scholarly journals Identification and characterization of the MUC2 (human intestinal mucin) gene 5′-flanking region: promoter activity in cultured cells

1997 ◽  
Vol 325 (1) ◽  
pp. 259-267 ◽  
Author(s):  
James R. GUM ◽  
James W. HICKS ◽  
Young S. KIM
Keyword(s):  
Cell Lines ◽  
Promoter Activity ◽  
Cultured Cells ◽  
Cell Types ◽  
Luciferase Reporter ◽  
Cell Type Specificity ◽  
Mobility Shift ◽  
Initiation Point ◽  
Mucin Gene ◽  
Flanking Region

The initiation point for MUC2 gene transcription is located within a 7000-base GC-rich region of the mucin gene cluster found on chromosome 11p15.5. The promoter activity of the 5′-flanking region of the MUC2 gene was examined following its cloning into the luciferase-producing pGL2-Basic reporter vector. A short segment comprising bases -91 to -73 relative to the start of transcription was found to be important for basal promoter activity in all cell lines tested. Electrophoretic mobility shift assays demonstrated nuclear protein binding to this region, which contains the consensus CACCC motif (5′-GCCACACCC). This element has been shown to be functionally important in several promoters that are active in diverse cell types. Competition experiments using an Sp1 oligonucleotide and antibody supershift experiments indicated that both Sp1 and other Sp1 family members bind to this element. Inclusion of the region between bases -228 and -171 in pGL2-Basic constructs increased normalized luciferase reporter activity by almost 3-fold in C1a cells, which produce relatively high levels of MUC2 mRNA. Significantly lower levels of normalized luciferase activity resulted when the same construct was transfected into cultured cell lines that express low or undetectable levels of MUC2, suggesting a possible role for this region in conferring cell-type specificity of expression. We also demonstrate, using actinomycin D, that the MUC2 mRNA is long-lived, at least in cultured cells. Moreover, no evidence was found that the MUC2 mRNA turned over more rapidly in LS174T cells, which produce relatively low levels of MUC2 mRNA, as compared with C1a cells, which produce high levels of mRNA. Thus a long mRNA half-life appears to be an important mechanism involved in achieving elevated levels of MUC2 mRNA.

scholarly journals Sequence of the 5′-flanking region and promoter activity of the human mucin gene MUC5B in different phenotypes of colon cancer cells

2000 ◽  
Vol 348 (3) ◽  
pp. 675-686 ◽  
Author(s):  
Isabelle VAN SEUNINGEN ◽  
Michaël PERRAIS ◽  
Pascal PIGNY ◽  
Nicole PORCHET ◽  
Jean-Pierre AUBERT
Keyword(s):  
Gene Expression ◽  
Transcription Start Site ◽  
Promoter Activity ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Start Site ◽  
Transcription Start ◽  
Mucin Gene ◽  
Intron 1 ◽  
Flanking Region

Control of gene expression in intestinal cells is poorly understood. Molecular mechanisms that regulate transcription of cellular genes are the foundation for understanding developmental and differentiation events. Mucin gene expression has been shown to be altered in many intestinal diseases and especially cancers of the gastrointestinal tract. Towards understanding the transcriptional regulation of a member of the 11p15.5 human mucin gene cluster, we have characterized 3.55 kb of the 5ʹ-flanking region of the human mucin gene MUC5B, including the promoter, the first two exons and the first intron. We report here the promoter activity of successively 5ʹ-truncated sections of 956 bases of this region by fusing it to the coding region of a luciferase reporter gene. The transcription start site was determined by primer-extension analysis. The region upstream of the transcription start site is characterized by the presence of a TATA box at bases -32/-26, DNA-binding elements for transcription factors c-Myc, N-Myc, Sp1 and nuclear factor ĸB as well as putative activator protein (AP)-1-, cAMP-response-element-binding protein (CREB)-, hepatocyte nuclear factor (HNF)-1-, HNF-3-, TGT3-, gut-enriched Krüppel factor (GKLF)-, thyroid transcription factor (TTF)-1- and glucocorticoid receptor element (GRE)-binding sites. Intron 1 of MUC5B was also characterized, it is 2511 nucleotides long and contains a DNA segment of 259 bp in which are clustered eight tandemly repeated GA boxes and a CACCC box that bind Sp1. AP-2α and GATA-1 nuclear factors were also shown to bind to their respective cognate elements in intron 1. In transfection studies the MUC5B promoter showed a cell-specific activity as it is very active in mucus-secreting LS174T cells, whereas it is inactive in Caco-2 enterocytes and HT-29 STD (standard) undifferentiated cells. Within the promoter, maximal transcription activity was found in a segment covering the first 223 bp upstream of the transcription start site. Finally, in co-transfection experiments a transactivating effect of Sp1 on to MUC5B promoter was seen in LS174T and Caco-2 cells.

scholarly journals Ca2+ administration stimulates the binding of AP-1 factor to the 5′-flanking region of the rat gene for the Ca2+-binding protein regucalcin

1998 ◽  
Vol 329 (1) ◽  
pp. 157-163 ◽  
Author(s):  
Tomiyasu MURATA ◽  
Masayoshi YAMAGUCHI
Keyword(s):  
Promoter Activity ◽  
Binding Protein ◽  
Luciferase Reporter ◽  
Rat Tissues ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Flanking Region ◽  
Gel Mobility Shift ◽  
Dna Complex ◽  
Regucalcin Gene

mRNA of the Ca2+-binding protein, regucalcin, is mainly expressed in the liver and only to a small extent in the kidney, and the expression of hepatic regucalcin mRNA is markedly stimulated by Ca2+ administration [Shimokawa and Yamaguchi (1992) FEBS Lett. 305, 151-154]. The existence of nuclear factors that bind to the 5ʹ-flanking region of the rat regucalcin gene was investigated. When nuclear proteins obtained from various rat tissues were used in gel mobility-shift assays, tissue-specific formation of a protein-DNA complex was found in the liver and kidney. An additional novel protein-DNA complex was formed when liver nuclear extracts obtained from Ca2+-administered rats (10 mg of Ca2+/100 g body weight) were used. Competition gel mobility-shift experiments using consensus and mutant oligonucleotides for AP-1 factor showed that the additional novel complex was formed from binding of the AP-1 factor to the regucalcin gene. Ca2+-induced binding of the AP-1 factor to the regucalcin gene was completely inhibited by simultaneous administration of trifluoperazine, an antagonist of calmodulin, suggesting that the activation of nuclear AP-1 protein is partly mediated through a Ca2+/calmodulin-dependent pathway. Moreover, the 5ʹ-flanking region of the rat regucalcin gene ligated to a luciferase reporter gene possessed the promoter activity in H4-II-E hepatoma cells. This promoter activity was enhanced by treatment with Bay K 8644, a Ca2+-channel agonist. The present study demonstrates that the Ca2+-response sequences are located within the 5ʹ-flanking region of the rat regucalcin gene.

scholarly journals Characterization of the 5′-flanking region of the mouse asparagine-linked glycosylation 12 homolog gene

2013 ◽  
Vol 18 (3) ◽  
Author(s):  
Kentaro Oh-Hashi ◽  
Tomomi Tejima ◽  
Yoko Hirata ◽  
Kazutoshi Kiuchi
Keyword(s):  
Cell Lines ◽  
Promoter Activity ◽  
Bidirectional Promoter ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Consensus Sequences ◽  
Reporter Activity ◽  
Distinct Cell ◽  
Flanking Region ◽  
Responsive Element

AbstractRecently, we characterized multiple roles of the endoplasmic reticulum stress responsive element (ERSE) in the promotion of a unique headto-head gene pair: mammalian asparagine-linked glycosylation 12 homolog (ALG12) and cysteine-rich with EGF-like domains 2 (CRELD2). This bidirectional promoter, which consists of fewer than 400 base pairs, separates the two genes. It has been demonstrated that the ALG12 promoter shows less transcriptional activity through ERSE, but its basic regulatory mechanism has not been characterized. In this study, we focused on well-conserved binding elements for the transcription factors for ATF6, NF-Y and YY1 and the Sp1 and Ets families in the 5’-flanking region of the mouse ALG12 gene. We characterized their dominant roles in regulating ALG12 promoter activities using several deletion and mutation luciferase reporter constructs. The ALG12 gene is expressed in three distinct cell lines: Neuro2a, C6 glioma and HeLa cells. The reporter activity in each cell line decreased similarly with serial deletions of the mouse ALG12 promoter. Mutations in the ERSE and adjacent NF-Y-binding element slightly affected reporter activity. Each of the mutations in the GC-rich sequence and YY1-binding element reduced ALG12 promoter activity, and the combination of these mutations additively decreased reporter activity. Each mutation in the tandem-arranged Ets-family consensus sequences partially attenuated ALG12 promoter activity, and mutations of all three Ets-binding elements decreased promoter activity by approximately 40%. Mutation of the three conserved regulatory elements (GC-rich, YY1 and Ets) in the ALG12 promoter decreased reporter activity by more than 90%. Our results suggest that the promoter activity of the mouse ALG12 gene is regulated in a similar manner in the three cell lines tested in this study. The well-conserved consensus sequences in the promoter of this gene synergistically contribute to maintaining basal gene expression.

Abstract 1384: Osteogenic Transcription Factor Runx2 Regulates Expression of RANKL during VSMC Calcification

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Chang Hyun Byon ◽  
Jay McDonald ◽  
Yabing Chen
Keyword(s):  
Oxidative Stress ◽  
Transcription Factor ◽  
Molecular Mechanism ◽  
Luciferase Reporter ◽  
Rankl Expression ◽  
Reporter System ◽  
Mobility Shift ◽  
Atherosclerotic Lesions ◽  
Flanking Region

The expression of receptor activator of nuclear factor κ B (RANKL) is up-regulated in calcified atherosclerotic lesions, whereas it is frequently undetectable in normal vessels. The underlying molecular mechanism of increased expression of RANKL in calcified vessels is not known. We have previously demonstrated that oxidative stress induces calcification of vascular smooth muscle cells (VSMC) in vitro . Therefore, we determined whether oxidative stress regulates RANKL expression in VSMC and the underlying molecular mechanism. Consistent with previous observations in vivo , we found that the expression of RANKL in VSMC isolated from mouse. However, hydrogen peroxide (H 2 O 2 ), which induces VSMC calcification, induced a 33-fold increase in the transcripts of RANKL as determined by real-time PCR. Increased expression of RANKL protein was further confirmed by ELISA. Using flow cytometry, we demonstrated that membrane-bound RANKL was increased by oxidative stress. To characterize the molecular mechanism underlying H 2 O 2 -induced RANKL expression, we employed the luciferase reporter system with a series of deletion mutants of the RANKL 5′-flanking region. The H 2 O 2 responsive region is located between −200 to −400 in the 5′-flanking region of RANKL gene. Analyses of the sequence of this region identified multiple binding sites for the key osteogenic transcription factor, Runx2, which we previously reported to be an essential regulator of VSMC calcification. Electrophoretic mobility shift analyses demonstrated increased binding of Runx2 on the RANKL promoter sequence in nuclear extracts from VSMC exposed to H 2 O 2 . To further determine the role of Runx2 in regulating RANKL expression, we generated stable Runx2 knockdown VSMC with the use of lentivirus-carrying shRNA for Runx2 gene. H 2 O 2 -induced RANKL expression was abrogated in VSMC with Runx2 knockdown. In addition, adenovirus-mediated overexpression of Runx2 in VSMC induced the expression of RANKL. In summary, we have demonstrated that H 2 O 2 induces the expression of RANKL in VSMC, which is regulated by the osteogenic transcription factor Runx2. These observations provide novel molecular insights into the regulation of RANKL and its role on the pathogenesis of calcified atherosclerotic lesions.

USF-1 and USF-2 trans-repress IL-1β-induced iNOS transcription in mesangial cells

2002 ◽  
Vol 283 (4) ◽  
pp. C1065-C1072 ◽  
Author(s):  
Ashish K. Gupta ◽  
Bruce C. Kone
Keyword(s):  
Dna Binding ◽  
Promoter Activity ◽  
Mesangial Cells ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Inos Gene ◽  
E Box ◽  
Inos Promoter ◽  
Gel Mobility Shift

Transcriptional activation of the inducible nitric oxide synthase (iNOS) gene requires multiple interactions of cis elements and trans-acting factors. Previous in vivo footprinting studies (Goldring CE, Reveneau S, Algarte M, and Jeannin JF. Nucleic Acids Res 24: 1682–1687, 1996) of the murine iNOS gene demonstrated lipopolysaccharide-inducible protection of guanines in the region −904/−883, which includes an E-box motif. In this report, by using site-directed mutagenesis of the −893/−888 E-box and correlating functional assays of the mutated iNOS promoter with upstream stimulatory factor (USF) DNA-binding activities, we demonstrate that the −893/−888 E-box motif is functionally required for iNOS regulation in murine mesangial cells and that USFs are in vivo components of the iNOS transcriptional response complex. Mutation of the E-box sequence augmented the iNOS response to interleukin-1β (IL-1β) in transiently transfected mesangial cells. Gel mobility shift assays demonstrated that USFs cannot bind to the −893/−888 E-box promoter region when the E-box is mutated. Cotransfection of USF-1 and USF-2 expression vectors with iNOS promoter-luciferase reporter constructs suppressed IL-1β-simulated iNOS promoter activity. Cotransfection of dominant-negative USF-2 mutants lacking the DNA binding domain or cis-element decoys containing concatamers of the −904/−883 region augmented IL-1β stimulation of iNOS promoter activity. Gel mobility shift assays showed that only USF-1 and USF-2 supershifted the USF protein-DNA complexes. These results demonstrated that USF binding to the E-box at −893/−888 serves to trans-repress basal expression and IL-1β induction of the iNOS promoter.

scholarly journals Endothelial-specific gene expression directed by the tie gene promoter in vivo

Blood ◽  
1995 ◽  
Vol 86 (5) ◽  
pp. 1828-1835 ◽  
Author(s):  
J Korhonen ◽  
I Lahtinen ◽  
M Halmekyto ◽  
L Alhonen ◽  
J Janne ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transgenic Mice ◽  
Reporter Gene ◽  
Promoter Activity ◽  
Cultured Cells ◽  
Luciferase Reporter ◽  
Specific Gene ◽  
Luciferase Reporter Gene ◽  
Human And Mouse

The tie gene encodes a receptor tyrosine kinase that is expressed in the endothelium of blood vessels, particularly during embryonic development and angiogenesis in adults. We have cloned and characterized the mouse tie gene and isolated the human and mouse tie promoters. The promoter activities of human and mouse tie were analyzed using luciferase reporter gene constructs in transfected cell lines and beta-galactosidase constructs in transgenic mice. In transfection assays of cultured cells, both human and mouse promoter DNA fragments showed activity that was not restricted to endothelial cells. In contrast, in transgenic mice both promoters directed expression of the reporter gene to endothelial cells undergoing vasculogenesis and angiogenesis. In adult mice, tie promoter activity in lung and many vessels of the kidney was as high as in the vessels of the corresponding embryonic tissues, whereas in the heart, brain and liver, tie promoter activity was downregulated and restricted to coronaries, cusps, capillaries, and arteries. Our results show that the endothelial cell-type specificity of the tie promoter in vivo can be transferred to heterologous genes by using relatively short promoter fragments. The tie promoter, thus, has useful properties for potential gene therapy.

scholarly journals A cell proliferation-dependent multiprotein complex NC-3A positively regulates the CD34 promoter via a TCATTT-containing element

Blood ◽  
1996 ◽  
Vol 88 (9) ◽  
pp. 3336-3348 ◽  
Author(s):  
D Perrotti ◽  
T Bellon ◽  
R Trotta ◽  
R Martinez ◽  
B Calabretta
Keyword(s):  
Promoter Activity ◽  
Hematopoietic Cells ◽  
Cell Types ◽  
Multiprotein Complex ◽  
Mobility Shift ◽  
Hematopoietic Stem ◽  
Enhancer Element ◽  
Human Cd34 ◽  
Stem And Progenitor Cells ◽  
Mobility Shift Assay

The CD34 cell surface antigen is a glycoprotein expressed by hematopoietic stem and progenitor cells and also by certain nonhematopoietic cell-types. Because CD34 expression is regulated both at the transcriptional and the posttranscriptional level, we attempted to identify factors that, by interacting with the 5′ flanking region of the human CD34 gene, may regulate its promoter activity in proliferating hematopoietic cells. By electrophoretic mobility shift assay, UV cross-linking and DNase I footprinting analyses, we identified a multiprotein complex, designated NC-3A, that specifically interacts with the CD34 promoter region from nucleotides -375 to -351. Sequence analysis of this region revealed the presence of a distinct motif, TCATTT. Chloramphenicol acetyl-transferase assays used to assess promoter activity in transiently transfected cells showed that this TCATTT-containing element, which is conserved in both the human and the murine CD34 genes, mediates positive regulatory activity in hematopoietic and nonhematopoietic cells, and acts as an enhancer when placed upstream of a heterologous promoter. Moreover, loss of CD34 promoter activity was caused by mutation of the TCATTT motif. In addition, the interaction of the nuclear multiprotein complex NC-3A with this enhancer element is proliferation-dependent. These data indicate that, although not cell-type specific, the formation of a multiprotein complex NC-3A interacting with the region from nucleotides -375 to 351 plays an important role in controlling CD34 promoter activity in proliferating hematopoietic cells.

scholarly journals Expression of small cytoplasmic transcripts of the rat identifier element in vivo and in cultured cells.

1987 ◽  
Vol 7 (6) ◽  
pp. 2148-2154 ◽  
Author(s):  
R D McKinnon ◽  
P Danielson ◽  
M A Brow ◽  
F E Bloom ◽  
J G Sutcliffe
Keyword(s):  
Rat Brain ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Cell Types ◽  
Culture Conditions ◽  
Specific Expression ◽  
Peripheral Tissues ◽  
Primate Cell

We examined the level of expression of small RNA transcripts hybridizing to a rodent repetitive DNA element, the identifier (ID) sequence, in a variety of cell types in vivo and in cultured mammalian cells. A 160-nucleotide (160n) cytoplasmic poly(A)+ RNA (BC1) appeared in late embryonic and early postnatal rat brain development, was enriched in the cerebral cortex, and appeared to be restricted to neural tissue and the anterior pituitary gland. A 110n RNA (BC2) was specifically enriched in brain, especially the postnatal cortex, but was detectable at low levels in peripheral tissues. A third, related 75n poly(A)- RNA (T3) was found in rat brain and at lower levels in peripheral tissues but was very abundant in the testes. The BC RNAs were found in a variety of rat cell lines, and their level of expression was dependent upon cell culture conditions. A rat ID probe detected BC-like RNAs in mouse brain but not liver and detected a 200n RNA in monkey brain but not liver at lower hybridization stringencies. These RNAs were expressed by mouse and primate cell lines. Thus, tissue-specific expression of small ID-sequence-related transcripts is conserved among mammals, but the tight regulation found in vivo is lost by cells in culture.

scholarly journals Gastrin regulates the TFF2 promoter through gastrin-responsive cis-acting elements and multiple signaling pathways

2007 ◽  
Vol 292 (6) ◽  
pp. G1726-G1737 ◽  
Author(s):  
Shuiping Tu ◽  
Alfred L. Chi ◽  
SeonHee Lim ◽  
Guanglin Cui ◽  
Zina Dubeykovskaya ◽  
...  
Keyword(s):  
Dna Binding ◽  
Promoter Activity ◽  
Growth Factor Receptor ◽  
Gastric Cancer Cell Line ◽  
Cancer Cell Line ◽  
Gastric Fundus ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Cis Acting

Trefoil family factor 2 (TFF2) is expressed in gastrointestinal epithelial cells where it serves to maintain mucosal integrity and promote epithelial repair. The peptide hormone, gastrin, stimulates acid secretion but also induces proliferation of the acid-secreting mucosa. Because the relationship between these peptides of overlapping function is not understood, we chose to investigate the regulatory effect of gastrin on TFF2 expression. The expression of mRNA and protein of TFF2 was determined by RT-PCR and immunohistochemical staining, respectively. A series of truncated and mutant murine TFF2 promoter constructs was generated. Promoter activity was assessed using dual luciferase reporter assays. Gastrin-responsive DNA-binding sites in the TFF2 promoter were evaluated by electrophoretic mobility shift assay. Gastrin significantly increased the level of endogenous mRNA of TFF2 in the gastrin receptor-expressing AGS-E gastric cancer cell line in a time- and dose-dependent manner. TFF2 protein expression in the gastric fundus was elevated in hypergastrinemic (INS-GAS) transgenic mice and reduced in gastrin-deficient mice. Gastrin treatment increased TFF2 promoter activity through cis-acting regions, containing CCAATA- and GC-rich enhancers. Pretreatment with Y-F476, a gastrin/CCKB receptor antagonist, abolished gastrin-dependent promoter activity. Inhibitors of protein kinase C (PKC), mitogen/extracellular signal-regulated kinase (MEK1), and phosphatidylinositol 3-kinase (PI 3-kinase) reduced gastrin-dependent TFF2 promoter activity, whereas an epithelial growth factor receptor (EGFR) inhibitor had no effect. We found that gastrin regulates TFF2 transcription through a GC-rich DNA-binding site and a PKC-, MEK1- and PI 3-kinase-dependent but EGFR-independent pathway. Regulation of TFF2 by gastrin may play a role in the maintenance and repair of the gastrointestinal mucosa.

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