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

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.

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

Blood ◽

10.1182/blood.v108.11.1603.1603 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1603-1603 ◽

Cited By ~ 1

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

Biochemical Journal ◽

10.1042/bj3470485 ◽

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.18.11.6191 ◽

1998 ◽

Vol 18 (11) ◽

pp. 6191-6200 ◽

Cited By ~ 59

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.

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

Journal of Virology ◽

10.1128/jvi.01338-10 ◽

2010 ◽

Vol 84 (21) ◽

pp. 11470-11478 ◽

Cited By ~ 7

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.

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Functional analysis of haplotypes and promoter activity at the 5' region of the DRD2 gene and associations with schizophrenia.

10.21203/rs.3.rs-15812/v1 ◽

2020 ◽

Author(s):

Xicen Zhang ◽

Mei Ding ◽

Yi Liu ◽

Yongping Liu ◽

Jiaxin Xing ◽

...

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Promoter Region ◽

Regulatory Region ◽

Gene Promoter ◽

Luciferase Reporter ◽

Drd2 Gene ◽

Functional Regions

Abstract Background: In previous studies, we researched the association of the DRD2 gene promoter region SNP loci rs7116768, rs1047479195, rs1799732, rs1799978 and schizophrenia using Sanger sequencing. rs7116768 and rs1799978 were found to be slightly associated with schizophrenia. This study investigated the effects of haplotypes consisted of the four SNPs on protein expression level in vitro and identified the functional sequence in the 5’ regulatory region of DRD2 gene which has a potential link with schizophrenia.Methods: Recombinant plasmids with haplotypes, SNPs and 13 recombinant vectors containing deletion fragments from the DRD2 gene 5' regulatory region were transfected into HEK293 and SK-N-SH cell lines. Relative luciferase activity of the haplotypes, SNPs and different sequences was compared using a dual luciferase reporter assay system.Results: Haplotype H4(G-C-InsC-G) could significantly increase the gene expression in SK-N-SH cell lines. Allele C of rs7116768, allele A of rs1047479195 and allele del C of rs1799732 could up-regulate the gene expression. There were 5~7 functional regions in the promoter region of DRD2 gene that could affect the level of gene expression.Conclusion: We cannot rule out the possibility that different haplotypes may influence DRD2 gene expression in vivo. We observed that allele C of rs7116768, allele A of rs1047479195 and allele del C of rs1799732 could up-regulate gene expression. The truncation results confirmed the existence of functional regions in the promoter region of DRD2 gene that could affect the level of gene expression.

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Regulation of the human biotin transporter hSMVT promoter by KLF-4 and AP-2: confirmation of promoter activity in vivo

AJP Cell Physiology ◽

10.1152/ajpcell.00360.2006 ◽

2007 ◽

Vol 292 (4) ◽

pp. C1305-C1312 ◽

Cited By ~ 17

Author(s):

Jack C. Reidling ◽

Hamid M. Said

Keyword(s):

Epithelial Cell ◽

Cell Lines ◽

Promoter Activity ◽

Regulatory Region ◽

Regulatory Elements ◽

Minimal Promoter ◽

Intestinal Cell ◽

Epithelial Cell Line ◽

Hek 293

The mechanism of biotin uptake in human intestine has been well characterized and involves the human sodium-dependent multivitamin transporter (hSMVT), yet little is known about the molecular/transcriptional regulation of the system. Previous investigations cloned the 5′ regulatory region of the hSMVT gene and identified the minimal promoter. To expand these investigations, we compared activity of the hSMVT promoter in three human intestinal epithelial cell lines (NCM460, Caco-2, and HuTu-80) and contrasted a renal epithelial cell line (HEK-293). We analyzed the role of putative cis-elements in regulating promoter activity and confirmed activity of the cloned hSMVT promoter in vivo. In vitro studies demonstrated that all cell lines utilized the same minimal promoter region, and mutation of specific cis-regulatory elements [Kruppel-like factor 4 (KLF-4) and activator protein-2 (AP-2)] led to a decrease in promoter activity in all intestinal cell types but not in renal cells. Using electrophoretic mobility shift assays, we identified two specific DNA/protein complexes. Using oligonucleotide competition and antibody supershift analysis, we determined that KLF-4 and AP-2 were involved in forming the complexes. In HEK-293 cells, overexpressing KLF-4 increased the endogenous hSMVT message levels threefold and activated a cotransfected hSMVT promoter-reporter construct. In vivo studies using hSMVT promoter-luciferase transgenic mice established physiological relevance and showed the pattern of hSMVT promoter expression to be similar to endogenous mouse SMVT mRNA expression. The results demonstrate, for the first time, the importance of KLF-4 and AP-2 in regulating the activity of the hSMVT promoter in the intestine and provide direct in vivo confirmation of hSMVT promoter activity.

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Transcriptional regulation of the cystathionine-β-synthase gene in Down syndrome and non–Down syndrome megakaryocytic leukemia cell lines

Blood ◽

10.1182/blood-2002-07-2337 ◽

2003 ◽

Vol 101 (4) ◽

pp. 1551-1557 ◽

Cited By ~ 35

Author(s):

Yubin Ge ◽

Tanya L. Jensen ◽

Larry H. Matherly ◽

Jeffrey W. Taub

Keyword(s):

Transcriptional Regulation ◽

Down Syndrome ◽

Cell Line ◽

Cell Lines ◽

Promoter Activity ◽

Leukemia Cell ◽

Chromosome 21 ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Nuclear Extracts

Children with Down syndrome (DS) with acute myeloid leukemia (AML) have significantly higher event-free survival rates compared to those with non-DS AML, linked to greater cytosine arabinoside (ara-C) sensitivity and higher transcript levels of the chromosome 21–localized gene, cystathionine-β-synthase(CBS), in DS myeloblasts. In this study, we examined the transcriptional regulation of the CBS gene in the DS megakaryocytic leukemia (AMkL) cell line, CMK, characterized by significantly higher CBS transcripts compared with the non-DS AMkL cell line, CMS. Rapid amplification of 5′-cDNA ends (5′-RACE) analysis demonstrated exclusive use of the CBS−1b promoter in the cell lines, and transient transfections with the full-length CBS −1b luciferase reporter gene construct showed 40-fold greater promoter activity in the CMK than CMS cells. Electrophoretic mobility shift assays showed enhanced binding of the transcription factors Sp1/Sp3 to 2 GC/GT-box elements (GC-f and GT-d) in the upstream regions of the CBS −1b promoter in CMK nuclear extracts and undetectable binding in CMS cells. Mutation of the GC-f– or GT-d–binding site resulted in an approximately 90% decrease of theCBS −1b promoter activity in transient transfections of CMK cells. Chromatin immunoprecipitation assays confirmed in vivo binding of Sp3, USF-1, and nuclear factor YA (NF-YA) to theCBS −1b promoter region in chromatin extracts of CMK and CMS cells. Decreased binding of Sp1/Sp3 in CMK nuclear extracts following treatment with calf alkaline phosphatase suggested a role for phosphorylation of Sp1/Sp3 in regulating CBS promoter activity and in the differential CBS expression between CMK and CMS cells. The results of this study with clinically relevant cell line models suggest potential mechanisms for disparate patterns ofCBS gene expression in DS and non-DS myeloblasts and may, in part, explain the greater sensitivity to chemotherapy shown by patients with DS AML.

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Functional mapping of the mouse hairless gene promoter region

10.1101/244426 ◽

2018 ◽

Author(s):

Eric G. Folco ◽

Stefan Nonchev

Keyword(s):

Hair Follicle ◽

Cell Lines ◽

Dna Sequences ◽

Promoter Region ◽

Regulatory Region ◽

Hair Follicles ◽

Luciferase Reporter ◽

Control Element ◽

Reporter Expression ◽

Hairless Gene

AbstractThe mouse hairless gene (Hr) encodes a protein of 127 kDa, acting as corepressor of nuclear hormone receptors. The Hairless protein (HR) is involved in the control of the cellular transition to the first hair cycle in adult Mammals. In its absence hair follicles disintegrate leading to a complete and irreversible hair loss with formation of cutaneous cysts. The hairless phenotype is therefore linked to defective proliferation and migration of the hair follicle stem cells apparently unable to respond to various signalling molecules. The Hr gene is expressed at high levels in skin and brain, and hairless transcripts were detected in gonads, thymus and colon. Although the patterns of Hr expression appear to be spatially and temporally regulated, very little is known about the molecular basis of the transcriptional control underlying Hr gene function. In this work we determine the precise transcriptional initiation start site of the mouse Hr gene and identify a new 1,1 kb cis-control element (RE1) that encompasses the promoter region and is able to drive luciferase reporter expression in skin and brain derived cell lines. We performed a deletion analysis and explored functionally regulatory motifs within this fragment to show that the role of this upstream regulatory region is linked to the presence of TRE and VDRE binding sites. We find that a TRE situated at –300 bp from the cap site is essential for gene expression in both skin NIH 3T3 and GHFT1 cells, while a VDRE positioned 94 bp upstream of the TRE modulates reporter expression specifically in skin derived cell lines. In addition, we define a novel cis-regulatory motif UE60, situated at the 5’-end of RE1 and likely to interact with both TRE and VDRE. Our data complete previous results on the possible existence of an autoregulatory pathway, implicated in Hr gene regulation. Taken together these findings reveal a complex molecular network that potentially links several signalling pathways in hair follicle formation. We discuss the organisation of the regulatory modules in the mouse Hr gene upstream DNA sequences in the light of the high homology of this region in mouse, rat and human.

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

Biochemical Journal ◽

10.1042/bj20070364 ◽

2007 ◽

Vol 407 (1) ◽

pp. 101-111 ◽

Cited By ~ 9

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.

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The Chilo iridescent virus DNA polymerase promoter contains an essential AAAAT motif

Journal of General Virology ◽

10.1099/vir.0.82947-0 ◽

2007 ◽

Vol 88 (9) ◽

pp. 2488-2494 ◽

Cited By ~ 18

Author(s):

Remziye Nalçacioğlu ◽

Ikbal Agah Ince ◽

Just M. Vlak ◽

Zihni Demirbağ ◽

Monique M. van Oers

Keyword(s):

Dna Polymerase ◽

Transcription Initiation ◽

Promoter Activity ◽

Transcriptional Start Site ◽

Point Mutations ◽

Luciferase Reporter ◽

Start Site ◽

Chilo Iridescent Virus ◽

Luciferase Reporter Gene ◽

Iridescent Virus

The delayed-early DNA polymerase promoter of Chilo iridescent virus (CIV), officially known as Invertebrate iridescent virus, was fine mapped by constructing a series of increasing deletions and by introducing point mutations. The effects of these mutations were examined in a luciferase reporter gene system using Bombyx mori cells transfected with promoter constructs and infected with CIV. When the size of the upstream element was reduced from position −19 to −15, relative to the transcriptional start site, the luciferase activity was reduced to almost zero. Point mutations showed that each of the 5 nt (AAAAT) located between –19 and –15 were equally essential for promoter activity. Mutations at individual bases around the transcription initiation site showed that the promoter extended until position −2 upstream of the transcription start site. South-Western analysis showed that a protein of approximately 100 kDa interacted with the −19 nt promoter fragment in CIV-infected cells. This binding did not occur with a point mutant that lacked promoter activity. The AAAAT motif was also found in the DNA polymerase promoter region of other iridoviruses and in other putative CIV delayed-early genes.

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