The Proto-Oncogene Myb Augments Transcription of the Neuropeptide Neuromedin U in Human Myeloid Leukemias.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2254-2254
Author(s):  
Susan E. Shetzline ◽  
Joseph Conlon ◽  
Cezary Swider ◽  
Lindsay Thalheim ◽  
Alan M. Gewirtz
Keyword(s):  
Transcription Start Site ◽  
Promoter Region ◽  
Hematopoietic Cells ◽  
Leukemic Cells ◽  
Luciferase Reporter ◽  
Start Site ◽  
Quantitative Real Time Pcr ◽  
Transcription Start ◽  
Binding Motifs

Abstract The c-myb proto-oncogene encodes a transcription factor, Myb, which is essential for the growth and survival of normal and malignant hematopoietic cells. We, and others, have previously shown that malignant hematopoietic cells are more dependent on c-Myb function than are normal hematopoietic cells. Based on these findings, we hypothesized that c-Myb regulates a unique set of genes in leukemic cells that are required for their growth. To identify Myb gene targets, we performed a transcriptome analysis using human myeloid leukemic cells engineered to express a conditionally active dominant negative Myb (MERT). Analysis of the microarray data derived from these experiments revealed that when Myb activity was inhibited, neuromedin U (NmU), a neuropeptide involved in energy homeostasis, decreased in expression 5 fold compared to control cells, a result that was confirmed by quantitative real-time PCR. Combined, the microarray and quantitative real-time PCR data suggested that Myb directly regulates NmU gene expression in hematopoietic cells. To address this question in the absence of a formally defined human NmU promoter, we examined the DNA sequence upstream of the predicted transcription start site (as noted in Genbank accession #NM_006681) for potential Myb transcription factor binding motifs. After scanning the DNA sequence (~2kb) upstream of the predicted transcription start site, eleven potential Myb response elements (MREs) were identified. Of these MREs, five were identified as canonical (PyAAC(G/C)G). Our search also identified potential AML1, PU.1, CBP, STAT3, and STAT5 binding motifs within the human NmU promoter region. To determine if any of the potential MREs within the NmU promoter were functional, we first completed in vitro assays using luciferase reporter constructs followed by in vivo assays using chromatin immunoprecipitation (ChIP) assays. The luciferase reporter constructs were generated after we determined the actual transcription start of human NmU by primer extension assays. Using a Fam-labeled NmU specific primer that annealed proximal to the predicted transcription start site, we observed a 20-nucleotide difference between the predicted and actual transcription start of NmU. When all eleven potential MREs within the NmU promoter were upstream of luciferase, a 6-fold increase in luciferase activity was observed compared to empty vector. We next systematically mutated the MREs to determine which one(s) Myb bound directly. Thus far, the in vitro luciferase assay has identified MREs at −446 and −626, which are proximal to NmU’s transcription start as important for Myb-mediated expression. To determine the physiologic relevance of our in vitro studies, we performed ChIP assays. When chromatin from K562 cells, a human myeloid leukemia cell line, was immunoprecipitated with anti-c-Myb, we observed the expected PCR product using primer pairs that flanked select MREs. These same results were obtained in our positive control ChIP experiment in which the chromatin was immunoprecipitated with anti-acetyl histone 4 indicating that the promoter region of NmU is poised for transcription. Further characterization of the regulation of NmU gene expression in normal and malignant hematopoietic cells may yield new clues to Myb’s role in leukemogenesis and could suggest new therapeutic targets in human leukemia cells.

Transcriptional Regulation of the Intra-S-Phase Regulator CDC7 in Human Myeloid Leukemic Cells: A New Downstream Target of the C-Myb Protooncogene.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1354-1354
Author(s):  
Lindsay B. Thalheim ◽  
Susan E. Shetzline ◽  
Cezary R. Swider ◽  
Alan M. Gewirtz
Keyword(s):  
Transcription Factor ◽  
Transcriptional Regulation ◽  
Promoter Region ◽  
Hematopoietic Cells ◽  
S Phase ◽  
Leukemic Cells ◽  
Start Site ◽  
Transcription Start ◽  
Binding Motifs ◽  
Pcr Product

Abstract The c-myb proto-oncogene encodes a transcription factor, Myb, which is essential for the growth and proliferation of normal and malignant hematopoietic cells. We, and others, have previously shown that malignant hematopoietic cells are much more dependent on c-myb function than are normal hematopoeitic cells, and that transient disruption of c-myb expression causes malignant cells to undergo apoptosis while normal cells are relatively spared. Based on these findings, we hypothesized that c-myb regulates a unique set of genes in leukemic cells that are required for cell survival. To identify Myb gene targets, we performed a transcriptome analysis using human myeloid leukemic cells engineered to express a conditionally active dominant negative Myb (MERT). Analysis of the microarray data revealed that when Myb activity was inhibited by tamoxifen in MERT cells, CDC7, an intra-S phase regulator, decreased in expression 2.8-fold compared to untreated control cells. To verify this, we utilized real-time PCR to quantitate the expression of CDC7, and found that it decreased 5-fold in tamoxifen treated MERT cells relative to control cells. In aggregate, the microarray and real-time PCR data suggested that Myb directly regulates CDC7 gene expression in hematopoietic cells. To address this question in the absence of a formally defined human CDC7 promoter, we examined the DNA sequence upstream of the predicted transcription start site (as noted in Genbank accession # AY585721) for potential Myb transcription factor binding motifs. After scanning the DNA sequence (~3kb) upstream of the predicted transcription start site, nine potential Myb response elements (MREs) were identified. The CDC7 sequences from mouse, chimp, and yeast were also analyzed for MREs and compared to those present in the putative human CDC7 promoter to identify conserved MREs. Using this strategy, we also identified potential AML1, PU.1, CBP, STAT3, and STAT5 binding motifs within the human CDC7 promoter region. To determine if any of the potential Myb binding sites with the CDC7 promoter were actually utilized in vivo, we carried out chromatin immunoprecopitation (ChIP) assays. When the chromatin from untreated MERT cells was immunoprecipitated with anti-c-Myb, we observed one PCR product using a primer pair that flanked each conserved MRE. These same results were obtained in our positive control ChIP experiment in which the chromatin was immunoprecipitated with anti-acetyl histone 4. When Myb transactivation activity was inhibited in tamoxifen treated MERT cells, no PCR product was detected following chromatin immunoprecipitation with the anti-Myb antibody suggesting that the ChIP binding results were not due to artifact. We have just completed a primer extension assay with a Fam-labeled primer that flanked the predicted CDC7 promoter region and will use the resulting sequence data to identify the actual CDC7 transcriptional start site. We will also shortly complete identification of functional regions within the human CDC7 promoter through use of Luciferase reporter assays. Investigation of the transcriptional regulation of CDC7 in hematopoietic cells may yield new clues to Myb’s role in leukemogenesis.

Increased Transcription of the Neuropeptide Neuromedin U by Ets-2 and C-Myb Not B-Myb in Human Myeloid Leukemia Cells Promotes Their Proliferation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2254-2254
Author(s):  
Julia Gambone ◽  
Stephanie Dusaban ◽  
Susan Shetzline
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Transcription Start Site ◽  
Myeloid Leukemia ◽  
Luciferase Activity ◽  
Hematopoietic Cells ◽  
Luciferase Reporter ◽  
Start Site ◽  
Transcription Start

Abstract Neuromedin U (NmU) is a 25-amino acid neuropeptide that is expressed at high levels in the brain, gut, and bone marrow. In the brain, NmU modulates the central control of feeding and bone mass. In the intestine, NmU regulates the contraction of smooth muscle, local blood flow, and ion transport. In the bone marrow, the physiological function of NmU remains ill-defined. To determine the physiological function of NmU in hematopoietic cells, we utilized K562-MERT cells, which express a tamoxifen-inducible dominant negative Myb (MERT), and cell culture assays. We chose to test the function of NmU in a cell line that expresses high levels of the proto-oncogene c-myb because c-Myb encodes a transcription factor that regulates cell proliferation, survival, and differentiation through the transactivation of its target genes. By inhibiting endogenous Myb activity in K562-MERT cells by tamoxifen in methylcellulose cultures, we observed a 6-fold decrease in cell number compared to untreated K562-MERT cells. Supplementing the tamoxifen treated methylcellulose cultures of K562-MERT cells with NmU resulted in a 3.6-fold increase in K562-MERT cell number compared to K562-MERT cells treated with only tamoxifen. In liquid cultures using primary cells from patients with acute myeloid leukemia, we observed more cells in the cultures with NmU than when NmU was absent from the cultures. Finally, silencing NmU gene expression in K562 cells via RNA interference decreased the proliferation of these cells. Collectively, these data demonstrate that NmU functions to promote the proliferation of hematopoietic cells. The ability of NmU to rescue cell growth in tamoxifen treated K562-MERT cells and Myb’s role in regulating hematopoietic cell proliferation led us to hypothesize that Myb mediates hematopoietic cell proliferation in part by directly regulating NmU gene expression. To test this hypothesis, we examined the DNA sequence upstream of NmU’s predicted transcription start site (as noted in Genbank accession #NM_006681) for potential Myb response elements (MREs). We identified eleven potential MREs within the first 2kb upstream of NmU’s transcription start site. Of these MREs, five were identified as canonical (PyAAC(G/T)G). Our search also identified potential Ets-2 binding motifs within the human NmU promoter region, which were of interest because Myb has been reported to cooperate with Ets-2 in the regulation of c-kit and CD34 gene expression. To determine if any of the potential MREs within the NmU promoter were functional, we first completed in vitro assays using luciferase reporter constructs followed by in vivo assays using chromatin immunoprecipitation (ChIP) assays. The luciferase reporter constructs were generated such that the first 2kb upstream of NmU’s transcription start site was inserted upstream of the luciferase gene in pGL3-basic to yield pGL3-NmU. When pGL3-NmU was co-transfected into 293T cells with a c-Myb expression construct, we observed an average of 15-fold induction of luciferase activity compared to empty vector. There was no change in luciferase activity when 293T cells were co-transfected with the c-Myb isoform B-Myb compared to empty vector, suggesting that c-Myb and not B-Myb interact with the MREs within the NmU promoter to induce its expression. Mutation of either MRE9 or 10, which are distal to NmU’s transcription start site, in pGL3-NmU prevented c-Myb from inducing luciferase gene expression, demonstrating that MRE9 and 10 are functional. When Ets-2 was co-transfected with pGL3-NmU into 293T cells, a 10-fold increase in luciferase activity was observed suggesting that Ets-2 and c-Myb may cooperate to increase the transcription of NmU. To determine the physiologic relevance of our in vitro luciferase studies, we performed ChIP assays. Following immunoprecipitation of the cross-linked chromatin with either anti-c-Myb or anti-Ets-2, a PCR product of the recovered DNA was observed using primers that flanked MRE9, MRE10, and one Ets-2 site, demonstrating that in vivo c-Myb and Ets-2 interact directly with NmU’s promoter. Studies are underway to determine whether c-Myb and Ets-2 cooperate to induce NmU gene expression. Further characterization of the regulation of NmU gene expression in normal and malignant hematopoietic cells may yield new clues to Myb’s role in leukemogenesis and could suggest new therapeutic targets in human myeloid leukemia cells.

scholarly journals Function and Transcriptional Regulation of Bovine TORC2 Gene in Adipocytes: Roles of C/EBP, XBP1, INSM1 and ZNF263

2019 ◽  
Vol 20 (18) ◽  
pp. 4338 ◽  
Author(s):  
Khan ◽  
Raza ◽  
Junjvlieke ◽  
Xiaoyu ◽  
Garcia ◽  
...  
Keyword(s):  
Transcription Start Site ◽  
Promoter Region ◽  
Binding Protein ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Marker Genes ◽  
Start Site ◽  
Transcription Start ◽  
Bovine Adipocytes

The TORC2 gene is a member of the transducer of the regulated cyclic adenosine monophosphate (cAMP) response element binding protein gene family, which plays a key role in metabolism and adipogenesis. In the present study, we confirmed the role of TORC2 in bovine preadipocyte proliferation through cell cycle staining flow cytometry, cell counting assay, 5-ethynyl-2′-deoxyuridine staining (EdU), and mRNA and protein expression analysis of proliferation-related marker genes. In addition, Oil red O staining analysis, immunofluorescence of adiponectin, mRNA and protein level expression of lipid related marker genes confirmed the role of TORC2 in the regulation of bovine adipocyte differentiation. Furthermore, the transcription start site and sub-cellular localization of the TORC2 gene was identified in bovine adipocytes. To investigate the underlying regulatory mechanism of the bovine TORC2, we cloned a 1990 bp of the 5' untranslated region (5′UTR) promoter region into a luciferase reporter vector and seven vector fragments were constructed through serial deletion of the 5′UTR flanking region. The core promoter region of the TORC2 gene was identified at location −314 to −69 bp upstream of the transcription start site. Based on the results of the transcriptional activities of the promoter vector fragments, luciferase activities of mutated fragments and siRNAs interference, four transcription factors (CCAAT/enhancer-binding protein C/BEP, X-box binding protein 1 XBP1, Insulinoma-associated 1 INSM1, and Zinc finger protein 263 ZNF263) were identified as the transcriptional regulators of TORC2 gene. These findings were further confirmed through Electrophoretic Mobility Shift Assay (EMSA) within nuclear extracts of bovine adipocytes. Furthermore, we also identified that C/EBP, XBP1, INSM1 and ZNF263 regulate TORC2 gene as activators in the promoter region. We can conclude that TORC2 gene is potentially a positive regulator of adipogenesis. These findings will not only provide an insight for the improvement of intramuscular fat in cattle, but will enhance our understanding regarding therapeutic intervention of metabolic syndrome and obesity in public health as well.

scholarly journals Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection

2016 ◽  
Vol 113 (21) ◽  
pp. E2899-E2905 ◽  
Author(s):  
Irina O. Vvedenskaya ◽  
Hanif Vahedian-Movahed ◽  
Yuanchao Zhang ◽  
Deanne M. Taylor ◽  
Richard H. Ebright ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Start Site ◽  
Transcription Initiation ◽  
Specific Protein ◽  
Start Site ◽  
Transcription Start ◽  
Transcription Bubble ◽  
Recognition Element

During transcription initiation, RNA polymerase (RNAP) holoenzyme unwinds ∼13 bp of promoter DNA, forming an RNAP-promoter open complex (RPo) containing a single-stranded transcription bubble, and selects a template-strand nucleotide to serve as the transcription start site (TSS). In RPo, RNAP core enzyme makes sequence-specific protein–DNA interactions with the downstream part of the nontemplate strand of the transcription bubble (“core recognition element,” CRE). Here, we investigated whether sequence-specific RNAP–CRE interactions affect TSS selection. To do this, we used two next-generation sequencing-based approaches to compare the TSS profile of WT RNAP to that of an RNAP derivative defective in sequence-specific RNAP–CRE interactions. First, using massively systematic transcript end readout, MASTER, we assessed effects of RNAP–CRE interactions on TSS selection in vitro and in vivo for a library of 47 (∼16,000) consensus promoters containing different TSS region sequences, and we observed that the TSS profile of the RNAP derivative defective in RNAP–CRE interactions differed from that of WT RNAP, in a manner that correlated with the presence of consensus CRE sequences in the TSS region. Second, using 5′ merodiploid native-elongating-transcript sequencing, 5′ mNET-seq, we assessed effects of RNAP–CRE interactions at natural promoters in Escherichia coli, and we identified 39 promoters at which RNAP–CRE interactions determine TSS selection. Our findings establish RNAP–CRE interactions are a functional determinant of TSS selection. We propose that RNAP–CRE interactions modulate the position of the downstream end of the transcription bubble in RPo, and thereby modulate TSS selection, which involves transcription bubble expansion or transcription bubble contraction (scrunching or antiscrunching).

scholarly journals Sequence of the 5′-flanking region and promoter activity of the human mucin gene MUC5B in different phenotypes of colon cancer cells

2000 ◽  
Vol 348 (3) ◽  
pp. 675-686 ◽  
Author(s):  
Isabelle VAN SEUNINGEN ◽  
Michaël PERRAIS ◽  
Pascal PIGNY ◽  
Nicole PORCHET ◽  
Jean-Pierre AUBERT
Keyword(s):  
Gene Expression ◽  
Transcription Start Site ◽  
Promoter Activity ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Start Site ◽  
Transcription Start ◽  
Mucin Gene ◽  
Intron 1 ◽  
Flanking Region

Control of gene expression in intestinal cells is poorly understood. Molecular mechanisms that regulate transcription of cellular genes are the foundation for understanding developmental and differentiation events. Mucin gene expression has been shown to be altered in many intestinal diseases and especially cancers of the gastrointestinal tract. Towards understanding the transcriptional regulation of a member of the 11p15.5 human mucin gene cluster, we have characterized 3.55 kb of the 5ʹ-flanking region of the human mucin gene MUC5B, including the promoter, the first two exons and the first intron. We report here the promoter activity of successively 5ʹ-truncated sections of 956 bases of this region by fusing it to the coding region of a luciferase reporter gene. The transcription start site was determined by primer-extension analysis. The region upstream of the transcription start site is characterized by the presence of a TATA box at bases -32/-26, DNA-binding elements for transcription factors c-Myc, N-Myc, Sp1 and nuclear factor ĸB as well as putative activator protein (AP)-1-, cAMP-response-element-binding protein (CREB)-, hepatocyte nuclear factor (HNF)-1-, HNF-3-, TGT3-, gut-enriched Krüppel factor (GKLF)-, thyroid transcription factor (TTF)-1- and glucocorticoid receptor element (GRE)-binding sites. Intron 1 of MUC5B was also characterized, it is 2511 nucleotides long and contains a DNA segment of 259 bp in which are clustered eight tandemly repeated GA boxes and a CACCC box that bind Sp1. AP-2α and GATA-1 nuclear factors were also shown to bind to their respective cognate elements in intron 1. In transfection studies the MUC5B promoter showed a cell-specific activity as it is very active in mucus-secreting LS174T cells, whereas it is inactive in Caco-2 enterocytes and HT-29 STD (standard) undifferentiated cells. Within the promoter, maximal transcription activity was found in a segment covering the first 223 bp upstream of the transcription start site. Finally, in co-transfection experiments a transactivating effect of Sp1 on to MUC5B promoter was seen in LS174T and Caco-2 cells.

scholarly journals Determination of the InvE Binding Site Required for Expression of IpaB of the Shigella sonnei Virulence Plasmid: Involvement of a ParB BoxA-Like Sequence

2003 ◽  
Vol 185 (17) ◽  
pp. 5158-5165 ◽  
Author(s):  
Takayuki Taniya ◽  
Jiro Mitobe ◽  
Shu-ichi Nakayama ◽  
Qi Mingshan ◽  
Kenji Okuda ◽  
...  
Keyword(s):  
Dna Binding ◽  
Transcription Start Site ◽  
Promoter Region ◽  
Binding Activity ◽  
Shigella Sonnei ◽  
Virulence Plasmid ◽  
Start Site ◽  
Transcription Start ◽  
Dna Binding Activity ◽  
K 12

ABSTRACT The InvE protein positively regulates the expression of virulence genes ipaBCD in Shigella sonnei. The InvE has significant homology with ParB of plasmid P1, which is known as a plasmid partitioning factor with DNA binding ability. Although the DNA binding activity of InvE has been predicted, it is not known whether the DNA binding activity is necessary for type III secretion system-associated gene expression. In this study, we determined the transcription start site of the icsB-ipaBCD operon (ipa operon) and constructed a series of deletions of the icsB promoter region in the Escherichia coli K-12 background. The deletion study revealed that an 86-bp region upstream of the icsB transcription start site was essential for expression of the ipa operon, where the ParB binding motif (ParB BoxA-like sequence) was observed. Purified glutathione S-transferase-InvE fusion protein bound directly to the −93 to −54 region (designating the icsB transcription start site as nucleotide +1) containing the ParB BoxA-like sequence. These results indicated that InvE bound directly to the promoter region.

scholarly journals Nascent RNA sequencing identifies a widespread sigma70-dependent pausing regulated by Gre factors in bacteria

2020 ◽  
Author(s):  
Zhe Sun ◽  
Alexander Yakhnin ◽  
Peter C. FitzGerald ◽  
Carl E. Mclntosh ◽  
Mikhail Kashlev
Keyword(s):  
Rna Polymerase ◽  
Transcription Start Site ◽  
Environmental Cues ◽  
Start Site ◽  
Transcription Start ◽  
E Coli ◽  
Eukaryotic Genes ◽  
Genome Wide ◽  
Transcript Cleavage

ABSTRACTPromoter-proximal pausing regulates expression of many eukaryotic genes and serves as checkpoints for assembly of elongation/splicing machinery. Little is known how broadly the pausing is employed in transcriptional regulation in bacteria. We applied NET-seq combined with RNase I footprinting for genome-wide analysis of σ70-dependent transcription pauses in Escherichia coli. Many E. coli genes appear to contain clusters of strong backtracked pauses at 10-20-bp distance from the transcription start site caused by retention of σ70 subunit in RNA polymerase. The pauses in 10-15-bp register of the promoter are dictated by binding of σ70 to canonical −10 element, 6-7 nt spacer and “YR+1Y” motif centered at transcription start site all characteristic for strong E. coli promoters. The promoters for the pauses in 16-20-bp register contain an additional −10-like sequence positioned on the same face of the DNA duplex as the original −10 element suggesting that σ70 hopping was responsible for these pauses. Our in vitro analysis reveals that RNA polymerase backtracking and DNA scrunching are involved in these pauses that are relieved by Gre transcript cleavage factors. The genes coding for transcription factors are enriched in these pauses suggesting that σ70 and Gre proteins regulate transcription in response to changing environmental cues.

MOLECULAR CLONING OF THE HUMAN GENE FOR VON WILLEBRAND FACTOR AND IDENTIFICATION OF THE TRANSCRIPTION INITIATION SITE

1987 ◽  
Author(s):  
Corolyn J Collins ◽  
Richard B Levene ◽  
Christina P Ravera ◽  
Marker J Dombalagian ◽  
David M Livingston ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transcription Start Site ◽  
Von Willebrand Factor ◽  
Transcription Initiation ◽  
Promoter Region ◽  
Start Site ◽  
Transcription Start ◽  
Von Willebrand's Disease ◽  
Von Willebrand ◽  
Willebrand Factor

Most patients with von Willebrand's disease appear to have a defect affecting the level of expression of the von Willebrand factor (vWf) gene. Thus, an understanding of the pathogenesis of von Willebrand's disease will require an analysis of the structure and function of the vWf gene in normals and in patients. To begin such analyses, we have screened a human genomic cosmid library with probes obtained from vWf cDNA and isolated a colinear segment spanning ≈175 kb in five overlapping clones. This segment extends ≈25 kb upstream and ≈5 kb downstream of the transcription start and stop sites for vWf mRNA, implying the vWf gene has a length of ≈150 kb. Within one of these clones, the vWf transcription initiation sites have been mapped. A portion of the promoter region has been sequenced, revealing a typical TATA box, a downstream CCAAT box, and a perfect downstream repeat of the 8 base pairs containing the major transcription start site. Primer extension analysis suggests that sequences contained within the downstream repeat of the transcription start site may be used as minor initiation sites in endothelial cells. Transfection studies are underway to evaluate the role of sequences within this promoter region in gene regulatory activity. Comparative restriction analyses of cloned and chromosomal DNA segments strongly suggests that no major alterations ocurred during cloning and that there is only one complete copy of the vWf gene in the human haploid genome. Similar analyses of DNA from vWf-expressing endothelial cells and non-expressing white blood cells suggests that no major rearrangements are associated with vWf gene expression. Finally, cross hybridization patterns among seven mammalian species suggests a strong conservation of genomic sequences encoding the plasma portion of vWf, but a lower degree of conservation of sequences encoding the N terminal region of provWf.

scholarly journals Functional analysis of the human annexin I and VI gene promoters

1998 ◽  
Vol 332 (3) ◽  
pp. 681-687 ◽  
Author(s):  
Shaun R. DONNELLY ◽  
Stephen E. MOSS
Keyword(s):  
Gene Expression ◽  
Transcription Start Site ◽  
Luciferase Reporter ◽  
Gene Promoters ◽  
Start Site ◽  
Luciferase Reporter Gene ◽  
Transcription Start ◽  
A431 Cells ◽  
Annexin I ◽  
Annexin Vi

To gain insight into the molecular basis of annexin gene expression we have analysed the annexin I and VI gene promoters. A previously described 881 bp sequence immediately upstream of the annexin I transcription start site and a similar size fragment proximal to the annexin VI transcription start site both drove expression of the luciferase reporter gene in fibroblasts and epithelial cells. Neither promoter displayed any sensitivity to dexamethasone, suggesting that the putative glucocorticoid response element in the annexin I promoter is non-functional. Consistent with this, endogenous annexin I gene expression was unaffected by dexamethasone at the mRNA and protein levels in A431 cells. A series of 5´ deletions of the two promoters were examined to define the minimal active sequences. For annexin I this corresponded to a sequence approx. 150 bp upstream of the transcription start site that included CAAT and TATA boxes. Unexpectedly, the annexin VI promoter, which also contains CAAT and TATA boxes, was fully active in the absence of these elements, a 53 bp sequence between these boxes and the transcription start site having maximal activity. Electrophoretic mobility-shift assays with nuclear extracts from A431 and HeLa cells with probes corresponding to this region revealed an SP1-binding site. These results show that the annexin I and VI genes have individual modes of transcriptional regulation and that if either annexin I or annexin VI has an anti-inflammatory role, then this is in the absence of steroid-induced gene expression.

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