marks ◽  
Linda J. Van Eldik ◽  
David O'Hanlon ◽  
Thomas G. Parker
Keyword(s):  
Signaling Pathway ◽  
Cardiac Myocytes ◽  
Transcription Initiation ◽  
Regulatory Elements ◽  
Negative Regulator ◽  
Neonatal Rat ◽  
Luciferase Reporter ◽  
Adrenergic Stimulation ◽  
Luciferase Reporter Gene ◽  
Pkc Signaling

We previously reported that S100B, a 20-kDa Ca2+-binding homodimer, inhibited the postinfarct myocardial hypertrophic response mediated by α1-adrenergic stimulation through the protein kinase C (PKC) signaling pathway. In the present study, we examined whether the same pathway induced the S100B gene, supporting the hypothesis that S100B is a feedback negative regulator of this pathway. We transfected cultured neonatal rat cardiac myocytes with a luciferase reporter gene driven by the maximal human S100B promoter and progressively shorter segments of this promoter sequentially deleted from the 5′ end. We identified a basic promoter essential for transcription spanning 162 bp upstream of the transcription initiation site and positive (at −782/−162 and −6,689/−4,463) and negative (at −4,463/−782) myocyte-selective regulatory elements. We showed that the basic and maximal S100B promoters were activated specifically by α1-adrenergic agonists through the α1A-adrenergic receptor, but not by any other trophic hormonal stimuli. The activation of the S100B promoter was mediated through the PKC signaling pathway. Transcription enhancer factor-1 (TEF-1) and related to TEF-1 (RTEF-1) influenced transcription from the maximal, but not the basic, promoter implicating active MCAT elements upstream from the basic promoter. Acting in opposing fashions, TEF-1 transrepressed the S100B promoter and RTEF-1 transactivated the promoter. Our results suggest that α1-adrenergic stimulation induces the S100B gene after myocardial infarction through the PKC signaling pathway and that this induction is modulated by TEF-1 and RTEF-1.

Isolation and characterization of kidney-specific ClC-K1 chloride channel gene promoter

1998 ◽  
Vol 274 (3) ◽  
pp. F602-F610 ◽  
Author(s):  
Shinichi Uchida ◽  
Tatemitsu Rai ◽  
Hiroshi Yatsushige ◽  
Yoshihiro Matsumura ◽  
Masanobu Kawasaki ◽  
...  
Keyword(s):  
Cell Lines ◽  
Chloride Channel ◽  
Gene Promoter ◽  
Simian Virus 40 ◽  
Regulatory Elements ◽  
Start Codon ◽  
Luciferase Reporter ◽  
Specific Expression ◽  
Isolation And Characterization ◽  
Flanking Region

The rat ClC-K1 chloride channel is a kidney-specific member of the ClC chloride channel family found exclusively in the thin ascending limb of Henle’s loop in the kidney. To gain insight into the mechanism(s) of kidney-specific expression of ClC-K1, a genomic clone that contains the 5′-flanking region of the rat ClC-K1 gene was isolated. A single transcription start site was located 84 bp upstream of the start codon. The sequence of the proximal 5′-flanking region contained an activator protein (AP)-3 site, a glucocorticoid-responsive element, several AP-2 sites, and several E-boxes, but it lacked a TATA box. To functionally express the promoter, the ∼2.5-kb pair 5′-flanking region was ligated to a luciferase reporter gene and transfected into inner medullary (IM) cells, a stable ClC-K1-expressing cell line derived from the inner medulla of simian virus 40 transgenic mouse, and ClC-K1-nonexpressing cell lines. Luciferase activity was 7- to 24-fold greater in IM cells than those in nonexpressing cell lines, suggesting that the ∼2.5-kb fragment contained cis-acting regulatory elements for cell-specific expression of the ClC-K1 gene. Deletion analysis revealed that this cell-specific promoter activity in IM cells was still present in the construct containing 51 bp of the 5′-flanking region but was lost in the −29 construct, clearly demonstrating that the 22 bp from −51 to −30 have a major role in the cell-specific activity of the ClC-K1 promoter. These 22 bp consist of purine-rich sequence (GGGGAGGGGGAGGGGAG), and gel-retardation analysis demonstrated the existence of a specific protein(s) binding to this element in IM cells. These results suggest that the novel purine-rich element may play a key role in the activity of the ClC-K1 gene promoter.

scholarly journals Gamma-Globin Gene Promoter Elements Required for Interaction With Globin Enhancers

Blood ◽  
1998 ◽  
Vol 91 (1) ◽  
pp. 309-318
Author(s):  
Scott D. Langdon ◽  
Russel E. Kaufman
Keyword(s):  
Binding Sites ◽  
Globin Gene ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Promoter Strength ◽  
Gene Promoters ◽  
Wild Type ◽  
Luciferase Reporter Gene

Normal expression of the human β-globin domain genes is dependent on at least three types of regulatory elements located within the β-globin domain: the locus control region (LCR), globin enhancer elements (3′β and 3′Aγ), and the individual globin gene promoter and upstream regions. It has been postulated that regulation occurs through physical interactions between factors bound to these elements, which are located at considerable distances from each other. To identify the elements required for promoter-enhancer interactions from a distance, we have investigated the expression of the wild-type, truncated, and mutated γ-globin promoters linked to the 5′HS2 enhancer. We show that in K562 cells, 5′HS2 increases activity approximately 20-fold from both a wild-type and truncated (-135 → +25) γ promoter and that truncation or site-directed mutagenesis of the tandem CCAAT boxes eliminated the enhancement by 5′HS2. Mutation of the γ-globin gene promoter GATA-1 binding sites did not decrease either promoter strength or enhancement of activity by 5′HS2. To determine if enhanced expression of γ-globin gene promoters carrying mutations associated with hereditary persistence of fetal hemoglobin (HPFH) was due to greater interactions with enhancers, we linked these HPFH γ-globin gene promoters to 5′HS2 and demonstrated a twofold to threefold higher expression than the corresponding wild-type promoter plus enhancer in MEL cells. Addition of the Aγ-globin gene 3′ enhancer to a plasmid containing the γ-globin gene promoter and 5′HS2 did not further enhance promoter strength. Furthermore, we have demonstrated that the previously identified core 5′HS2 enhancer (46-bp tandem AP-1/NF-E2 sites) increased expression only when located 5′, but not 3′, to the γ-globin-luciferase reporter gene, suggesting that its enhancer effect is not by DNA looping. Our results suggest that CCAAT boxes, but not GATA or CACCC binding sites, are required for interaction between the γ-globin promoter and the LCR/5′HS2 and that regulatory elements in addition to the core enhancer may be required for the enhancer to act from a distance.

scholarly journals Characterization of the Human Corticotropin-Releasing Factor2(a) Receptor Promoter: Regulation by Glucocorticoids and the Cyclic Adenosine 5′-Monophosphate Pathway

Endocrinology ◽  
2004 ◽  
Vol 145 (12) ◽  
pp. 5605-5615 ◽  
Author(s):  
Steven A. Nanda ◽  
Patrick H. Roseboom ◽  
George A. Nash ◽  
James M. Speers ◽  
Ned H. Kalin
Keyword(s):  
Gene Expression ◽  
Promoter Region ◽  
Chinese Hamster ◽  
Regulatory Elements ◽  
Site Directed Mutagenesis ◽  
Model Systems ◽  
Luciferase Reporter ◽  
Intracellular Camp ◽  
Luciferase Reporter Gene ◽  
Response Element

Abstract Corticotropin-releasing factor (CRF) is a neurotransmitter and hormone believed to integrate responses to stress. Evidence suggests central CRF systems are overactive in some individuals suffering from depression and anxiety disorders. CRF receptor antagonism blocks stress-induced endocrine, autonomic, and behavioral effects in animal models, and studies have implicated the CRF2 receptor in anxiety-related behaviors. Greater understanding of the regulation of CRF2 expression may facilitate understanding mechanisms underlying anxiety. The present studies are the first to characterize the transcriptional regulation of the human CRF2(a), the predominant CRF2 isoform in brain. Four kilobase pairs of sequence immediately upstream of the first exon of CRF2(a) represented our full-length promoter region. Sequentially smaller fragments of the CRF2(a) promoter region were generated by PCR and cloned upstream of a luciferase reporter gene. Expression was monitored from these constructs within Chinese hamster ovary-K1 cells and within rat aortic A7R5 cells that express CRF2. Glucocorticoid treatment decreased expression and elevating intracellular cAMP increased expression from the human CRF2(a) promoter. The regions of the CRF2(a) promoter that regulate the inducible expression were determined, and the functional cAMP response element and glucocorticoid response element cis-regulatory elements within these regions were identified using a combination of site-directed mutagenesis and EMSAs. Given the possibility of species-specific differences in gene expression, interpretation of gene expression studies from rat and mouse model systems is difficult. Examination of expression from the human CRF2(a) promoter will provide insight into these model systems and may translate more readily to the development of therapeutics to treat human psychiatric illness.

scholarly journals SalA Attenuates Hypoxia-Induced Endothelial Endoplasmic Reticulum Stress and Apoptosis via Down-Regulation of VLDL Receptor Expression

2015 ◽  
Vol 35 (1) ◽  
pp. 17-28 ◽  
Author(s):  
Ping Xie ◽  
Yingchun Duan ◽  
Xianzhi Guo ◽  
Lina Hu ◽  
Minghua Yu
Keyword(s):  
Endothelial Cells ◽  
Er Stress ◽  
Umbilical Vein ◽  
Gene Promoter ◽  
Immunoblot Analysis ◽  
Receptor Expression ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Luciferase Reporter Gene ◽  
Vldl Receptor

Background: Salvianolic acid A (SalA) has been shown to display robust protection against endothelial injury. VLDL receptor (VLDLr) is expressed at high levels in the endothelial cells. However its endothelial biological function has not been completely elucidated. Here, we investigated molecular effects of SalA on endothelial VLDLr expression, ER stress, and apoptosis under hypoxia condition. Methods: Human umbilical vein endothelial cells (HUVECs) pretreated with SalA were subjected to hypoxia stimulation. Endothelial ER stress and apoptosis were examined. The mRNA levels were tested by real-time RT-PCR, and the protein levels were determined by immunoblot analysis. Results: Pretreatment of HUVECs with SalA markedly attenuated hypoxia-induced endothelial ER stress and apoptosis. Hypoxia resulted in enhancement of VLDLr expression, which was effectively inhibited by SalA pretreatment. Furthermore, luciferase reporter gene assays indicated that SalA inhibited vldlr gene promoter activity, and ChIP assays showed that hypoxia increase the recruitment of HIF-1α to the vldlr gene promoter, and this process was hampered markedly by pretreatment of SalA. Finally, overexpression of VLDLr abolished SalA-mediated protection of endothelial cells from ER stress and apoptosis. Knockdown of VLDLr mimicked SalA protective effect. Conclusion: These results for the first time demonstrate that SalA protects against hypoxia-induced endothelial ER stress and apoptosis through inhibiting recruitment of HIF-1α to vldlr gene promoter and thus suppressing VLDLr expression.

scholarly journals Genome-Wide Identification and Analysis of the APETALA2 (AP2) Transcription Factor in Dendrobium officinale

2021 ◽  
Vol 22 (10) ◽  
pp. 5221
Author(s):  
Danqi Zeng ◽  
Jaime A. Teixeira da Silva ◽  
Mingze Zhang ◽  
Zhenming Yu ◽  
Can Si ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Flower Development ◽  
Regulatory Elements ◽  
Negative Control ◽  
Dendrobium Officinale ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Luciferase Reporter Gene ◽  
Genome Wide ◽  
Ap2 Transcription Factor

The APETALA2 (AP2) transcription factors (TFs) play crucial roles in regulating development in plants. However, a comprehensive analysis of the AP2 family members in a valuable Chinese herbal orchid, Dendrobium officinale, or in other orchids, is limited. In this study, the 14 DoAP2 TFs that were identified from the D. officinale genome and named DoAP2-1 to DoAP2-14 were divided into three clades: euAP2, euANT, and basalANT. The promoters of all DoAP2 genes contained cis-regulatory elements related to plant development and also responsive to plant hormones and stress. qRT-PCR analysis showed the abundant expression of DoAP2-2, DoAP2-5, DoAP2-7, DoAP2-8 and DoAP2-12 genes in protocorm-like bodies (PLBs), while DoAP2-3, DoAP2-4, DoAP2-6, DoAP2-9, DoAP2-10 and DoAP2-11 expression was strong in plantlets. In addition, the expression of some DoAP2 genes was down-regulated during flower development. These results suggest that DoAP2 genes may play roles in plant regeneration and flower development in D. officinale. Four DoAP2 genes (DoAP2-1 from euAP2, DoAP2-2 from euANT, and DoAP2-6 and DoAP2-11 from basal ANT) were selected for further analyses. The transcriptional activation of DoAP2-1, DoAP2-2, DoAP2-6 and DoAP2-11 proteins, which were localized in the nucleus of Arabidopsis thaliana mesophyll protoplasts, was further analyzed by a dual-luciferase reporter gene system in Nicotiana benthamiana leaves. Our data showed that pBD-DoAP2-1, pBD-DoAP2-2, pBD-DoAP2-6 and pBD-DoAP2-11 significantly repressed the expression of the LUC reporter compared with the negative control (pBD), suggesting that these DoAP2 proteins may act as transcriptional repressors in the nucleus of plant cells. Our findings on AP2 genes in D. officinale shed light on the function of AP2 genes in this orchid and other plant species.

Sign in / Sign up

Export Citation Format

Share Document