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.

PLAG1 and USF2 Regulate Primitive Hematopoietic Expression of Musashi-2

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3583-3583
Author(s):  
Muluken S Belew ◽  
Stefan Rentas ◽  
Laura de Rooij ◽  
Kristin J Hope
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Cell Lines ◽  
Regulatory Elements ◽  
Hematopoietic Cell ◽  
Minimal Promoter ◽  
Luciferase Reporter ◽  
Specific Expression ◽  
Self Renewal ◽  
Model Cell

Abstract The Musashi-2 (MSI2) RNA binding protein is now recognized as a key regulator of hematopoietic stem cells (HSCs). Its expression is most elevated in the primitive HSC compartment and progressively decreases with differentiation. In mouse models of CML, ectopic expression of MSI2 drives progression from the chronic to the blast crisis state while in the human context its aberrantly high expression correlates with more aggressive CML disease states and is associated with poor prognosis in AML. These studies suggest that the precise molecular regulation of MSI2 gene expression may be among the critical mechanisms underlying balanced HSC self-renewal and differentiation and as a result, the prevention of leukemic transformation/progression. Despite the clear importance of understanding how Msi2 maintains an appropriate stem cell-specific expression level, very little is understood of the transcription factors (TFs) that mediate this. To define those factors that govern MSI2 expression and function specifically in the HSC compartment we undertook a systematic approach to map and define relevant regulatory elements of the MSI2 minimal promoter. We dissected a 3.5 kb region 5' upstream of MSI2's translational start site (TSS) shared between mouse and human and thus having the greatest potential of containing regulatory elements key to a conserved MSI2 stem-cell-specific gene expression program. Progressive 5'-terminal deletions of this region cloned upstream of a luciferase reporter gene and transfected into K562 and 293T model cell lines allowed us to define a minimal conserved promoter region from -588 to -203 bp upstream of the TSS that reports accurately on endogenous MSI2 expression. Coupled with in silico prediction of TF that bind this region, systematic TF binding site mutagenesis and luciferase reporter assays in model cell lines identified USF2 and PLAG1 as TFs whose direct binding to the MSI2 minimal promoter direct reporter activity. Loss and gain of function studies in K562 cells confirm that these factors co-regulate the transactivation of endogenous MSI2. Moreover we show in the most relevant primary human CD34+ hematopoietic cell context that these factors bind the MSI2 minimal promoter. While USF2 is a ubiquitously expressed TF across the hematopoietic hierarchy, the uniquely restricted expression of PLAG1 within only the most primitive of hematopoietic cells suggests that it specifically contributes to the heightened stem cell-specific expression of MSI2. Consistent with its role as a key driver of MSI2 and thus an enforcer of its pro-self-renewal functions, we found that overexpression of PLAG1 in human Lin-CD34+ cord blood cells enhanced MSI2 transcription and increased total Colony Forming Unit (CFU) output and re-plating efficiency of primitive CFU progenitors. PLAG1 overexpression also offered a pro-survival advantage to these cells as evidenced by a more than two-fold reduction in Annexin V positive cells compared to negative controls. We have thus described important transcriptional circuitry that governs stem-cell specific expression of MSI2 while at the same time functionally validated PLAG1 as a novel factor capable of modulating primitive hematopoietic cell self-renewal and survival. Disclosures No relevant conflicts of interest to declare.

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.

Molecular mechanisms of epithelial cell-specific expression and regulation of the human anion exchanger (pendrin) gene

2008 ◽  
Vol 294 (5) ◽  
pp. C1261-C1276 ◽  
Author(s):  
Lior Adler ◽  
Edna Efrati ◽  
Israel Zelikovic
Keyword(s):  
Epithelial Cell ◽  
Inner Ear ◽  
Cell Lines ◽  
Anion Exchanger ◽  
Promoter Activity ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Acidic Ph ◽  
Alkaline Ph ◽  
Specific Expression

Pendrin, a Cl−/anion exchanger encoded by the gene PDS, is highly expressed in the kidney, thyroid, and inner ear epithelia and is essential for bicarbonate secretion, iodide accumulation, and endolymph ion balance, respectively. This study aimed to define promoter regulatory elements essential for renal, thyroid, and inner ear epithelial cell-specific expression of human PDS (hPDS) and to explore the effect of ambient pH and aldosterone on hPDS promoter activity. Endogenous pendrin mRNA and protein were detected in renal HEK293, thyroid LA2, and inner ear VOT36 epithelial cell lines, but not in the fibroblast cell line, NIH3T3. A 4.2-kb hPDS 5′-flanking DNA sequence and consecutive 5′-deletion products were cloned into luciferase reporter vectors and transiently transfected into the above cell lines. Distinct differences in expression/activity of deduced positive/negative regulatory elements within the hPDS promoter between HEK293, LA2, and VOT36 cells were demonstrated, with only basal activity in NIH3T3 cells. Acidic pH (7.0–7.1) decreased and alkaline pH (7.6–7.7) increased hPDS promoter activity in transfected HEK293 and VOT36, but not in LA2 cells. Aldosterone (10−8 M) reduced hPDS promoter activity in HEK293 but had no effect in LA2 and VOT36 cells. These pH and aldosterone-induced effects on the hPDS promoter occurred within 96-bp and 89-bp regions, respectively, which likely contain distinct response elements to these modulators. Acidic pH and aldosterone decreased, and alkaline pH increased, endogenous pendrin mRNA level in HEK293 cells. In conclusion, pendrin-mediated HCO3− secretion in the renal tubule and anion transport in the endolymph may be regulated transcriptionally by systemic pH and aldosterone.

Structure of the 5′ region of the Hst70 gene transcription unit: presence of an intron and multiple transcription initiation sites

2001 ◽  
Vol 359 (1) ◽  
pp. 129-137 ◽  
Author(s):  
Dorota ŚCIEGLIŃSKA ◽  
Wiesława WID̄ŁAK ◽  
Witold KONOPKA ◽  
Matti POUTANEN ◽  
Nafis RAHMAN ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Transcription Unit ◽  
Northern Blotting ◽  
Start Codon ◽  
Specific Expression ◽  
Rnase Protection ◽  
Exon 1 ◽  
Testis Specific Expression

The rat Hst70 gene and its mouse counterpart Hsp70.2 belong to the family of Hsp70 heat shock genes and are specifically expressed in male germ cells. Previous studies regarding the structure of the 5′ region of the transcription unit of these genes as well as localization of the ‘cis’ elements conferring their testis-specific expression gave contradictory results [Widłak, Markkula, Krawczyk, Kananen and Huhtaniemi (1995) Biochim. Biophys. Acta 1264, 191–200; Dix, Rosario-Herrle, Gotoh, Mori, Goulding, Barret and Eddy (1996) Dev. Biol. 174, 310–321]. In the present paper we solve these controversies and show that the 5′ untranslated region (UTR) of the Hst70 gene contains an intron which is localized similar to that of the mouse Hsp70.2 gene. Reverse transcriptase-mediated PCR, Northern blotting and RNase protection analysis revealed that the transcription initiation of both genes starts at two main distant sites, and one of them is localized within the intron. As a result two populations of Hst70 gene transcripts with similar sizes but different 5′ UTR structures can be detected in total testicular RNA. Functional analysis of the Hst70 gene promoter in transgenic mice and transient transfection assays proved that the DNA fragment of approx. 360bp localized upstream of the ATG transcription start codon is the minimal promoter required for testis-specific expression of the HST70/chloramphenicol acetyltransferase transgene. These experiments also suggest that the expression of the gene may depend on ‘cis’ regulatory elements localized within exon 1 and the intron sequences.

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.

Identification of upstream and intragenic regulatory elements that confer cell-type-restricted and differentiation-specific expression on the muscle creatine kinase gene

1988 ◽  
Vol 8 (7) ◽  
pp. 2896-2909 ◽  
Author(s):  
E A Sternberg ◽  
G Spizz ◽  
W M Perry ◽  
D Vizard ◽  
T Weil ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Regulatory Element ◽  
Simian Virus 40 ◽  
Muscle Cells ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Simian Virus ◽  
Cell Type ◽  
Specific Expression ◽  
Developing Muscle

Terminal differentiation of skeletal myoblasts is accompanied by induction of a series of tissue-specific gene products, which includes the muscle isoenzyme of creatine kinase (MCK). To begin to define the sequences and signals involved in MCK regulation in developing muscle cells, the mouse MCK gene has been isolated. Sequence analysis of 4,147 bases of DNA surrounding the transcription initiation site revealed several interesting structural features, some of which are common to other muscle-specific genes and to cellular and viral enhancers. To test for sequences required for regulated expression, a region upstream of the MCK gene from -4800 to +1 base pairs, relative to the transcription initiation site, was linked to the coding sequences of the bacterial chloramphenicol acetyltransferase (CAT) gene. Introduction of this MCK-CAT fusion gene into C2 muscle cells resulted in high-level expression of CAT activity in differentiated myotubes and no detectable expression in proliferating undifferentiated myoblasts or in nonmyogenic cell lines. Deletion mutagenesis of sequences between -4800 and the transcription start site showed that the region between -1351 and -1050 was sufficient to confer cell type-specific and developmentally regulated expression on the MCK promoter. This upstream regulatory element functioned independently of position, orientation, or distance from the promoter and therefore exhibited the properties of a classical enhancer. This upstream enhancer also was able to confer muscle-specific regulation on the simian virus 40 promoter, although it exhibited a 3- to 5-fold preference for its own promoter. In contrast to the cell type- and differentiation-specific expression of the upstream enhancer, the MCK promoter was able to function in myoblasts and myotubes and in nonmyogenic cell lines when combined with the simian virus 40 enhancer. An additional positive regulatory element was identified within the first intron of the MCK gene. Like the upstream enhancer, this intragenic element functioned independently of position, orientation, and distance with respect to the MCK promoter and was active in differentiated myotubes but not in myoblasts. These results demonstrate that expression of the MCK gene in developing muscle cells is controlled by complex interactions among multiple upstream and intragenic regulatory elements that are functional only in the appropriate cellular context.

scholarly journals Characterization of two atypical promoters and alternate mRNA processing in the mouse Thy-1.2 glycoprotein gene.

1986 ◽  
Vol 6 (8) ◽  
pp. 2923-2931 ◽  
Author(s):  
H A Ingraham ◽  
G A Evans
Keyword(s):  
Genomic Sequence ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Lymphoid Cells ◽  
Gene Encoding ◽  
Specific Expression ◽  
Transcriptional Initiation ◽  
Glycoprotein Gene ◽  
Cell Type Specific Expression ◽  
Flanking Region

The promoter and 5' flanking region of the mouse Thy-1.2 glycoprotein gene were characterized by DNA sequencing, primer extension analysis, and deletion analysis. Transcriptional initiation sites were identified which corresponded to two separate exons upstream of the portion of the gene encoding the Thy-1.2 glycoprotein. We demonstrated that the mouse Thy-1.2 gene was transcribed from two atypical promoters separated by 260 base pairs in the genomic sequence. These promoters contained neither TATAAG nor GGPyCCAATCT homologous sequences but defined a conserved nonamer CTCCCTGCT at -48 from each initiation site. Two Thy-1.2 mRNA species of 1,835 and 1,939 nucleotides, differing in the 5' untranslated region of the mRNA, were thus transcribed from the single Thy-1.2 gene by mRNA splicing to the same downstream exon. Recombinant genomes in which the bacterial chloramphenicol acetyltransferase gene was expressed from either of the two Thy-1.2 promoters demonstrated that each promoter functioned independently and did not direct cell-specific expression in lymphoid cells. The 5' flanking region of the Thy-1.2 gene upstream of -68 could be eliminated without altering cell-type-specific expression. This suggests that regulatory elements responsible for tissue and developmental stage-specific expression of the Thy-1.2 gene are not present in the 5' flanking DNA but may reside downstream of the promoters.

scholarly journals Regulatory elements of the erythropoietin gene

Blood ◽  
1991 ◽  
Vol 77 (2) ◽  
pp. 278-285 ◽  
Author(s):  
S Imagawa ◽  
MA Goldberg ◽  
J Doweiko ◽  
HF Bunn
Keyword(s):  
Transfection Efficiency ◽  
Hepatoma Cell ◽  
Internal Standard ◽  
Regulatory Elements ◽  
Site Directed Mutagenesis ◽  
Start Codon ◽  
Hepatoma Cell Line ◽  
First Intron ◽  
Initiation Of Translation ◽  
Flanking Region

Abstract Because the human hepatoma cell line Hep3B produces erythropoietin (Epo) in a regulated fashion, it can be used to investigate the cis- acting regulatory elements of the Epo gene. Comparison of primate and mouse sequences shows strong homology not only in the coding sequence but also within the 5′ flanking region, the first intron, and the 3′ flanking region. These portions of the Epo gene were inserted 5′ and 3′ to a reporter gene, human growth hormone (GH). 5A is a 1,192-base pair (bp) HindIII-Xbal fragment that extends from 378 bp 5′ to the cap site through the first intron. To obviate the problem of false initiation of translation from the Epo ATG start codon, this site was changed to TAG by site-directed mutagenesis. 3A is a 255-bp Accl-BglII fragment that extends 67 bp upstream from the Epo termination codon and covers most of the 3′ noncoding region of homology. The plasmid DNAs were transfected by electroporation into Hep3B cells with RSVCAT as an internal standard to correct for transfection efficiency. One aliquot of cells was exposed to 50 mumol/L CoCl2 or to 1% O2. At the end of the incubations, GH and Epo were measured in the cell media and the cell pellet was assayed for CAT. Production of GH was stimulated 1.7-fold by cobalt or hypoxia. Furthermore, addition of 3A to the GH gene, irrespective of orientation, stimulated GH production 2.6-fold with CoCl2 and 2.3-fold with hypoxia. Stable cell lines were produced by cotransfection of the above constructions, along with the selectable marker pSV-Neo. In two clones, exposure to hypoxia resulted in much more marked (16-fold) induction of GH. Stimulus of both GH and Epo production by hypoxia was partially abrogated by carbon monoxide. These results demonstrate the presence of promoter and enhancer elements within the human Epo gene that are appropriately responsive to hypoxia and cobalt.

Isolation and characterization of the human CLC-5 chloride channel gene promoter

Gene ◽  
2000 ◽  
Vol 261 (2) ◽  
pp. 355-364 ◽  
Author(s):  
Atsushi Hayama ◽  
Shinichi Uchida ◽  
Sei Sasaki ◽  
Fumiaki Marumo
Keyword(s):  
Chloride Channel ◽  
Gene Promoter ◽  
Channel Gene ◽  
Isolation And Characterization

scholarly journals Site-specific methylation of the rat prolactin and growth hormone promoters correlates with gene expression.

1996 ◽  
Vol 16 (7) ◽  
pp. 3245-3254 ◽  
Author(s):  
V Ngô ◽  
D Gourdji ◽  
J N Laverrière
Keyword(s):  
Gene Expression ◽  
Growth Hormone ◽  
Cell Lines ◽  
Anterior Pituitary ◽  
Regulatory Elements ◽  
Specific Expression ◽  
Site Specific ◽  
Cpg Dinucleotides ◽  
Cpg Sites

The methylation patterns of the rat prolactin (rPRL) (positions -440 to -20) and growth hormone (rGH) (positions -360 to -110) promoters were analyzed by bisulfite genomic sequencing. Two normal tissues, the anterior pituitary and the liver, and three rat pituitary GH3 cell lines that differ considerably in their abilities to express both genes were tested. High levels of rPRL gene expression were correlated with hypomethylation of the CpG dinucleotides located at positions -277 and -97, near or within positive cis-acting regulatory elements. For the nine CpG sites analyzed in the rGH promoter, an overall hypomethylation-expression coupling was also observed for the anterior pituitary, the liver, and two of the cell lines. The effect of DNA methylation was tested by measuring the transient expression of the chloramphenicol acetyltransferase reporter gene driven by a regionally methylated rPRL promoter. CpG methylation resulted in a decrease in the activity of the rPRL promoter which was proportional to the number of modified CpG sites. The extent of the inhibition was also found to be dependent on the position of methylated sites. Taken together, these data suggest that site-specific methylation may modulate the action of transcription factors that dictate the tissue-specific expression of the rPRL and rGH genes in vivo.

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