Transcriptional regulation of the KEL gene and Kell protein expression in erythroid and non-erythroid cells

2001 ◽  
Vol 356 (1) ◽  
pp. 171-180 ◽  
Author(s):  
Valérie CAMARA-CLAYETTE ◽  
Cécile RAHUEL ◽  
Claude LOPEZ ◽  
Claude HATTAB ◽  
Virginie VERKARRE ◽  
...  
Keyword(s):  
Hela Cells ◽  
Blot Analysis ◽  
Promoter Activity ◽  
K562 Cells ◽  
Site Directed Mutagenesis ◽  
Transcriptional Analysis ◽  
Lymphoid Tissues ◽  
Dot Blot ◽  
Mobility Shift ◽  
Exon 1

The Kell blood-group antigen was originally reported to be a protein expressed in erythroid tissue only. Transcriptional analysis of the KEL promoter activity in human erythroleukaemia K562 and epithelial HeLa cells by electrophoretic mobility-shift and supershift assays, chloramphenicol acetyltransferase assays, co-transfection studies and site-directed mutagenesis provided the following results: (i) the KEL promoter exhibits a strong transcriptional activity in K562 cells and, unexpectedly, a basal non-erythroid activity in HeLa cells, (ii) up-regulation of the 5′ distal promoter activity occurs only in the erythroid context, and (iii) two motifs localized in the exon 1 region, which bind the Sp1/Sp3 and the human GATA-1/Ku70/80 factors, were required for down-regulation of the promoter activity, but inhibition of the promoter activity by the repressing factors in HeLa cells was incomplete. KEL expression in HeLa cells was performed further by primer-extension analysis, which revealed the presence of a low amount of Kell transcript correlating with basal expression of the Kell protein in these cells, as shown by immunopurification and Western-blot analysis. DNA sequencing of the transcript revealed a sequence identical to that obtained from erythroid tissue. In human tissues, KEL expression was investigated by dot-blot analysis and revealed high levels of Kell mRNAs, particularly in brain tissues, testis and lymphoid tissues. Moreover, most tissues analysed exhibited low levels of Kell transcripts. The Kell protein was also detected by immunohistochemistry in the Sertoli cells of the testis and in lymphoid tissues like spleen and tonsil, specifically localized in the follicular dendritic cells. Altogether, the results indicated that KEL expression is not restricted to erythroid tissue.

scholarly journals Cloning and characterization of the human beta2-glycoprotein I (beta2-GPI) gene promoter: roles of the atypical TATA box and hepatic nuclear factor-1alpha in regulating beta2-GPI promoter activity

2004 ◽  
Vol 380 (2) ◽  
pp. 455-463 ◽  
Author(s):  
Hsueh-Hsiao WANG ◽  
An-Na CHIANG
Keyword(s):  
Gene Expression ◽  
Hela Cells ◽  
Promoter Activity ◽  
Hepatoma Cell ◽  
Site Directed Mutagenesis ◽  
Tata Box ◽  
Proximal Promoter ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
Glycoprotein I

β2-Glycoprotein I (β2-GPI) is a plasma glycoprotein primarily synthesized in the liver. The interindividual variability of β2-GPI expression in subjects with various metabolic syndromes and disease states suggests that it may have clinical importance. However, the regulation of β2-GPI gene expression has not been clarified. To gain more insight into the control of β2-GPI gene expression, we cloned the 4.1-kb 5´-flanking region and characterized the proximal promoter of the β2-GPI gene in this study. Cis-acting elements required for β2-GPI promoter activity were identified with transient transfection assays in the hepatoma cell lines HepG2 and Huh7 and in non-hepatic HeLa cells. Serial deletion analyses of the β2-GPI 5´-flanking sequence revealed that the region from −197 to +7 had strong promoter activity in hepatoma cells but not in HeLa cells. Truncation and site-directed mutagenesis of putative cis-elements within this region showing an atypical TATA box and a HNF-1 (hepatic nuclear factor-1) element were both essential for the β2-GPI promoter activity. Subsequent gel mobility shift assays confirmed the interaction of HNF-1α with the HNF-1 site residing downstream of the TATA box. Co-transfection of β2-GPI promoter-luciferase vector with HNF-1α expression vector in Huh7 and HNF-1-deficient HeLa cells demonstrated the transactivation effect of HNF-1α on β2-GPI promoter activity. In addition, overexpression of HNF-1α enhanced the endogenous β2-GPI expression. These results suggest that the atypical TATA box and HNF-1 cis-element are critical for β2-GPI transcription and HNF-1α may play an important role in cell-specific regulation of β2-GPI gene expression.

GATA-1 Binding to Exon 1 Directs High Level Erythroid-Specific Expression of the Human Protoporphyrinogen Oxidase Gene.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1682-1682
Author(s):  
Karen M.K. de Vooght ◽  
Richard van Wijk ◽  
Wouter W. van Solinge
Keyword(s):  
Transcriptional Regulation ◽  
Promoter Activity ◽  
K562 Cells ◽  
Promoter Strength ◽  
Specific Expression ◽  
Oxidase Gene ◽  
Protoporphyrinogen Oxidase ◽  
Exon 1 ◽  
High Level

Abstract Protoporphyrinogen oxidase (PPOX), the penultimate enzyme in the heme biosynthetic pathway, catalyzes the six-electron oxidation of protoporphyrinogen IX to protoporphyrin IX. Like other heme biosynthetic proteins, PPOX is involved in synthesizing heme for red cells (erythroid-specific expression) and as a cofactor for the respiratory cytochromes (housekeeping expression). To date, little is known about transcriptional regulation of the human PPOX gene (PPOX). We established the molecular basis for erythroid-specific expression of PPOX. Using transient in vitro transfection assays in human erythroleukemic K562 cells we studied tissue-specific expression of PPOX. We found that reporter constructs lacking exon 1 showed a 75% reduction in promoter strength in K562 cells (Figure, no 1 and 2). Hence, in vitro high-level erythroid-specific expression of PPOX is dependent on the presence of exon 1. Examination of erythroid-specific regulatory elements in exon 1 revealed two GATA-1 sites, one consensus (A/T)GATA(A/G) site (GATA-1_II AGATAA) and one non-consensus site, deviating at the first nucleotide (GATA-1_I, CGATAG). To study the relative contribution of these two GATA-1 sites to erythroid-specific transcriptional regulation, we performed in vitro transfections of wild-type and mutant (GATA → GTTA) reporter plasmids in K562 cells. We found that the highest level of transcription depended on the integrity of both sites (Figure, no 5). The consensus GATA-1_II site contributed the most to promoter strength (Figure, no 4). Subsequent electrophoretic mobility shift assay and supershift experiments using K562 nuclear extracts demonstrated that both GATA sites were able to bind GATA-1 in vitro. Our experiments showed that exon 1 was dispensable for PPOX promoter activity in human hepatoma HepG2 cells. Interestingly, in HeLa human cervical carcinoma cells the presence of exon 1 decreased promoter activity. Conclusively, exon 1 of the human PPOX gene contains two GATA-1 binding sites, which are required for high level erythroid-specific expression of PPOX and, in addition, bind GATA-1 in vitro. Our results contribute to a better understanding of the molecular mechanisms involved in differential regulation of the human PPOX promoter in erythroid and non-erythroid cells. Figure Figure

scholarly journals Sp3 regulates Fas expression in lung epithelial cells

1998 ◽  
Vol 333 (1) ◽  
pp. 209-213 ◽  
Author(s):  
Huiling PANG ◽  
Kathleen MIRANDA ◽  
Alan FINE
Keyword(s):  
Epithelial Cells ◽  
Promoter Activity ◽  
Functional Results ◽  
Lung Epithelial Cells ◽  
Site Directed Mutagenesis ◽  
Mouse Lung ◽  
Epithelial Cell Line ◽  
Upstream Sequence ◽  
Mobility Shift ◽  
Lung Epithelial

By transducing an apoptotic signal in immune effector cells, Fas has been directly implicated in the control of immunological activity. Expression and functional results, however, have also suggested a role for Fas in regulating cell turnover in specific epithelial populations. To characterize factors responsible for Fas expression in epithelial cells, approximately 3 kb of the 5´ flanking region of the mouse Fas gene was isolated. By rapid amplification of cDNA ends and primer extension, transcriptional start sites were identified within 50 bp upstream of the translation start site. Transient transfection of promoter–luciferase constructs in a mouse lung epithelial cell line, MLE-15, localized promoter activity to the first 77 bp of upstream sequence. By using a 60 bp DNA probe (-18 to -77) in electrophoretic mobility-shift assays, three shifted complexes were found. Incubation with excess cold Sp1 oligonucleotide or an anti-Sp3 antibody inhibited complex formation. Site-directed mutagenesis of the Sp1 site resulted in 60–70% loss of promoter activity. In Drosophila SL-2 cells, promoter activity was markedly increased by co-transfection of an Sp3 expression construct. These results show that the Sp3 protein is involved in regulating Fas gene expression in lung epithelial cells.

scholarly journals Identification of a major positive regulatory element located 5' to the human zeta-globin gene

Blood ◽  
1995 ◽  
Vol 85 (9) ◽  
pp. 2587-2597 ◽  
Author(s):  
DE Sabath ◽  
KM Koehler ◽  
WQ Yang ◽  
K Patton ◽  
G Stamatoyannopoulos
Keyword(s):  
Point Mutation ◽  
Promoter Activity ◽  
Regulatory Element ◽  
Globin Gene ◽  
K562 Cells ◽  
Luciferase Expression ◽  
Globin Mrna ◽  
Mobility Shift ◽  
Alpha Globin ◽  
Globin Promoter

The function of the zeta-globin promoter was studied using a series of zeta-globin promoter deletion constructs to drive luciferase expression in transiently transfected human erythroleukemia cells. The promoters were used without enhancers, or with enhancers derived from the beta-globin locus control region and the alpha-globin HS-40 enhancer. When transfected into K562 cells, which express zeta-globin, comparable amounts of activity were obtained from the -557 and -417 zeta-luciferase constructs and the alpha-luciferase constructs when no enhancers or the alpha-globin locus enhancers were used. When the constructs were transfected into OCIM1 cells, which do not express zeta-globin, the zeta-globin promoters were at best 20% as active as the alpha-globin promoters. When sequences from -417 to -207 5′ to the zeta-globin mRNA cap site were deleted, up to 95% of the zeta-globin promoter activity was lost in K562 cells. Reinsertion of these sequences into zeta-luciferase constructs missing the -417 to -207 region showed that the sequences lack classical enhancer activity. Point mutation of a GATA-1 site at -230 reduced promoter activity by 37%. Point mutation of a CCACC site at -240 had no effect. Electrophoretic mobility shift assays indicated that the -230 GATA-1 site has a relatively low affinity for GATA-1. These experiments show the presence of a strong positive-acting element, located between -417 and -207 bp 5′ to the zeta-globin mRNA cap site, is necessary for high-level promoter activity in K562 cells. This element requires GATA-1 and additional unknown factors for maximal activity.

scholarly journals Expression of wnt4/5 during reproductive cycle of catfish and wnt5 promoter analysis

2017 ◽  
Vol 232 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Yarikipati Prathibha ◽  
Balasubramanian Senthilkumaran
Keyword(s):  
Reproductive Cycle ◽  
Promoter Activity ◽  
Ovarian Development ◽  
Developmental Stages ◽  
Protein Localization ◽  
Site Directed Mutagenesis ◽  
Cloning And Expression ◽  
Mobility Shift ◽  
Mammalian Cell Lines

Signaling molecules, Wnt4 and Wnt5, are essential for ovarian growth during developmental stages in mammals. Although these molecules were identified in several teleosts, their precise expression and role in reproductive processes have not yet been explored in any lower vertebrates. In view of this, using catfish, Clarias batrachus as an animal model, cloning and expression analysis of wnt4 and wnt5 were analyzed in different tissues, at various developmental stages, during ovarian reproductive cycle and after gonadotropin induction. These studies indicate a plausible influence of Wnts in ovarian development and recrudescence. Transcript and protein localization revealed their presence in peri-nucleolar, pre-vitellogenic, vitellogenic and follicular layer of post-vitellogenic oocytes. Synchronous expression of pax2 and wnt5 during the ovarian development and recrudescence of catfish led us to analyze the importance of putative binding element of Pax2 in the 5′-promoter motif of wnt5. Promoter activity of wnt5 was analyzed by luciferase assays after transfecting progressive deletion constructs in pGL3 basic vector into the mammalian cell lines (HEK 293 and CHO). The constructs having putative Pax2 motif showed high promoter activity compared with controls. Likewise, the constructs with site-directed mutagenesis showed increased activity after supplementing recombinant Pax2 indicating the prominence of this motif in wnt5 promoter, in vitro. Electrophoretic gel mobility shift, supershift and chromatin immunoprecipitation assays confirmed the binding of Pax2 to its corresponding cis-acting element in the upstream of wnt5. This study is the first of its kind to report the critical transcriptional interaction of Pax2 on wnt5 vis-à-vis ovarian development in teleosts.

scholarly journals Purification, reconstitution, and I kappa B association of the c-Rel-p65 (RelA) complex, a strong activator of transcription.

1994 ◽  
Vol 14 (4) ◽  
pp. 2593-2603 ◽  
Author(s):  
S K Hansen ◽  
P A Baeuerle ◽  
F Blasi
Keyword(s):  
Dna Binding ◽  
Hela Cells ◽  
Hepg2 Cells ◽  
Nuclear Translocation ◽  
K562 Cells ◽  
Binding Activity ◽  
In Vitro Translation ◽  
Nf Kappa B ◽  
Mobility Shift ◽  
Dna Binding Activity

HeLa cells contain a DNA-binding activity which associates with a kappa B-like DNA element, termed Rel-related protein-binding element (RRBE), localized upstream of the human urokinase promoter. We have purified this activity from the HeLa cell cytosol and have shown that it represents a performed heteromeric complex between p65 (RelA) and c-Rel. Coexpression of c-Rel and p65 (RelA) by in vitro translation formed a DNA-binding complex indistinguishable from purified cellular c-Rel-p65 (RelA) in mobility shift assays. The c-Rel-p65 (RelA) complex was also formed in COS7 cells upon coexpression of c-Rel and p65 (RelA) cDNAs. Cotransfection experiments with COS7 cells, using expression plasmids encoding p50, p65 (RelA), or c-Rel and reporter constructs containing a trimerized RRBE, revealed that c-Rel-p65 (RelA) is a potent activator of the RRBE, giving rise to transcriptional activity higher than that observed with NF-kappa B (p50-p65). In the cytosol, the c-Rel-p65 (RelA) complex existed in a latent, non-DNA-binding form but could be activated by detergent treatment, suggesting that it was associated with an I kappa B protein. Recombinant I kappa B-alpha inhibited the DNA-binding activity of c-Rel-p65 (RelA) via association with either c-Rel or p65 (RelA). Finally, NF-kappa B and c-Rel-p65 (RelA) complexes were found to be differentially expressed and regulated in different cells. The two complexes were present in equimolar amounts in HeLa cells and K562 cells. Stimulation with tetradecanoyl phorbol acetate (TPA) resulted in the nuclear translocation of both NF-kappa B and c-Rel-p65 (RelA) in HeLa cells and of NF-kappa B in HepG2 cells but had no effect on either complex in K562 cells. In addition, TPA stimulation of HepG2 cells induced the expression of a cytosolic latent c-Rel-p65 (RelA) complex which, however, was not translocated to the nucleus. In conclusion, our findings show that c-Rel-p65 (RelA) is an inducible and very potent transcriptional activator which is differentially activated in a cell-type-specific manner.

scholarly journals Different Sp1 family members differentially affect transcription from the human elongation factor 1 A-1 gene promoter

1998 ◽  
Vol 333 (3) ◽  
pp. 511-517 ◽  
Author(s):  
S⊘ren J. NIELSEN ◽  
Morten PRÆSTEGAARD ◽  
Helle F. J. ØRGENSEN ◽  
Brian F. C. CLARK
Keyword(s):  
Hela Cells ◽  
Promoter Activity ◽  
Binding Proteins ◽  
Regulatory Element ◽  
Elongation Factor ◽  
Proximal Promoter ◽  
Mobility Shift ◽  
Gc Box ◽  
Elongation Factor 1 ◽  
Human Elongation Factor

The GC box is an important transcriptional regulatory element present in the promoters of many mammalian genes. In the present study we examine the effect of known GC-box-binding proteins on the promoter of the human elongation factor 1 A-1 (hEF1A-1) gene in human HeLa cells and Drosophila SL2 cells. In HeLa cells co-transfection with the GC-box-binding protein BTEB resulted in a 4–10-fold increase in hEF1A-1 promoter activity. This stimulation was dependent on a single GC box located between positions -69 and -50 of the promoter. Little or no effect was observed of other GC-box-binding proteins including Sp1, Sp3, Sp4 and BTEB2. In SL2 cells stimulation by Sp1 and Sp3 through the single GC box of the proximal promoter led to 13-fold and 21-fold increases respectively in promoter activity. Inclusion of further upstream sequences resulted in high levels of expression when Sp1 or Sp3 was co-transfected with the reporter plasmid. In this setting Sp1 stimulated transcription by 750-fold, whereas Sp3 was even more potent, yielding a 1150-fold stimulation. Mobility-shift assays performed with the promoter-proximal GC box demonstrated the binding of Sp1, Sp3 and Sp4 to this sequence. To our knowledge, the present study represents the first comparison of all known GC-box-binding proteins on a natural promoter.

Purification, reconstitution, and I kappa B association of the c-Rel-p65 (RelA) complex, a strong activator of transcription

1994 ◽  
Vol 14 (4) ◽  
pp. 2593-2603
Author(s):  
S K Hansen ◽  
P A Baeuerle ◽  
F Blasi
Keyword(s):  
Dna Binding ◽  
Hela Cells ◽  
Hepg2 Cells ◽  
Nuclear Translocation ◽  
K562 Cells ◽  
Binding Activity ◽  
In Vitro Translation ◽  
Nf Kappa B ◽  
Mobility Shift ◽  
Dna Binding Activity

HeLa cells contain a DNA-binding activity which associates with a kappa B-like DNA element, termed Rel-related protein-binding element (RRBE), localized upstream of the human urokinase promoter. We have purified this activity from the HeLa cell cytosol and have shown that it represents a performed heteromeric complex between p65 (RelA) and c-Rel. Coexpression of c-Rel and p65 (RelA) by in vitro translation formed a DNA-binding complex indistinguishable from purified cellular c-Rel-p65 (RelA) in mobility shift assays. The c-Rel-p65 (RelA) complex was also formed in COS7 cells upon coexpression of c-Rel and p65 (RelA) cDNAs. Cotransfection experiments with COS7 cells, using expression plasmids encoding p50, p65 (RelA), or c-Rel and reporter constructs containing a trimerized RRBE, revealed that c-Rel-p65 (RelA) is a potent activator of the RRBE, giving rise to transcriptional activity higher than that observed with NF-kappa B (p50-p65). In the cytosol, the c-Rel-p65 (RelA) complex existed in a latent, non-DNA-binding form but could be activated by detergent treatment, suggesting that it was associated with an I kappa B protein. Recombinant I kappa B-alpha inhibited the DNA-binding activity of c-Rel-p65 (RelA) via association with either c-Rel or p65 (RelA). Finally, NF-kappa B and c-Rel-p65 (RelA) complexes were found to be differentially expressed and regulated in different cells. The two complexes were present in equimolar amounts in HeLa cells and K562 cells. Stimulation with tetradecanoyl phorbol acetate (TPA) resulted in the nuclear translocation of both NF-kappa B and c-Rel-p65 (RelA) in HeLa cells and of NF-kappa B in HepG2 cells but had no effect on either complex in K562 cells. In addition, TPA stimulation of HepG2 cells induced the expression of a cytosolic latent c-Rel-p65 (RelA) complex which, however, was not translocated to the nucleus. In conclusion, our findings show that c-Rel-p65 (RelA) is an inducible and very potent transcriptional activator which is differentially activated in a cell-type-specific manner.

5-Aminolaevulinate synthase gene promoter contains two cAMP-response element (CRE)-like sites that confer positive and negative responsiveness to CRE-binding protein (CREB)

2001 ◽  
Vol 353 (2) ◽  
pp. 307-316 ◽  
Author(s):  
Luciana E. GIONO ◽  
Cecilia L. VARONE ◽  
Eduardo T. CÁNEPA
Keyword(s):  
Gene Expression ◽  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Binding Protein ◽  
Phosphorylation Site ◽  
Gene Promoter ◽  
Site Directed Mutagenesis ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Putative Transcription Factor

The first and rate-controlling step of the haem biosynthetic pathway in mammals and fungi is catalysed by the mitochondrial-matrix enzyme 5-aminolaevulinate synthase (ALAS). The purpose of this work was to explore the molecular mechanisms involved in the cAMP regulation of rat housekeeping ALAS gene expression. Thus we have examined the ALAS promoter for putative transcription-factor-binding sites that may regulate transcription in a cAMP-dependent protein kinase (PKA)-induced context. Applying both transient transfection assays with a chloramphenicol acetyltransferase reporter gene driven by progressive ALAS promoter deletions in HepG2, and electrophoresis mobility-shift assays we have identified two putative cAMP-response elements (CREs) at positions -38 and -142. Functional analysis showed that both CRE-like sites were necessary for complete PKA induction, but only one for basal expression. Co-transfection with a CRE-binding protein (CREB) expression vector increased PKA-mediated induction of ALAS promoter transcriptional activity. However, in the absence of co-transfected PKA, CREB worked as a specific repressor for ALAS promoter activity. A CREB mutant deficient in a PKA phosphorylation site was unable to induce expression of the ALAS gene but could inhibit non-stimulated promoter activity. Furthermore, a DNA-binding mutant of CREB did not interfere with ALAS promoter basal activity. Site-directed-mutagenesis studies showed that only the nearest element to the transcription start site was able to inhibit the activity of the promoter. Therefore, we conclude that CREB, through its binding to CRE-like sites, mediates the effect of cAMP on ALAS gene expression. Moreover, we propose that CREB could also act as a repressor of ALAS transcription, but is able to reverse its role after PKA activation. Dephosphorylated CREB would interfere in a spatial-disposition-dependent manner with the transcriptional machinery driving inhibition of gene expression.

scholarly journals Thyroid transcription factor 1 and Pax8 synergistically activate the promoter of the human thyroglobulin gene

2001 ◽  
Vol 27 (1) ◽  
pp. 59-67 ◽  
Author(s):  
CR Espinoza ◽  
TL Schmitt ◽  
U Loos
Keyword(s):  
Gene Expression ◽  
Promoter Activity ◽  
Transcriptional Start Site ◽  
Promoter Sequence ◽  
Specific Protein ◽  
Site Directed Mutagenesis ◽  
Expression Vectors ◽  
Mobility Shift ◽  
Thyroid Transcription Factor 1 ◽  
Thyroid Hormone Biosynthesis

Thyroglobulin (Tg) is an essential thyroid-specific protein, which serves as the matrix for thyroid hormone biosynthesis. To obtain new insights in the regulation of Tg gene expression, we investigated the interaction of the human Tg promoter with the thyroid-specific transcription factors TTF-1 and Pax8. A reporter gene, containing a 202 bp fragment from the human Tg 5'-flanking region including the promoter sequence and the transcriptional start site, and expression vectors containing the cDNAs for human TTF-1 and Pax8 were used in cotransfection experiments, in the non-thyroidal cell lines COS-7 and HeLa. Pax8 increased the specific transcriptional activity of the Tg promoter about threefold, whereas cotransfection with the homeodomain-containing protein TTF-1 stimulated promoter activity from six- to tenfold. The simultaneous expression of both factors stimulated the Tg promoter activity in a multiplicative manner up to 25-fold. TTF-1 binding sites could be localized precisely by lectron mobility shift assay. The two binding elements corresponded to sites A and C in the rat Tg promoter. Site-directed mutagenesis of three nucleotides in each binding element inhibited binding of TTF-1 to the two oligonucleotides. In cotransfection experiments, the mutant site C decreased TTF-1 transactivation to 26% of the wild-type, whereas an additional mutation in the site A reduced this value to almost zero, thus proving the physiological relevance of these sites. The present results demonstrate that the activity of the human Tg promoter is closely dependent on the function of TTF-1 and Pax8, opening the field for further investigations of pathological alterations of Tg gene expression.

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