scholarly journals Identification of a Previously Unrecognized Promoter That Drives Expression of the UXP Transcription Unit in the Human Adenovirus Type 5 Genome

2010 ◽  
Vol 84 (21) ◽  
pp. 11470-11478 ◽  
Author(s):  
Baoling Ying ◽  
Ann E. Tollefson ◽  
William S. M. Wold
Keyword(s):  
Transcription Initiation ◽  
Promoter Activity ◽  
Mrna Level ◽  
A549 Cells ◽  
Initiation Site ◽  
Transcription Unit ◽  
Transcription Initiation Site ◽  
Luciferase Reporter ◽  
Rnase Protection ◽  
Ccaat Box

ABSTRACT We previously identified an adenovirus (Ad) protein named U exon protein (UXP) encoded by a leftward-strand (l-strand) transcription unit. Here we identify and characterize the UXP promoter. Primer extension and RNase protection assays mapped the transcription initiation site at 32 nucleotides upstream of the UXP gene initiation codon. A series of viral mutants with mutations at two putative inverted CCAAT (I-CCAAT) boxes and two E2F sites were generated. With mutants lacking the proximal I-CCAAT box, the UXP mRNA level decreased significantly to 30% of the Ad type 5 (Ad5) mRNA level as measured by quantitative reverse transcription-PCR. Decreased UXP was also observed by immunoblotting and immunofluorescence. UXP mRNA and protein levels were similar to those of Ad5 for mutants lacking the distal I-CCAAT box or both putative E2F sites. Ad DNA levels were similar in mutant- and wild-type Ad5-infected cells during the late stage of infection, strongly suggesting that the decreased UXP mRNA and protein from mutants lacking the proximal I-CCAAT box was due to decreased promoter activity. Electrophoretic mobility shift assays (EMSA) indicated that a cellular factor binds specifically to the proximal I-CCAAT box of the UXP promoter. An in vitro luciferase reporter assay demonstrated that basal promoter activity lies between bp −158 and +30 of the transcription initiation site. No E1A-mediated promoter transactivation was observed in 293 cells compared with A549 cells. Thus, we propose that there is a previously unidentified Ad5 promoter that drives expression of the UXP transcription unit. This promoter is embedded within the gene for fiber, and it contains a proximal I-CCAAT box critical for UXP mRNA transcription.

Characterization of a chicken retinoid X receptor-γ gene promoter and identification of sequences that direct expression in retinal cells

2000 ◽  
Vol 347 (2) ◽  
pp. 485-490
Author(s):  
Clara AMEIXA ◽  
Paul M. BRICKELL
Keyword(s):  
Transcription Initiation ◽  
Promoter Activity ◽  
Initiation Site ◽  
Neural Retina ◽  
Specific Protein ◽  
Retinoid X Receptor ◽  
Transcription Initiation Site ◽  
Luciferase Reporter ◽  
Enhancer Element ◽  
Extracellular Signals

Development of the cellular complexity of the vertebrate neural retina relies on an intricate interplay between extracellular signals and intracellular factors. In particular, transcription factors play a key role in determining the competence of cells to respond to extracellular signals. We have previously shown that, in the developing chick neural retina, expression of the retinoid X receptor-γ (RXR-γ2) nuclear receptor gene is restricted to photoreceptors. To characterize the mechanisms that regulate expression of this gene in the neural retina, we isolated a chicken RXR-γ genomic clone containing the RXR-γ2 promoter and mapped the transcription initiation site by means of ribonuclease protection. We analysed promoter activity by transient transfection of luciferase reporter gene constructs into cultured cells isolated from embryonic-chick neural retina or facial mesenchyme, which does not normally express detectable RXR-γ2 transcripts. The DNA fragment lying between nucleotides -657 and +37 with respect to the transcription initiation site had basal promoter activity in both cell types. The fragment lying between nucleotides -1198 and -991 directed 10-20-fold higher levels of luciferase activity in neural retina cells, but only basal levels in facial mesenchyme cells. This 208 bp fragment also enhanced the activity of the simian-virus-40 promoter, when placed upstream in either orientation. Electrophoretic-mobility-shift assays using this 208 bp fragment demonstrated the formation of four neural retina-specific protein-DNA complexes. These results indicate that regulation of RXR-γ2 transcription in the developing chick neural retina involves the binding of one or more neural retina-specific protein factors to an enhancer element located approx. 1 kbp upstream of the transcription initiation site.

scholarly journals The gene structure and promoter sequence of mouse hyaluronan synthase 1 (mHAS1)

1998 ◽  
Vol 330 (3) ◽  
pp. 1223-1227 ◽  
Author(s):  
Yoichi YAMADA ◽  
Naoki ITANO ◽  
Masahiro ZAKO ◽  
Mamoru YOSHIDA ◽  
Petros LENAS ◽  
...  
Keyword(s):  
Gene Structure ◽  
Transcription Initiation ◽  
Direct Sequencing ◽  
Promoter Sequence ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Hyaluronan Synthase ◽  
Pcr Method ◽  
Rapid Amplification ◽  
Ccaat Box

The structure and organization of mouse hyaluronan synthase 1 gene, HAS1 were determined by direct sequencing of λ phage clones carrying the entire gene and by application of the long and accurate (LA)-PCR method to amplify regions encompassing the exon-intron boundaries and all of the exons. This gene spans about 11 kb of genomic DNA and consists of 5 exons and 4 introns. A similarity in the exon-intron organization was found between the genes of mouse HAS1 and Xenopus laevis DG42 which was recently identified as Xenopus hyaluronan synthase. The transcription initiation site was determined by rapid amplification of the cDNA ends (5ʹ-RACE). Position +1 is located 55 nucleotides upstream of the ATG initiation codon. The promoter region of the HAS1 gene has no typical TATA box, but contains a CCAAT box located 190 nucleotides upstream of the transcription initiation site. Further analysis of 1.4 kb of the 5ʹ flanking region revealed several potential binding motifs for transcription factors. This information about the gene structure may be useful for further studies on the promoter activity.

Selective Sp1 Binding Is Critical for Maximal Activity of the Human c-kit Promoter

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4138-4149
Author(s):  
Gyeong H. Park ◽  
Howard K. Plummer ◽  
Geoffrey W. Krystal
Keyword(s):  
Binding Site ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Core Promoter ◽  
Maximal Activity ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Rnase Protection ◽  
Kit Gene ◽  
Core Promoter Activity

The receptor tyrosine kinase c-kit is necessary for normal hematopoiesis, the development of germ cells and melanocytes, and the pathogenesis of certain hematologic and nonhematologic malignancies. To better understand the regulation of the c-kit gene, a detailed analysis of the core promoter was performed. Rapid amplification of cDNA ends (RACE) and RNase protection methods showed two major transcriptional initiation sites. Luciferase reporter assays using 5′ promoter deletion-reporter constructs containing up to 3 kb of 5′ sequence were performed in hematopoietic and small-cell lung cancer cell lines which either did or did not express the endogenous c-kit gene. This analysis showed the region 83 to 124 bp upstream of the 5′ transcription initiation site was crucial for maximal core promoter activity. Sequence analysis showed several potential Sp1 binding sites within this highly GC-rich region. Gel shift and DNase footprinting showed that Sp1 selectively bound to a single site within this region. Supershift studies using an anti-Sp1 antibody confirmed specific Sp1 binding. Site-directed mutagenesis of the −93/−84 Sp1 binding site reduced promoter-reporter activity to basal levels in c-kit–expressing cells. Cotransfection into DrosophilaSL2 cells of a c-kit promoter-reporter construct with an Sp1 expression vector showed an Sp1 dose-dependent enhancement of expression that was markedly attenuated by mutation of the −93/−84 site. These results indicate that despite the fact that the human c-kit promoter contains multiple potential Sp1 sites, Sp1 binding is a selective process that is essential for core promoter activity.

scholarly journals Sp1-Mediated Transcription of the Werner Helicase Gene Is Modulated by Rb and p53

1998 ◽  
Vol 18 (11) ◽  
pp. 6191-6200 ◽  
Author(s):  
Yukako Yamabe ◽  
Akira Shimamoto ◽  
Makoto Goto ◽  
Jun Yokota ◽  
Minoru Sugawara ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Promoter Activity ◽  
Transcriptional Control ◽  
Housekeeping Genes ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Control Element ◽  
Mrna Levels

ABSTRACT The regulation of Werner’s syndrome gene (WRN) expression was studied by characterizing the cis-regulatory elements in the promoter region and the trans-activating factors that bind to them. First, we defined the transcription initiation sites and the sequence of the 5′ upstream region (2.8 kb) ofWRN that contains a number of cis-regulatory elements, including 7 Sp1, 9 retinoblastoma control element (RCE), and 14 AP2 motifs. A region consisting of nucleotides −67 to +160 was identified as the principal promoter of WRN by reporter gene assays in HeLa cells, using a series of WRNpromoter-luciferase reporter (WRN-Luc) plasmids that contained the 5′-truncated or mutated WRN upstream regions. In particular, two Sp1 elements proximal to the transcription initiation site are indispensable for WRN promoter activity and bind specifically to Sp1 proteins. The RCE enhances WRN promoter activity. Coexpression of the WRN-Luc plasmids with various dosages of plasmids expressing Rb or p53 in Saos2 cells lacking active Rb and p53 proteins showed that the introduced Rb upregulates WRN promoter activity a maximum of 2.5-fold, while p53 downregulates it a maximum of 7-fold, both dose dependently. Consistently, the overexpressed Rb and p53 proteins also affected the endogenous WRN mRNA levels in Saos2 cells, resulting in an increase with Rb and a decrease with p53. These findings suggest that WRN expression, like that of other housekeeping genes, is directed mainly by the Sp1 transcriptional control system but is also further modulated by transcription factors, including Rb and p53, that are implicated in the cell cycle, cell senescence, and genomic instability.

scholarly journals IL-4 and IL-13 InduceSOCS-1Gene Expression in A549 Cells by Three Functional STAT6-Binding Motifs Located Upstream of the Transcription Initiation Site

2003 ◽  
Vol 171 (11) ◽  
pp. 5901-5907 ◽  
Author(s):  
Daniel Hebenstreit ◽  
Petra Luft ◽  
Angela Schmiedlechner ◽  
Gerhard Regl ◽  
Anna-Maria Frischauf ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
A549 Cells ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Binding Motifs

scholarly journals Identification and analysis of the promoter region of the human methionine sulphoxide reductase A gene

2005 ◽  
Vol 393 (1) ◽  
pp. 321-329 ◽  
Author(s):  
Antonella De Luca ◽  
Paolo Sacchetta ◽  
Carmine Di Ilio ◽  
Bartolo Favaloro
Keyword(s):  
Cell Lines ◽  
Transcription Initiation ◽  
Promoter Region ◽  
Promoter Activity ◽  
Regulatory Region ◽  
Initiation Site ◽  
Luciferase Reporter ◽  
Mcf7 Cells ◽  
Luciferase Reporter Gene ◽  
Hek 293

MsrA (methionine sulphoxide reductase A) is an antioxidant repair enzyme that reduces oxidized methionine to methionine. Moreover, the oxidation of methionine residues in proteins is considered to be an important consequence of oxidative damage to cells. To understand mechanisms of human msrA gene expression and regulation, we cloned and characterized the 5′ promoter region of the human msrA gene. Using 5′-RACE (rapid amplification of cDNA ends) analysis of purified mRNA from human cells, we located the transcription initiation site 59 nt upstream of the reference MsrA mRNA sequence, GenBank® accession number BC 054033. The 1.3 kb of sequence located upstream of the first exon of msrA gene was placed upstream of the luciferase reporter gene in a pGL3-Basic vector and transfected into different cell lines. Sequentially smaller fragments of the msrA promoter region were generated by PCR, and expression levels were monitored from these constructs within HEK-293 and MCF7 human cell lines. Analysis of deletion constructs revealed differences in promoter activity in these cell lines. In HEK-293 cells, the promoter activity was constant from the minimal promoter region to the longest fragment obtained. On the other hand, in MCF7 cells we detected a down-regulation in the longest fragment. Mutation of a putative negative regulatory region that is located between −209 and −212 bp (the CCAA box) restored promoter activity in MCF7 cells. The location of the msrA promoter will facilitate analysis of the transcriptional regulation of this gene in a variety of pathological contexts.

scholarly journals Sp1 and Sp3 control constitutive expression of the human NHE2 promoter by interactions with the proximal promoter and the transcription initiation site

2007 ◽  
Vol 407 (1) ◽  
pp. 101-111 ◽  
Author(s):  
Ian Pearse ◽  
Ying X. Zhu ◽  
Eleanor J. Murray ◽  
Pradeep K. Dudeja ◽  
Krishnamurthy Ramaswamy ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Promoter Region ◽  
Promoter Activity ◽  
Critical Role ◽  
Constitutive Expression ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Proximal Promoter ◽  
Gc Box

We have previously cloned the human Na+/H+ exchanger NHE2 gene and its promoter region. In the present study, the regulatory elements responsible for the constitutive expression of NHE2 were studied. Transient transfection assays revealed that the −40/+150 promoter region contains the core promoter responsible for the optimal promoter activity. A smaller fragment, −10/+40, containing the TIS (transcription initiation site) showed minimal activity. We identified a palindrome that overlaps the TIS and binds to the transcription factors Sp1 and Sp3. Mutations in the 5′ flank of the palindrome abolished the Sp1/Sp3 interaction and reduced promoter activity by approx. 45%. In addition, a conserved GC-box centered at −25 was found to play a critical role in basal promoter activity and also interacted with Sp1 and Sp3. An internal deletion in the GC-box severely reduced the promoter activity. Sp1/Sp3 binding to these elements was established using gel-mobility shift assays, confirmed by chromatin immunoprecipitation and co-transfections in Drosophila SL2 cells. Furthermore, we identified two positive regulatory elements in the DNA region corresponding to the 5′-UTR (5′-untranslated region). The results in the present study indicate that Sp1 and Sp3 are required for constitutive NHE2 expression and that the positive regulatory elements of the 5′-UTR may co-operate with the 5′-flanking region to achieve the optimal promoter activity.

scholarly journals c-mos expression in mouse oocytes is controlled by initiator-related sequences immediately downstream of the transcription initiation site

1991 ◽  
Vol 11 (10) ◽  
pp. 5190-5196
Author(s):  
S K Pal ◽  
S S Zinkel ◽  
A A Kiessling ◽  
G M Cooper
Keyword(s):  
Transcription Start Site ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Consensus Sequence ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Transcriptional Level ◽  
Start Site ◽  
Transcription Start ◽  
Mouse Oocytes

We have employed transient expression assays to analyze the sequences that direct c-mos transcription in mouse oocytes. Plasmids containing the chloramphenicol acetyltransferase (CAT) gene fused to either a 2.4-kb or a 731-bp fragment from the 5'-flanking region of c-mos produced similar levels of CAT activity when injected into nuclei of growing oocytes. BAL 31 deletions revealed that sequences up to 20 bp upstream of the major transcription start site could be removed without any significant loss of CAT activity. Promoter activity only decreased when these deletions closely approached the transcription start site, which was mapped at 53 nucleotides upstream of the first ATG in the c-mos open reading frame. On the other hand, deletion of sequences within 20 nucleotides downstream of the transcription initiation site resulted in a 10-fold reduction in CAT expression. A similar decrease in promoter activity was observed as a result of point mutations in these 5' untranslated sequences. Thus, sequences immediately downstream of the transcription start site, including a consensus sequence (PyPyCAPyPyPyPyPy) present in the initiator elements of several genes, appear to regulate c-mos expression in mouse oocytes. Reverse transcription-polymerase chain reaction analysis of RNA from injected oocytes showed that this regulation is manifest at the transcriptional level. Expression of c-mos in mouse oocytes thus appears to be directed by a simple promoter consisting only of sequences immediately surrounding the transcription start site, including an initiator element in the untranslated leader.

Isolation and Characterization of Human ADAMTS13 Gene Promoter Region - Control of ADAMTS13 Gene Expression by Hepatic Transcription Factors and Inflammatory Cytokines.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1603-1603 ◽  
Author(s):  
Xingwu Zheng ◽  
Masami Niiya ◽  
X. Long Zheng ◽  
Eleanor S. Pollak
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Inflammatory Cytokines ◽  
Binding Sites ◽  
Transcription Initiation ◽  
Promoter Region ◽  
Promoter Activity ◽  
Initiation Site ◽  
Luciferase Reporter ◽  
Tnf Alpha

Abstract ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats-13) controls von Willebrand factor multimer sizes by cleaving the Tyr1605-Met1606 bond in the central A2 domain. Deficiency of plasma ADAMTS13 activity can result in a lethal syndrome, thrombotic thrombocytopenic purpura (TTP). ADAMTS13 is primarily synthesized in hepatic stellate cells (HSCs), endothelial cells and megakaryocytes. We determined the transcription initiation site, the core region for promoter activity, the putative transcription factor binding sites as well as the influence of inflammatory cytokines on ADAMTS13 promoter activity. To explore the transcriptional control of ADAMTS13 gene expression, we constructed reporter genes containing 991 base pairs (bp) of the ADAMTS13 5′ untranslated (UT) region. We showed by deletion mutagenesis and luciferase reporter expression that the proximal-most 197 bp region was required for maximal luciferase activity in transfected cells in the human hepatic stellate cell line (LX-2) and in the human hepatocyte-like cell line (HepG2); the major transcription initiation site determined by 5′ - RACE was found at 77 bp upstream from the translation start site (ATG). However, the minimal sequences that were required for the promoter activity varied depending on the cells, with required sequences of approximately 147 and 127 bp in LX-2 and HepG2 cells, respectively. The proximal ADAMTS13 promoter region is evolutionally conserved between humans, mice and rats. This region is rich in GC content (72%) and contains putative binding sites for the transcription factors heat shock factor-2 (HSF2), FOXa2 [also named hepatocyte nuclear factor 3beta (HNF-3b)] and AP-1. A footprint assay demonstrated that the region between −116 and −126, containing the putative FOXa2 binding site, was largely protected by Dnase I digestion. The luciferase reporter activity was suppressed in cells transfected with the plasmid containing the proximal 314 bp human 5′ UT ADAMTS13 sequence in parallel with the inflammatory cytokines found to be elevated in patients with TTP: IL-4, TNF-alpha and INF-gamma. These inflammatory cytokines inhibited the Adamts13 mRNA and protein expression in rat primary HSCs in culture in a dose dependent manner. Approximately 70%, 71% and 80% of Adamts13 mRNA (by real time RT-PCR) and 77%, 78% and 92% of Adamts13 proteolytic activity (by FRETS-VWF73) were suppressed at 48 hours by IL-4 (10 ng/ml), TNF-alpha (10 ng/ml) and INF-gamma (100 ng/ml), respectively. We conclude that under physiological conditions ADAMTS13 synthesis may be strictly maintained at relatively low levels by binding transcription factors, whereas under pathological conditions inflammatory cytokines, released due to systemic inflammation, may further suppress ADAMTS13 gene expression, which may result in thrombotic complications. However, the mechanism regarding how the inflammatory cytokines negatively regulate ADAMTS13 (or Adamts13) synthesis remains to be determined.

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