scholarly journals The myeloid-cell-specific c-fes promoter is regulated by Sp1, PU.1, and a novel transcription factor.

1996 ◽  
Vol 16 (4) ◽  
pp. 1676-1686 ◽  
Author(s):  
A Heydemann ◽  
G Juang ◽  
K Hennessy ◽  
M S Parmacek ◽  
M C Simon
Keyword(s):  
Transcription Factor ◽  
Vascular Endothelial Cells ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Luciferase Reporter ◽  
Specific Gene ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Specific Expression ◽  
Flanking Sequences

The protein product of the c-fps/fes (c-fes) proto-oncogene has been implicated in the normal development of myeloid cells (macrophages and neutrophils). mRNA for c-fes has been detected exclusively in myeloid cells and vascular endothelial cells in adult mammals. Although a 13-kilobase-pair (kb) human c-fes transgene exhibits high levels of expression in mice, the sequences that confer myeloid-cell-specific expression of the human c-fes gene have not been defined. Transient-transfection experiments demonstrated that plasmids containing 446 bp of c-fes 5'-flanking sequences linked to a luciferase reporter gene were active exclusively in myeloid cells. No other DNA element within the 13-kb human c-fes locus contained positive cis-acting elements, with the exception of a weakly active region within the 3'-flanking sequences. DNase I footprinting assays revealed four distinct sites that bind myeloid nuclear proteins (-408 to -386, -293 to -254, -76 to -65, and -34 to +3). However, the first two footprints resided in sequences that were largely dispensable for transient activity. Plasmids containing 151 bp of 5'-flanking sequences confer myeloid-cell-specific gene expression. Electrophoretic mobility shift analyses demonstrated that the 151-bp region contains nuclear protein binding sites for Sp1, PU.1, and/or Elf-1, and a novel factor. This unidentified factor binds immediately 3' of the PU.1/Elf-1 sites and appears to be myeloid cell specific. Mutation of the PU.1/Elf-1 site or the 3' site (FP4-3') within the context of the c-fes promoter resulted in substantially reduced activity in transient transfections. Furthermore, transient-cotransfection assay demonstrated that PU.1 (and not Elf-1) can transactivate the c-fes promoter in nonmyeloid cell lines. We conclude that the human c-fes gene contains a strong myeloid-cell-specific promoter that is regulated by Sp1, PU.1, and a novel transcription factor.

scholarly journals An E-box-containing region is involved in the tissue-specific expression of the human MC2R gene

2004 ◽  
Vol 32 (3) ◽  
pp. 811-823 ◽  
Author(s):  
A Blondet ◽  
M Doghman ◽  
P Durand ◽  
M Begeot ◽  
D Naville
Keyword(s):  
Granulosa Cells ◽  
Gene Promoter ◽  
Cell Types ◽  
Melanocortin Receptor ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Specific Expression ◽  
E Box ◽  
Mobility Shift Assay

Expression of the melanocortin receptor (MC2R) gene is limited to adrenocortical cells and the aim of this study was to determine the factors responsible for this tissue specificity. We used different fragments of the human (h) MC2R gene promoter, inserted in a vector upstream of the luciferase reporter gene, to transiently transfect either bovine adrenocortical (BAC) cells or granulosa cells from bovine ovaries (B-Gran). Similar promoter activities were obtained in both cell types using constructs containing fragments up to 1017 bp of the hMC2R gene promoter. On the contrary, a 2-fold decrease was obtained after transfection of the B-Gran cells with vectors containing 1069 bp and more of the promoter. Results obtained here using BAC cells confirmed our previous data on human cells showing that steroidogenic factor 1 is the major transactivating factor involved in the basal expression of the hMC2R gene in adrenal cells. However, we showed that this factor did not permit, by itself, the expression of the hMC2R gene in B-Gran cells despite its expression in these cells. This study demonstrated for the first time that an E-box (located at -1020 bp) is involved in the repression of hMC2R gene expression in granulosa cells through interactions with several factors, such as activator protein 4, as suggested by electrophoretic mobility shift assay analyses.

Regulation of the human brain natriuretic peptide gene by GATA-4

2002 ◽  
Vol 283 (1) ◽  
pp. E50-E57 ◽  
Author(s):  
Quan He ◽  
Mariela Mendez ◽  
Margot C. LaPointe
Keyword(s):  
Brain Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Promoter Activity ◽  
Nuclear Protein ◽  
Ventricular Myocytes ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Specific Expression

Brain natriuretic peptide (BNP) is a cardiac hormone constitutively expressed in the adult heart. We previously showed that the human BNP (hBNP) proximal promoter region from −127 to −40 confers myocyte-specific expression. The proximal hBNP promoter contains several putative cis elements. Here we tested whether the proximal GATA element plays a role in basal and inducible regulation of the hBNP promoter. The hBNP promoter was coupled to a luciferase reporter gene (1818hBNPLuc) and transferred into neonatal ventricular myocytes (NVM), and luciferase activity was measured as an index of hBNP promoter activity. Mutation of the putative GATA element at −85 of the hBNP promoter [1818(mGATA)hBNPLuc] reduced activity by 97%. To study transactivation of the hBNP promoter, we co-transfected 1818hBNPLuc with the GATA-4 expression vector. GATA-4 activated 1818hBNPLuc, and this effect was eliminated by mutation of the proximal GATA element. Electrophoretic mobility shift assay showed that an oligonucleotide containing the hBNP GATA motif bound to cardiomyocyte nuclear protein, which was competed for by a consensus GATA oligonucleotide but not a mutated hBNP GATA element. The β-adrenergic agonist isoproterenol and its second messenger cAMP stimulated hBNP promoter activity and binding of nuclear protein to the proximal GATA element. Thus the GATA element in the proximal hBNP promoter is involved in both basal and inducible transcriptional regulation in cardiac myocytes.

scholarly journals Impact of Promoter Polymorphisms on the Transcriptional Regulation of the Organic Cation Transporter OCT1 (SLC22A1)

2018 ◽  
Vol 8 (4) ◽  
pp. 42 ◽  
Author(s):  
Kristin Bokelmann ◽  
Jürgen Brockmöller ◽  
Mladen Tzvetkov
Keyword(s):  
Transcription Factor ◽  
Promoter Activity ◽  
Organic Cation ◽  
Specific Binding ◽  
Organic Cation Transporter ◽  
Luciferase Reporter ◽  
Potential Contribution ◽  
Luciferase Reporter Gene ◽  
Promoter Polymorphisms ◽  
Mobility Shift

The organic cation transporter 1 (OCT1, SLC22A1) is strongly expressed in the human liver and facilitates the hepatic uptake of drugs such as morphine, metformin, tropisetron, sumatriptan and fenoterol and of endogenous substances such as thiamine. OCT1 expression is inter-individually highly variable. Here, we analyzed SNPs in the OCT1 promoter concerning their potential contribution to the variability in OCT1 expression. Using electrophoretic mobility shift and luciferase reporter gene assays in HepG2, Hep3B, and Huh7 cell lines, we identified the SNPs −1795G>A (rs6935207) and −201C>G (rs58812592) as having effects on transcription factor binding and/or promoter activity. The A-allele of the −1795G>A SNP showed allele-specific binding of the transcription factor NF-Y leading to 2.5-fold increased enhancer activity of the artificial SV40 promoter. However, the −1795G>A SNP showed no significant effects on the native OCT1 promoter activity. Furthermore, the −1795G>A SNP was not associated with the pharmacokinetics of metformin, fenoterol, sumatriptan and proguanil in healthy individuals or tropisetron efficacy in patients undergoing chemotherapy. Allele-dependent differences in USF1/2 binding and nearly total loss in OCT1 promoter activity were detected for the G-allele of −201C>G, but the SNP is apparently very rare. In conclusion, common OCT1 promoter SNPs have only minor effects on OCT1 expression.

In vitro and in vivo analysis of murine lipoprotein lipase gene promoter: tissue-specific expression

1995 ◽  
Vol 268 (2) ◽  
pp. E213-E218 ◽  
Author(s):  
J. M. Gimble ◽  
X. Hua ◽  
F. Wanker ◽  
C. Morgan ◽  
C. Robinson ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Lipoprotein Lipase ◽  
Reporter Gene ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Specific Expression ◽  
Tissue Specific ◽  
Brown Adipose ◽  
Octamer Motif

Lipoprotein lipase, an enzyme of central importance to lipid metabolism, is most abundant in adipose tissues, cardiac and skeletal muscle, and portions of the brain. The current work examined the murine lipoprotein lipase promoter using transient transfection, gel-retention analyses, and transgenic mice. Maximum expression of the luciferase reporter gene in transfected cells was observed with -101 bp of the promoter. Nuclear extracts from tissues expressing lipoprotein lipase contained DNA binding proteins that recognize the CCAAT box (-64 bp) and an octamer motif (-46 bp); this combination of factors was absent in nonexpressing tissues. Transgenic mice from three of five founders prepared with -1,824-bp promoter constructs expressed the luciferase reporter gene at highest levels in brown adipose tissue and brain. These findings suggest that the -1,824-bp promoter region contains sequence elements responsible for the tissue-specific transcription of lipoprotein lipase in vivo.

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.

Abstract 267: Hemoglobin, Endothelial Cells, and TLRs. Common Connection?

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Christina Lisk ◽  
David Irwin
Keyword(s):  
Endothelial Cells ◽  
Messenger Rna ◽  
Time Course ◽  
Vascular Endothelial Cells ◽  
Vascular Diseases ◽  
Luciferase Reporter ◽  
Signaling Cascade ◽  
Pulmonary Vascular Disease ◽  
Luciferase Reporter Gene ◽  
Pulmonary Arterial

Introduction: Patients suffering from chronic hereditary hemolytic anemic syndromes, such as sickle cell disease (SCD) and thalassemia, are often at risk for systemic and pulmonary vascular disease. It has been suggested that chronic exposure to cell free hemoglobin (CFH) may contribute to some vascular diseases associated with these syndromes such as pulmonary arterial hypertension. To date, the vasculotoxic effects of CFH have mostly been attributed to its pro-oxidant and nitric oxide scavenging characteristics. However, emerging evidence suggests CFH may contribute to inflammation by directly activating a signaling cascade event by binding to a pattern recognition receptor (PRR) or a toll like receptor (TLR) on vascular endothelial cells. Hypothesis: We hypothesized that CFH would increase the activity of transcription factors, NF-κb and HIF-1α, via a MyD88-dependent pathway. Methods: Human microvascular endothelial cells (HMEC) were transfected with either an NF-κB or HIF-1α luciferase reporter gene and treated with CFH (ferrous, ferric, and ferryl forms) in the presence or absence of SOD, catalase, dexamethasome, MyD88 inhibitor, or, the PHD inhibitor, DMOG. Messenger RNA for HIF-1α and HIF-2 were also measured after treatments. Results: All three states of hemoglobin increased NF-κB and HIF-1α activity in a dose response fashion, with ferryl inducing the greatest activity of both NF-κB and HIF-1α. Time course studies showed that NF-κB and HIF-1α activity tracked together. A unique synergy was noted with co-treatment of ferryl and DMOG. Co-treatment with SOD or catalase did not inhibit the CFH-induced NF-κB or HIF-1α response. Dexamthasome and MyD88 inhibition reduced the CFH-induced NF-κB and HIF-1α activity. Conclusion: Our results support the hypothesis, that CFH may activate a TLR or PRR signaling cascade subsequently activating MyD88-NF-κB and HIF-1α. Our data, that showed SOD and/or catalase did not block CFH effects, suggests that this event is not mediated by CFH pro-oxidant characteristics. CFH-induced HIF-1α was blocked by NF-κB inhibition with either, Dexamethasome or MyD88 inhibition emphasizing the importance of NF-κB in the HIF-1α pathway.

Angiopoietin-2 promotes myeloid cell infiltration in a β2-integrin–dependent manner

Blood ◽  
2011 ◽  
Vol 118 (18) ◽  
pp. 5050-5059 ◽  
Author(s):  
Alexander Scholz ◽  
Victoria Lang ◽  
Reinhard Henschler ◽  
Marcus Czabanka ◽  
Peter Vajkoczy ◽  
...  
Keyword(s):  
Inflammatory Diseases ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Delayed Type Hypersensitivity ◽  
Cell Infiltration ◽  
Dependent Manner ◽  
Specific Expression ◽  
Β2 Integrin ◽  
Angiopoietin 2

Abstract In human inflammatory diseases, we identified endothelial angiopoietin-2 (Ang-2) expression to be strongly associated with inflammations mediated by myeloid cells but not lymphocytes. To identify the underlying mechanism, we made use of a transgenic mouse model with inducible endothelial cell-specific expression of Ang-2. In this model, in the absence of inflammatory stimuli, long-term expression of Ang-2 led to a time-dependent accumulation of myeloid cells in numerous organs, suggesting that Ang-2 is sufficient to recruit myeloid cells. In models of acute inflammation, such as delayed-type hypersensitivity and peritonitis, Ang-2 transgenic animals showed an increased responsiveness. Intravital fluorescence video microscopy revealed augmented cell adhesion as an underlying event. Consequently, we demonstrated that Ang-2 is able to induce strong monocyte adhesion under shear in vitro, which could be blocked by antibodies to β2-integrin. Taken together, our results describe Ang-2 as a novel, endothelial-derived regulator of myeloid cell infiltration that modulates β2-integrin–mediated adhesion in a paracrine manner.

Canine H(+)-K(+)-ATPase alpha-subunit gene promoter: studies with canine parietal cells in primary culture

1996 ◽  
Vol 271 (6) ◽  
pp. G1104-G1113 ◽  
Author(s):  
A. Muraoka ◽  
M. Kaise ◽  
Y. J. Guo ◽  
J. Yamada ◽  
I. Song ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Primary Culture ◽  
Transcriptional Activity ◽  
Parietal Cells ◽  
Luciferase Reporter ◽  
Cell Type ◽  
High Concentration ◽  
Specific Expression ◽  
Atpase Gene ◽  
Cell Type Specific

H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) is the principal enzyme responsible for the process of gastric acid secretion. This enzyme is expressed in a cell-type-specific manner in gastric parietal cells. To explore the mechanisms regulating its expression, we transfected differentiated canine parietal cells in primary culture with H(+)-K(+)-ATPase-luciferase reporter genes and assessed transcriptional activities. Deletional analysis of the 5'-flanking region of this gene demonstrated a remarkable increment in transcriptional activity associated with a segment between bases -54 to -45 (5' GCTCCGCCTC 3') relative to the transcriptional initiation site. Gel shift assays with competition and supershift analysis demonstrated that this segment is specifically bound by the transcription factor Sp1. A point mutation, eliminating Sp1 binding, diminished basal transcriptional activity by 80%, indicating that this Sp1 binding site is important for constitutive transcriptional activity. Although these studies indicate that Sp1 is required to maintain a high concentration of the H(+)-K(+)-ATPase gene in the parietal cell, its cell-type-specific expression must rely on other elements because Sp1 is a ubiquitously expressed transcription factor.

scholarly journals Xiao Yao San against Corticosterone-Induced Stress Injury via Upregulating Glucocorticoid Receptor Reaction Element Transcriptional Activity

2016 ◽  
Vol 2016 ◽  
pp. 1-14 ◽  
Author(s):  
Guoping Cao ◽  
Shenglan Gong ◽  
Fengxue Zhang ◽  
Wenjun Fu
Keyword(s):  
Hippocampal Neurons ◽  
Transcriptional Activity ◽  
Nuclear Translocation ◽  
Luciferase Reporter ◽  
Potential Candidate ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Stress Injury ◽  
Induced Stress

Previous studies have revealed that uncontrollable stress can impair the synaptic plasticity and firing property of hippocampal neurons, which influenced various hippocampal-dependent tasks including memory, cognition, behavior, and mood. In this work, we had investigated the effects and mechanisms of the Chinese herbal medicine Xiao Yao San (XYS) against corticosterone-induced stress injury in primary hippocampal neurons (PHN) cells. We found that XYS and RU38486 could increase cell viabilities and decrease cell apoptosis by MTT, immunofluorescence, and flow cytometry assays. In addition, we observed that XYS notably inhibited the nuclear translocation of GR and upregulated the mRNA and protein expressions levels of Caveolin-1, GR, BDNF, TrkB, and FKBP4. However, XYS downregulated the FKBP51 expressions. Furthermore, the results of the electrophoretic mobility shift assay (EMSA) and double luciferase reporter gene detection indicated that FKBP4 promotes the transcriptional activity of GR reaction element (GRE) by binding with GR, and FKBP51 processed the opposite action. Thein vivoexperiment also proved the functions of XYS. These results suggested that XYS showed an efficient neuroprotection against corticosterone-induced stress injury in PHN cells by upregulating GRE transcriptional activity, which should be developed as a potential candidate for treating stress injury in the future.

scholarly journals Transcriptomic Analysis and Specific Expression of Transcription Factor Genes in the Root and Sporophyll of Dryopteris fragrans (L.) Schott

2020 ◽  
Vol 21 (19) ◽  
pp. 7296
Author(s):  
Lingling Chen ◽  
Dongrui Zhang ◽  
Chunhua Song ◽  
Hemeng Wang ◽  
Xun Tang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Glandular Trichomes ◽  
Transcriptomic Analysis ◽  
Specific Gene ◽  
Specific Expression ◽  
Tissue Specific ◽  
Transcription Factor Genes ◽  
Dryopteris Fragrans ◽  
Different Tissues

Background: Dryopteris fragrans, which is densely covered with glandular trichomes, is considered to be one of the ferns with the most medicinal potential. The transcriptomes from selected tissues of D. fragrans were collected and analyzed for functional and comparative genomic studies. The aim of this study was to determine the transcriptomic characteristics of wild D. fragrans sporangium in tissues from the SR (root), SL (sporophyll), and TRL (sporophyll with glandular trichomes removed). Results: Cluster analysis identified genes that were highly expressed in an organ-specific manner according to read mapping, feature counting, and normalization. The functional map identified gene clusters that can uniquely describe the function of each tissue. We identified a group of three tissue-specific transcription factors targeting the SL, SR, and TRL. In addition, highly expressed transcription factors (TFs) were found in each tissue-specific gene cluster, where ERF and bHLH transcription factors were the two types showing the most distinct expression patterns between the three different tissues. The specific expression of transcription factor genes varied between the different types of tissues. The numbers of transcription factors specifically expressed in the roots and sporophylls were 60 and 30, respectively, while only seven were found for the sporophylls with glandular trichomes removed. The expression of genes known to be associated with the development of glandular trichomes in flowering plants, including MIXTA, ATML1, and MYB106, were also validated and are discussed. In particular, a unigene encoding MIXTA was identified and exhibited the highest expression level in SL in D. fragrans. Conclusions: This study is the first report of global transcriptomic analysis in different tissues of D. fragrans, and the first to discuss these findings in the context of the development of homologous glandular trichomes. These results set the stage for further research on the development, stress resistance, and secondary metabolism of D. fragrans glandular trichomes.

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