scholarly journals Mechanisms of Transcription in Eosinophils: GATA-1, but not GATA-2, Transactivates the Promoter of the Eosinophil Granule Major Basic Protein Gene

Blood ◽  
1998 ◽  
Vol 91 (9) ◽  
pp. 3447-3458 ◽  
Author(s):  
Yuji Yamaguchi ◽  
Steven J. Ackerman ◽  
Naoko Minegishi ◽  
Masaki Takiguchi ◽  
Masayuki Yamamoto ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Promoter Activity ◽  
Basic Protein ◽  
Transcriptional Start Site ◽  
Luciferase Reporter ◽  
Major Basic Protein ◽  
Mobility Shift ◽  
P2 Promoter ◽  
Eosinophil Granule ◽  
Electrophoretic Mobility Shift Assays

Granule major basic protein (MBP) is expressed exclusively in eosinophils, basophils, and placental trophoblasts. To identify thecis-elements and transcription factors involved in regulating MBP expression, we subcloned 3.2 kb of sequence upstream of the exon 9 transcriptional start site (P2 promoter) and serial 5′ deletions into the pXP2 luciferase reporter vector. An 80% decrement in promoter activity was obtained when MBP sequences between bp −117 to −67 were deleted. To identify transcription factors that bind to and transactivate through the bp −117 to −67 region, we first compared the upstream genomic sequences of human and murine MBP; a potential GATA binding consensus site was conserved in the 50-bp region between the two genes. To determine which GATA proteins bind this consensus site, we performed electrophoretic mobility shift assays (EMSAs), which showed that both GATA-1 and GATA-2 can bind to this consensus site. To determine the functionality of this site, we tested whether GATA-1 and GATA-2, either individually or in combination, can transactivate the MBP promoter in the Jurkat T cell line. Cotransfection with a GATA-1 expression vector produced 20-fold augmentation of MBP promoter activity, whereas GATA-2 had no activity. In contrast, combined cotransfection of GATA-1 and GATA-2 decreased the ability of GATA-1 to transactivate the MBP promoter by approximately 50%. Our results provide the first evidence for a GATA-1 target gene in eosinophils, a negative regulatory role for GATA-2 in MBP expression, and possibly eosinophil gene transcription in general during myelopoiesis.

scholarly journals Mechanisms of Transcription in Eosinophils: GATA-1, but not GATA-2, Transactivates the Promoter of the Eosinophil Granule Major Basic Protein Gene

Blood ◽  
1998 ◽  
Vol 91 (9) ◽  
pp. 3447-3458 ◽  
Author(s):  
Yuji Yamaguchi ◽  
Steven J. Ackerman ◽  
Naoko Minegishi ◽  
Masaki Takiguchi ◽  
Masayuki Yamamoto ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Promoter Activity ◽  
Basic Protein ◽  
Transcriptional Start Site ◽  
Luciferase Reporter ◽  
Major Basic Protein ◽  
Mobility Shift ◽  
P2 Promoter ◽  
Eosinophil Granule ◽  
Electrophoretic Mobility Shift Assays

Abstract Granule major basic protein (MBP) is expressed exclusively in eosinophils, basophils, and placental trophoblasts. To identify thecis-elements and transcription factors involved in regulating MBP expression, we subcloned 3.2 kb of sequence upstream of the exon 9 transcriptional start site (P2 promoter) and serial 5′ deletions into the pXP2 luciferase reporter vector. An 80% decrement in promoter activity was obtained when MBP sequences between bp −117 to −67 were deleted. To identify transcription factors that bind to and transactivate through the bp −117 to −67 region, we first compared the upstream genomic sequences of human and murine MBP; a potential GATA binding consensus site was conserved in the 50-bp region between the two genes. To determine which GATA proteins bind this consensus site, we performed electrophoretic mobility shift assays (EMSAs), which showed that both GATA-1 and GATA-2 can bind to this consensus site. To determine the functionality of this site, we tested whether GATA-1 and GATA-2, either individually or in combination, can transactivate the MBP promoter in the Jurkat T cell line. Cotransfection with a GATA-1 expression vector produced 20-fold augmentation of MBP promoter activity, whereas GATA-2 had no activity. In contrast, combined cotransfection of GATA-1 and GATA-2 decreased the ability of GATA-1 to transactivate the MBP promoter by approximately 50%. Our results provide the first evidence for a GATA-1 target gene in eosinophils, a negative regulatory role for GATA-2 in MBP expression, and possibly eosinophil gene transcription in general during myelopoiesis.

Regulation of plasma-membrane-associated sialidase NEU3 gene by Sp1/Sp3 transcription factors

2010 ◽  
Vol 430 (1) ◽  
pp. 107-117 ◽  
Author(s):  
Kazunori Yamaguchi ◽  
Koichi Koseki ◽  
Momo Shiozaki ◽  
Yukiko Shimada ◽  
Tadashi Wada ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Plasma Membrane ◽  
Promoter Activity ◽  
Neuronal Cell ◽  
Aberrant Crypt Foci ◽  
Luciferase Reporter ◽  
Alternative Promoters ◽  
Mobility Shift ◽  
Key Enzyme

Gene expression of the human plasma membrane-associated sialidase (NEU3), a key enzyme for ganglioside degradation, is relatively high in brain and is modulated in response to many cellular processes, including neuronal cell differentiation and tumorigenesis. We demonstrated previously that NEU3 is markedly up-regulated in various human cancers and showed that NEU3 transgenic mice developed a diabetic phenotype and were susceptible to azoxymethane-induced aberrant crypt foci in their colon tissues. These results suggest that appropriate control of NEU3 gene expression is required for homoeostasis of cellular functions. To gain insights into regulation mechanisms, we determined the gene structure and assessed transcription factor involvement. Oligo-capping analysis indicated the existence of alternative promoters for the NEU3 gene. Transcription started from two clusters of multiple TSSs (transcription start sites); one cluster is preferentially utilized in brain and another in other tissues and cells. Luciferase reporter assays showed further that the region neighbouring the two clusters has promoter activity in the human cell lines analysed. The promoter lacks TATA, but contains CCAAT and CAAC, elements, whose deletions led to a decrease in promoter activity. Electrophoretic mobility-shift assays and chromatin immunoprecipitation demonstrated binding of transcription factors Sp (specificity protein) 1 and Sp3 to the promoter region. Down-regulation of the factors by siRNAs (short interfering RNAs) increased transcription from brain-type TSSs and decreased transcription from other TSSs, suggesting a role for Sp1 and Sp3 in selection of the TSSs. These results indicate that NEU3 expression is diversely regulated by Sp1/Sp3 transcription factors binding to alternative promoters, which might account for multiple modulation of gene expression.

scholarly journals Sp1-mediated transcription is involved in the induction of leptin by insulin-stimulated glucose metabolism

2007 ◽  
Vol 38 (5) ◽  
pp. 537-546 ◽  
Author(s):  
M J Moreno-Aliaga ◽  
M M Swarbrick ◽  
S Lorente-Cebrián ◽  
K L Stanhope ◽  
P J Havel ◽  
...  
Keyword(s):  
Glucose Metabolism ◽  
Transcriptional Activation ◽  
Transcriptional Start Site ◽  
Specific Inhibitor ◽  
Gene Promoter ◽  
Competitive Inhibitor ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Rat Adipocytes ◽  
Electrophoretic Mobility Shift Assays

We have previously demonstrated that insulin-stimulated glucose metabolism, and not insulin per se, mediates the effects of insulin to increase the transcriptional activity of the leptin promoter in adipocytes. Here, we sought to identify the specific cis-acting DNA elements required for the upregulation of leptin gene transcription in response to insulin-mediated glucose metabolism. To accomplish this, 3T3-L1 cells and primary rat adipocytes were transfected with a series of luciferase reporter genes containing portions of the mouse leptin promoter. Using this method, we identified an element between −135 and −95 bp (relative to the transcriptional start site) that mediated transcription in response to insulin-stimulated glucose metabolism in adipocytes. This effect was abolished by incubation with 2-deoxy-d-glucose, a competitive inhibitor of glucose metabolism. Gel shift electrophoretic mobility shift assays confirmed that the stimulatory effect of insulin-mediated glucose metabolism on leptin transcription was mediated by a previously identified Sp1 site. Consistent with these findings, incubation of primary rat adipocytes with WP631, a specific inhibitor of specificity protein (Sp)1-dependent transcription, inhibited glucose- and insulin-stimulated, but not basal, leptin secretion. Together, these findings support a key role for Sp1 in the transcriptional activation of the leptin gene promoter by insulin-mediated glucose metabolism.

scholarly journals Biochemical and amino acid sequence analysis of human eosinophil granule major basic protein.

1988 ◽  
Vol 263 (25) ◽  
pp. 12559-12563
Author(s):  
T L Wasmoen ◽  
M P Bell ◽  
D A Loegering ◽  
G J Gleich ◽  
F G Prendergast ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequence ◽  
Basic Protein ◽  
Major Basic Protein ◽  
Amino Acid Sequence Analysis ◽  
Human Eosinophil ◽  
Eosinophil Granule

scholarly journals A Novel and Highly Divergent Homolog of Human Eosinophil Granule Major Basic Protein

1999 ◽  
Vol 274 (20) ◽  
pp. 14464-14473 ◽  
Author(s):  
Douglas A. Plager ◽  
David A. Loegering ◽  
Deborah A. Weiler ◽  
James L. Checkel ◽  
Jill M. Wagner ◽  
...  
Keyword(s):  
Basic Protein ◽  
Major Basic Protein ◽  
Human Eosinophil ◽  
Eosinophil Granule

C/EBPβ and GATA-1 Synergistically Regulate Activity of the Eosinophil Granule Major Basic Protein Promoter: Implication for C/EBPβ Activity in Eosinophil Gene Expression

Blood ◽  
1999 ◽  
Vol 94 (4) ◽  
pp. 1429-1439 ◽  
Author(s):  
Yuji Yamaguchi ◽  
Hitoshi Nishio ◽  
Kenji Kishi ◽  
Steven J. Ackerman ◽  
Toshio Suda
Keyword(s):  
Binding Site ◽  
Cell Line ◽  
Binding Sites ◽  
Basic Protein ◽  
Regulatory Role ◽  
Expression Vectors ◽  
Regulatory Function ◽  
Dependent Manner ◽  
Major Basic Protein ◽  
Eosinophil Granule

Abstract Eosinophil granule major basic protein (MBP) is expressed exclusively in eosinophils and basophils in hematopoietic cells. In our previous study, we demonstrated a major positive regulatory role for GATA-1 and a negative regulatory role for GATA-2 in MBP gene transcription. Further analysis of the MBP promoter region identified a C/EBP (CCAAT/enhancer-binding protein) consensus binding site 6 bp upstream of the functional GATA-binding site in the MBP gene. In the cell line HT93A, which is capable of differentiating towards both the eosinophil and neutrophil lineages in response to retinoic acid (RA), C/EBP mRNA expression decreased significantly concomitant with eosinophilic and neutrophilic differentiation, whereas C/EBPβ expression was markedly increased. Electrophoretic mobility shift assays (EMSAs) showed that recombinant C/EBPβ protein could bind to the potential C/EBP-binding site (bp −90 to −82) in the MBP promoter. Furthermore, we have demonstrated that both C/EBPβ and GATA-1 can bind simultaneously to the C/EBP- and GATA-binding sites in the MBP promoter. To determine the functionality of both the C/EBP- and GATA-binding sites, we analyzed whether C/EBPβ and GATA-1 can stimulate the MBP promoter in the C/EBPβ and GATA-1 negative Jurkat T-cell line. Cotransfection with C/EBPβ and GATA-1 expression vectors produced a 5-fold increase compared with cotransfection with the C/EBPβ or GATA-1 expression vectors individually. In addition, GST pull-down experiments demonstrated a physical interaction between human GATA-1 and C/EBPβ. Expression of FOG (F̲riendo̲fG̲ATA), which binds to GATA-1 and acts as a cofactor for GATA-binding proteins, decreased transactivation activity of GATA-1 for the MBP promoter in a dose-dependent manner. Our results provide the first evidence that both GATA-1 and C/EBPβ synergistically transactivate the promoter of an eosinophil-specific granule protein gene and that FOG may act as a negative cofactor for the eosinophil lineage, unlike its positively regulatory function for the erythroid and megakaryocyte lineages.

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 Acidic precursor revealed in human eosinophil granule major basic protein cDNA.

1988 ◽  
Vol 168 (4) ◽  
pp. 1493-1498 ◽  
Author(s):  
R L Barker ◽  
G J Gleich ◽  
L R Pease
Keyword(s):  
Endoplasmic Reticulum ◽  
Nucleotide Sequence ◽  
Cdna Library ◽  
Basic Protein ◽  
Cell Types ◽  
Major Basic Protein ◽  
Human Eosinophil ◽  
Damage Cell ◽  
Eosinophil Granule ◽  
Single Polypeptide

Eosinophil granule major basic protein (MBP), a potent toxin for helminths and various cell types, is a 13.8-kD single polypeptide rich in arginine with a calculated isoelectric point (pI) of 10.9. A cDNA for human MBP was isolated from a gamma GT10 HL-60 cDNA library. The nucleotide sequence of the MBP cDNA indicates that MBP is translated as a 25.2-kD preproprotein. The 9.9-kD pro-portion of proMBP is rich in glutamic and aspartic acids and has a calculated pI of 3.9, while proMBP itself has a calculated pI of 6.2. We suggest that MBP is translated as a nontoxic precursor that protects the eosinophil from damage while the protein is processed through the endoplasmic reticulum to its sequestered site in the granule core toxic MBP, and we present results from the literature suggesting that other cationic toxins, which damage cell membranes, may also be processed from nontoxic precursors containing distinct anionic and cationic regions.

Role of Cjun in Gγ-Globin Gene Trans-Activation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1870-1870
Author(s):  
Sirisha Kodeboyina ◽  
Sima Zein ◽  
Moosueng Lee ◽  
Parimaladevi Balamurugan ◽  
Xiao Yao ◽  
...  
Keyword(s):  
Electrophoretic Mobility ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Luciferase Reporter ◽  
Complete Analysis ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Electrophoretic Mobility Shift Assays

Abstract Previous studies from our laboratory demonstrated the role of the G-CRE (Gγ-globin cAMP response element) in drug-mediated fetal hemoglobin induction. The G-CRE located at −1222 to −1229 in the promoter of Gγ-globin gene, contains binding site for trans-factors CREB1, ATF-2 and cJun. We previously demonstrated binding of phosphorylated CREB1 and ATF-2 to this element via p38 MAPK signaling triggered by sodium butyrate (NaB) and trichostatin A (TSA). Electrophoretic mobility shift assays with a probe containing the AC → TG mutation in the G-CRE (TGTGGTCA, m2) abolished trans-factor binding to the G-CRE. Furthermore, Gγ promoter activity was abolished in the PGL3 luciferase reporter vector driven by the Gγ promoter (−1500 to +36) carrying the m2 mutation. (Sangerman et al. Blood108:3590–9, 2006). Subsequent studies in our laboratory were aimed at understanding the role of trans-factor cJun, an AP-1 family member, as a regulator of Gγ-globin expression via the G-CRE site. In K562 cells treated with 2mM NaB or 0.3μM TSA for 48 hrs, cJun phosphorylation increased 2.8-fold and 6.4-fold respectively by western blot analysis. Chromatin immunoprecipitation studies showed 16-fold chromatin enrichment in the −1225 Gγ-globin region compared to IgG control studies indicative of significant cJun binding in vivo at steady state. Electrophoretic mobility shift assays using cJun monoclonal antibody demonstrated a supershifted DNA-protein complex confirming binding of cJun to the G-CRE probe. To gain evidence for a functional role of cJun, we performed enforced expression studies using the pLen-cJun vector. In a concentration dependent manner, over-expression of cJun increased luciferase activity up to 350-fold in the luciferase reporter plasmid controlled by the Gγ-promoter (−1500 to +36). As predicted from binding studies, the m2 mutation in this promoter abolished the cJunmediated trans-activation confirming that the G-CRE is required to mediate effects of cJun. We are currently investigating the ability of cJun to trans-activate the endogenous Gγ-globin gene in K562 cells. To achieve this goal, K562 stable lines were established with the expression vectors pLen-cJun and empty vector. A complete analysis of the stable lines is in progress. Future investigations to identify other components of the functional CREB1/ATF2/cJun enhanceosome complex bound to the G-CRE will be performed using affinity chromatography and mass spectrometry. This information will be used to develop strategies for fetal hemoglobin induction.

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