Early Growth Response Factor EGR-1 Regulates Gαq Gene in Megakaryocytic Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1518-1518
Author(s):  
Gauthami Jalagadugula ◽  
Dhanasekaran N. Danny ◽  
Kim Soochong ◽  
Satya P. Kunapuli ◽  
A. Koneti Rao
Keyword(s):  
Protein Binding ◽  
Reporter Gene ◽  
Cellular Proliferation ◽  
Immunoblot Analysis ◽  
Nuclear Extract ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Luciferase Reporter Gene ◽  
Cell Extracts ◽  
Hel Cells

Abstract Gαq (Gene GNAQ) plays a major role in platelet signal transduction but little is known regarding its transcriptional regulation. We studied Gαq promoter activity using luciferase reporter gene assays in human erythroleukemia (HEL) cells treated with 10 nM of phorbol 12-myristate 13-acetate (PMA) for 24 h to induce megakaryocytic (MK) transformation. RT-PCR analysis of HEL cell RNA revealed that Gαq mRNA was relatively low in untreated cells and it increased after PMA treatment with a peak at 5 h. Immunoblot analysis of HEL lysates showed enhanced Gαq expression with PMA. Luciferase reporter gene studies on full length construct (upto −1116 bp from ATG) and its serial 5′ truncations revealed a negative regulatory site at −238/−202 and two positive sites at −203/−138 and −1116/−731. In the region −238/−202 consensus sites were noted for two transcription factors PU.1 and GATA-1 that are known to regulate several megakaryocytic genes. Deletions of these sites alone or together revealed no change in the transcriptional activity of the gene in reporter studies. The positive region −203/−138 contained two overlapping Sp1/AP-2/EGR-1 consensus sites at −202/−189 and −164/−150. Gel shift studies were performed on oligonucleotides 1 (−203/−175) and 2 (−174/−152) using HEL cell extracts. Protein binding occured with Gαq oligonucleotides 1 and 2, which was competed with excess unlabeled oligos but not by unlabeled Sp1 or AP-2 consensus oligos. Supershift assay using antibody against Sp1 revealed neither competition nor supershift, suggesting that Sp1 does not bind to these oligonucleotides. No protein binding was noted when Gαq oligos 1 or 2 were incubated with extracts known to contain Sp1 or AP-2. These results indicate that Sp1 and AP-2 do not bind to the Gαq oligonucleotide regions. Protein binding to oligonucleotides 1 and 2 was abolished by excess unlabeled consensus EGR-1 oligo, and by immunodepletion of the EGR-1 protein from the nuclear extract with anti-EGR-1 antibody. These experiments reveal that EGR-1 binds to both Gαq oligonucleotides −203/−175 and −174/−152. In luciferase reporter studies mutations in EGR-1 sites present in both oligonucleotides 1 and 2 markedly decreased gene activity indicating functional relevance. In further studies, reduction in endogenous EGR-1 expression with antisense oligonucleotide to EGR-1 inhibited PMA induced Gαq transcription and protein in HEL cells. Lastly, EGR-1 deficient mouse platelets also showed ~50% reduction in the Gαq protein (immunoblotting) relative to wild type platelets. These studies suggest that Gαq gene is regulated during PMA induced differentiation by EGR-1, a transcription factor that regulates a wide array of genes involved in cellular proliferation, differentiation, and apoptosis, and in vascular response to injury and atherosclerosis.

PMA Responsive Sequence (s) in Gαq Promoter during Megakaryocytic Differentiation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4246-4246
Author(s):  
Gauthami S. Jalagadugula ◽  
Danny Dhanasekharan ◽  
A.Koneti Rao
Keyword(s):  
Transcription Factor ◽  
Protein Binding ◽  
Reporter Gene ◽  
Transcriptional Control ◽  
Nuclear Protein ◽  
Genomic Region ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Megakaryocytic Differentiation ◽  
Hel Cells

Abstract Human erthroleukemia cells (HEL) differentiate towards megakaryocytic (MK) phenotype when stimulated with phorbol 12-myristate-13-acetate (PMA). We observed that the expression of Gq, a protein that plays a major role in platelet signal transduction, is increased in PMA-treated HEL cells. Western blotting revealed that Gq is upregulated in PMA-treated cells relative to untreated cells. Gq gene induction by PMA treatment was investigated with respect to transcriptional control. Serial 5′-truncations of the upstream region (upto 2727 bp from the ATG) of Gq gene were fused to a luciferase (Luc) reporter gene vector, PGL-3 Basic, and were transiently transfected into HEL cells in the absence and presence of PMA (10 nM). After 24 h, reporter gene activities were measured using Dual Luciferase Reporter Assay System (Promega). A reporter plasmid −1042 bp-Luc with a genomic region −1042/−1 showed a 12 fold activity in PMA treated cells and 4 fold activity in untreated cells. Its truncated plasmid with the genomic region −1036/−1 showed a decrease in luciferase activity by 50% in treated cells; and the activity became identical to that in untreated cells. Further truncation between −1036 and −1011 caused a complete loss of activity in both the cells. Thus, a PMA responsive element was localized to a region between −1042 and −1037 bp. Transcription factor data base search (TFSEARCH) predicted two consensus sites for early growth response factor EGR-1 at -1042/−1031 and −1026/−1015. Gel shift studies were performed with two oligos, −1042/−1012 and −1036/−1012, and nuclear extracts from PMA- treated and untreated cells. The studies with −1042/−1012 probe and extracts from treated cells showed that there was nuclear protein binding, which was abolished by competition with the consensus EGR-1 sequence. In extracts from untreated cells, the protein binding was observed but was not competed with consensus EGR-1 sequence. This suggests EGR-1 binding to the region −1042/−1012 in PMA-treated cells and role for this transcription factor in inducing Gq promoter activity. Moreover, studies on the region −1036/−1012 showed nuclear protein binding that was identical between extracts of untreated and treated cells, and it was not competed with consensus EGR-1 sequence. These findings suggest that, EGR-1 binding is localized to −1042/−1037, but not to −1036/−1012. Conclusion: A PMA responsive sequence (−1042/−1037) was identified in the Gq promoter. Our studies suggest that EGR-1 binding to this sequence confers the PMA responsive activity. These studies provide further evidence that EGR-1 plays an important role in the upregulation of Gq expression during PMA induced megakaryocytic differentiation.

RUNX1/CBFA2 Regulates Myosin Light Chain 9 (MYL9) in Megakaryocytic Cells: Decreased MYL9 Expression in Human RUNX1 Haplodeficiency.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1831-1831
Author(s):  
Gauthami S Jalagadugula ◽  
Gurpreet Kaur ◽  
Guangfen Mao ◽  
Danny Dhanasekaran ◽  
A. Koneti Rao
Keyword(s):  
Transcription Factor ◽  
Protein Binding ◽  
Platelet Function ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Luciferase Reporter ◽  
Wild Type ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Construct ◽  
Hel Cells

Abstract RUNX1 (also known as CBFA2 or AML1) is a transcription factor that plays a major role in hematopoiesis. Haplodeficiency of RUNX1 has been associated with familial thrombocytopenia, impaired megakaryopoiesis, impaired platelet function and predisposition to acute myeloid leukemia. We have reported a patient with inherited thrombocytopenia and abnormal platelet function (Gabbeta et al, Blood87:1368–76, 1996). The patient platelets showed impaired phosphorylation of pleckstrin and myosin light chain, diminished GPIIb-IIIa activation and decreased platelet protein kinase C-𝛉. This was associated with a heterozygous nonsense mutation in transcription factor RUNX1 (Sun et al, Blood103: 948–54, 2004). Platelet transcript profiling showed a striking downregulation of myosin light chain 9 (MYL9) by ~77-fold relative to normal platelets (Sun et al, J. Thromb Haemost.5: 146–54, 2007). Myosin light chains (MLCs) play an important role in platelet responses to activation, in platelet biogenesis, and are involved in cellular processes such as cytokinesis, cell adhesion, cell contraction, cell migration. We have addressed the hypothesis that MYL9 is a direct transcriptional target of RUNX1. Studies were performed in human erythroleukemia (HEL) cells treated with phorbol 12-myristate 13-acetate (PMA) for 24 h to induce megakaryocytic transformation. To determine endogenous interaction of RUNX1 with MYL9 promoter, we performed chromatin immunoprecipitation (ChIP) assay using anti-RUNX1 antibody. These studies revealed RUNX1 binding to MYL9 chromatin at −742/−529 bp upstream of the ATG codon. TFSEARCH revealed four RUNX1 sites within this region. We performed electrophoretic mobility shift assay (EMSA) using probes containing each of the RUNX1 motifs and PMA-treated nuclear extracts from HEL cells. With each probe, protein binding was observed that was competed by excess unlabelled probe and inhibited by anti-RUNX1 antibody indicating RUNX1 as the protein involved. This protein binding was not competed by oligos containing mutations in the specific RUNX1 sites. No binding was noted directly to the mutant probes. To further corroborate our findings, we performed transient-ChIP analysis where wild type luciferase reporter construct −691/+4 and constructs with each of the RUNX1 sites individually mutated were transiently transfected into HEL cells. ChIP was performed using these cells and anti-RUNX1 antibody, and the expression analyzed by PCR amplification with a forward primer from MYL9 promoter sequence and reverse primer from luciferase vector sequence. Amplification was observed with immunoprecipitated wild type construct but not with any of the mutant constructs. Thus, RUNX1 interacts in vivo with MYL9 promoter, and the multiple RUNX1 sites interact with each other, as also shown for other genes. To test the functional relevance, the wild type construct −691/+4 containing all 4 RUNX1 sites or mutant constructs with each site individually deleted were cloned into firefly luciferase reporter gene vector and transfected into HEL cells. Deletion of RUNX1 site 1, 2, 3 or 4 caused ~60–90% reduction in the activity indicating that each site was functional. Lastly, siRNA mediated knock down of RUNX1 in HEL cells was associated with a decrease in both RUNX1 and MYL9 protein. Conclusions: Our results provide the first evidence that MYL9 gene is transcriptionally regulated by RUNX1. They provide evidence for the presence of multiple RUNX1 sites in MYL9 promoter, as also observed in other genes. Moreover, these studies provide a cogent mechanism for the MYL9 transcript downregulation and the impaired MLC-phosphorylation we have previously described in association with RUNX1 haplodeficiency.

scholarly journals Analysis of Promoter Function in Aspergillus fumigatus

2012 ◽  
Vol 11 (9) ◽  
pp. 1167-1177 ◽  
Author(s):  
Sanjoy Paul ◽  
J. Stacey Klutts ◽  
W. Scott Moye-Rowley
Keyword(s):  
Aspergillus Fumigatus ◽  
Reporter Gene ◽  
Reporter Genes ◽  
Antifungal Drugs ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Luciferase Reporter Gene ◽  
Content Type ◽  
Gene System ◽  
Reporter Gene System

ABSTRACTThe filamentous fungusAspergillus fumigatusis an important opportunistic pathogen that can cause high mortality levels in susceptible patient populations. The increasing dependence on antifungal drugs to controlA. fumigatushas led to the inevitable acquisition of drug-resistant forms of this pathogen. In other fungal pathogens, drug resistance is often associated with an increase in transcription of genes such as ATP-binding cassette (ABC) transporters that directly lead to tolerance to commonly employed antifungal drugs. InA. fumigatus, tolerance to azole drugs (the major class of antifungal) is often associated with changes in the sequence of the azole target enzyme as well as changes in the transcription level of this gene. The target gene for azole drugs inA. fumigatusis referred to ascyp51A. In order to dissect transcription ofcyp51Atranscription and other genes of interest, we constructed a set of firefly luciferase reporter genes designed for use inA. fumigatus. These reporter genes can either replicate autonomously or be targeted to thepyrGlocus, generating an easily assayable uracil auxotrophy. We fused eight differentA. fumigatuspromoters to luciferase. Faithful behaviors of these reporter gene fusions compared to their chromosomal equivalents were evaluated by 5′ rapid amplification of cDNA ends (RACE) and quantitative reverse transcription-PCR (qRT-PCR) analysis. We used this reporter gene system to study stress-regulated transcription of a Hsp70-encoding gene, map an important promoter element in thecyp51Agene, and correct an annotation error in the actin gene. We anticipate that this luciferase reporter gene system will be broadly applicable in analyses of gene expression inA. fumigatus.

Influence of chlorophyll and tannins in plant extracts on cell-based luciferase reporter gene assays

Planta Medica ◽  
2009 ◽  
Vol 75 (09) ◽  
Author(s):  
S Vogl ◽  
P Picker ◽  
N Fakhrudin ◽  
A Atanasov ◽  
E Heiß ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Plant Extracts ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Reporter Gene Assays

scholarly journals Genome-Wide Identification and Analysis of the APETALA2 (AP2) Transcription Factor in Dendrobium officinale

2021 ◽  
Vol 22 (10) ◽  
pp. 5221
Author(s):  
Danqi Zeng ◽  
Jaime A. Teixeira da Silva ◽  
Mingze Zhang ◽  
Zhenming Yu ◽  
Can Si ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Flower Development ◽  
Regulatory Elements ◽  
Negative Control ◽  
Dendrobium Officinale ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Luciferase Reporter Gene ◽  
Genome Wide ◽  
Ap2 Transcription Factor

The APETALA2 (AP2) transcription factors (TFs) play crucial roles in regulating development in plants. However, a comprehensive analysis of the AP2 family members in a valuable Chinese herbal orchid, Dendrobium officinale, or in other orchids, is limited. In this study, the 14 DoAP2 TFs that were identified from the D. officinale genome and named DoAP2-1 to DoAP2-14 were divided into three clades: euAP2, euANT, and basalANT. The promoters of all DoAP2 genes contained cis-regulatory elements related to plant development and also responsive to plant hormones and stress. qRT-PCR analysis showed the abundant expression of DoAP2-2, DoAP2-5, DoAP2-7, DoAP2-8 and DoAP2-12 genes in protocorm-like bodies (PLBs), while DoAP2-3, DoAP2-4, DoAP2-6, DoAP2-9, DoAP2-10 and DoAP2-11 expression was strong in plantlets. In addition, the expression of some DoAP2 genes was down-regulated during flower development. These results suggest that DoAP2 genes may play roles in plant regeneration and flower development in D. officinale. Four DoAP2 genes (DoAP2-1 from euAP2, DoAP2-2 from euANT, and DoAP2-6 and DoAP2-11 from basal ANT) were selected for further analyses. The transcriptional activation of DoAP2-1, DoAP2-2, DoAP2-6 and DoAP2-11 proteins, which were localized in the nucleus of Arabidopsis thaliana mesophyll protoplasts, was further analyzed by a dual-luciferase reporter gene system in Nicotiana benthamiana leaves. Our data showed that pBD-DoAP2-1, pBD-DoAP2-2, pBD-DoAP2-6 and pBD-DoAP2-11 significantly repressed the expression of the LUC reporter compared with the negative control (pBD), suggesting that these DoAP2 proteins may act as transcriptional repressors in the nucleus of plant cells. Our findings on AP2 genes in D. officinale shed light on the function of AP2 genes in this orchid and other plant species.

Potency of isothiocyanates to induce luciferase reporter gene expression via the electrophile-responsive element from murine glutathione S-transferase Ya

2009 ◽  
Vol 23 (4) ◽  
pp. 617-621 ◽  
Author(s):  
Martijn Vermeulen ◽  
Anne-Marie M.J.F. Boerboom ◽  
Barry M.G. Blankvoort ◽  
Jac M.M.J.G. Aarts ◽  
Ivonne M.C.M. Rietjens ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Reporter Gene Expression ◽  
Luciferase Reporter ◽  
Glutathione S Transferase ◽  
Luciferase Reporter Gene ◽  
Responsive Element

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.

scholarly journals Non-invasive somatotransgenic bioimaging in living animals

F1000Research ◽  
2020 ◽  
Vol 9 ◽  
pp. 1216 ◽  
Author(s):  
Juliette M. Delhove ◽  
Rajvinder Karda ◽  
Lorna M. FitzPatrick ◽  
Suzanne M.K. Buckley ◽  
Simon N. Waddington ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Viral Vectors ◽  
Es Cells ◽  
Embryonic Stem ◽  
Whole Body ◽  
Luciferase Reporter ◽  
Gene Promoters ◽  
Luciferase Reporter Gene ◽  
Endogenous Gene

Bioluminescence imaging enables noninvasive quantification of luciferase reporter gene expression in transgenic tissues of living rodents. Luciferase transgene expression can be regulated by endogenous gene promoters after targeted knock-in of the reporter gene, usually within the first intron of the gene. Even using CRISPR/Cas9 mediated genome editing this can be a time consuming and costly process. The generation of germline transgenic (GLT) rodents by targeted genomic integration of a gene expression cassette in embryonic stem (ES) cells is commonplace but results in the wastage of large numbers of animals during colony generation, back-crossing and maintenance. Using a synthetic/truncated promoter-driven luciferase gene to study promoter activity in a given tissue or organ of a GLT also often results in unwanted background luciferase activity during whole-body bioluminescent imaging as every cell contains the reporter. We have developed somatotransgenic bioimaging; a method to generate tissue-restricted transcription factor activated luciferase reporter (TFAR) cassettes in rodents that substantially reduces the number of animals required for experimentation. Bespoke designed TFARs are delivered to newborn pups using viral vectors targeted to specific organs by tissue-tropic pseudotypes. Retention and proliferation of TFARs is facilitated by stem/progenitor cell transduction and immune tolerance to luciferase due to the naïve neonatal immune system. We have successfully applied both lentiviral and adeno-associated virus (AAV) vectors in longitudinal rodent studies, targeting TFARs to the liver and brain during normal development and in well-established disease models. Development of somatotransgenic animals has broad applicability to non-invasively determine mechanistic insights into homeostatic and disease states and assess toxicology and efficacy testing. Somatotransgenic bioimaging technology is superior to current whole-body, light-emitting transgenic models as it reduces the numbers of animals used by generating only the required number of animals. It is also a refinement over current technologies given the ability to use conscious, unrestrained animals.

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