scholarly journals Populus euphratica WRKY1 binds the promoter of H+-ATPase gene to enhance gene expression and salt tolerance

2019 ◽  
Vol 71 (4) ◽  
pp. 1527-1539 ◽  
Author(s):  
Jun Yao ◽  
Zedan Shen ◽  
Yanli Zhang ◽  
Xia Wu ◽  
Jianhui Wang ◽  
...  
Keyword(s):  
Salt Stress ◽  
Promoter Region ◽  
Affinity Purification ◽  
Regulatory Region ◽  
Populus Euphratica ◽  
Luciferase Reporter ◽  
Proton Pumps ◽  
Tobacco Plants ◽  
Atpase Gene ◽  
Pumping Activity

Abstract Plasma membrane proton pumps play a crucial role in maintaining ionic homeostasis in salt-resistant Populus euphratica under saline conditions. High levels of NaCl (200 mM) induced PeHA1 expression in P. euphratica roots and leaves. We isolated a 2022 bp promoter fragment upstream of the translational start of PeHA1 from P. euphratica. The promoter–reporter construct PeHA1-pro::GUS was transferred to tobacco plants, demonstrating that β-glucuronidase activities increased in root, leaf, and stem tissues under salt stress. DNA affinity purification sequencing revealed that PeWRKY1 protein targeted the PeHA1 gene. We assessed the salt-induced transcriptional response of PeWRKY1 and its interaction with PeHA1 in P. euphratica. PeWRKY1 binding to the PeHA1 W-box in the promoter region was verified by a yeast one-hybrid assay, EMSA, luciferase reporter assay, and virus-induced gene silencing. Transgenic tobacco plants overexpressing PeWRKY1 had improved expression of NtHA4, which has a cis-acting W-box in the regulatory region, and improved H+ pumping activity in both in vivo and in vitro assays. We conclude that salt stress up-regulated PeHA1 transcription due to the binding of PeWRKY1 to the W-box in the promoter region of PeHA1. Thus, we conclude that enhanced H+ pumping activity enabled salt-stressed plants to retain Na+ homeostasis.

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

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.

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 Functional mapping of the mouse hairless gene promoter region

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.

scholarly journals Identification and Characterization of the OCT4 Upstream Regulatory Region in Sus scrofa

2019 ◽  
Vol 2019 ◽  
pp. 1-11
Author(s):  
Seung-Hun Kim ◽  
Kwang-Hwan Choi ◽  
Dong-Kyung Lee ◽  
Mingyun Lee ◽  
Jae Yeon Hwang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Promoter Region ◽  
Regulatory Region ◽  
Embryonic Stem ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Fluorescence Signal ◽  
Reporter Assay ◽  
Reporter System

OCT4 plays pivotal roles in maintaining pluripotency during early mammalian embryonic development and in embryonic stem cells. It is essential to establish a reporter system based on the OCT4 promoter region to study pluripotency. However, there is still a lack of information about the porcine OCT4 upstream reporter system. To improve our understanding of the porcine OCT4 regulatory region, we identified conserved regions in the porcine OCT4 promoter upstream region by sequence-based comparative analysis using various mammalian genome sequences. The similarity of nucleotide sequences in the 5′ upstream region was low among mammalian species. However, the OCT4 promoter and four regulatory regions, including distal and proximal enhancer elements, had high similarity. Next, a functional analysis of the porcine OCT4 promoter region was conducted. Luciferase reporter assay results indicated that the porcine OCT4 distal enhancer and proximal enhancer were highly activated in mouse embryonic stem cells and embryonic carcinoma cells, respectively. A comparison analysis of naïve and primed state marker gene expression in a dual-reporter assay showed that the expression levels of naïve and primed markers differed in fluorescence signal between high-expressing cells and low-expressing cells. Similar to OCT4 upstream-based reporter systems derived from other species, the porcine OCT4 upstream region-based reporter constructs showed exclusive expression patterns depending on the state of pluripotency. This work provides basic information about the porcine OCT4 upstream region and various porcine OCT4 fluorescence reporter constructs, which can be applied to study species-specific pluripotency in early embryo development and the establishment of embryonic stem cells in pigs.

NaCl-altered oxygen flux profiles and H+-ATPase activity in roots of two contrasting poplar species

2020 ◽  
Author(s):  
Xiuying Ma ◽  
Jinke Li ◽  
Chen Deng ◽  
Jian Sun ◽  
Jian Liu ◽  
...  
Keyword(s):  
Salt Stress ◽  
Atpase Activity ◽  
Mitochondrial Respiration ◽  
Populus Euphratica ◽  
Nacl Stress ◽  
Proton Pumps ◽  
O2 Uptake ◽  
Transient Kinetics ◽  
Root Cells ◽  
Cytochemical Analysis

Abstract Maintaining mitochondrial respiration is crucial for proving ATP for H+ pumps to continuously exclude Na+ under salt stress. NaCl-altered O2 uptake, mitochondrial respiration, and the relevance to H+-ATPase activity were investigated in two contrasting poplar species, Populus euphratica (salt-tolerant) and P. popularis 35–44 (salt-sensitive). Compared with P. popularis, P. euphratica roots exhibited a greater capacity to extrude Na+ under NaCl stress (150 mM). The cytochemical analysis with Pb(NO3)2 staining revealed that P. euphratica root cells retained higher H+ hydrolysis activity than the salt-sensitive poplar during a long-term (LT) of increasing salt stress (50 to 200 mM NaCl, 4 weeks). Long-sustained activation of proton pumps require long-lasting supply of energy (ATP), delivered by aerobic respiration. Taking advantage of the vibrating-electrodes technology combined with the use of membrane-tipped, polarographic oxygen microelectrodes, the species, spatial, and temporal differences in root O2 uptake were characterized under conditions of salt stress. Oxygen uptake upon NaCl shock (150 mM) was less declined in P. euphratica than in P. popularis, although the salt-induced transient kinetics were distinct from the drastic drop of O2 caused by hyperosmotic shock (255 mM mannitol). Short-term (ST) treatment (150 mM NaCl, 24 h) stimulated O2 influx in P. euphratica roots, and LT-treated P. euphratica displayed an increased O2 influx along root axis, whereas O2 influx declined with increasing salinity in P. popularis roots. The spatial localization of O2 influxes revealed that the apical zone was more susceptible than elongation region upon high NaCl (150, 200 mM) during ST and LT stress. Pharmacological experiments showed that the Na+ extrusion and H+-ATPase activity in salinized roots were correspondingly suppressed when O2 uptake was inhibited by a mitochondrial respiration inhibitor, NaN3. Therefore, we conclude that the stable mitochondrial respiration energized H+-ATPase of P. euphratica root cells for maintaining Na+ homeostasis under salt environments.

Molecular Characterization of a Tonoplast Na+/H+ Antiporter from Iris Lactea

2018 ◽  
Author(s):  
Qiang Guo ◽  
Xiaoxia Tian ◽  
Peichun Mao ◽  
Lin Meng
Keyword(s):  
Salt Stress ◽  
Transgenic Tobacco ◽  
Membrane Integrity ◽  
Nacl Stress ◽  
Proton Pumps ◽  
Transgenic Tobacco Plants ◽  
Tobacco Plants ◽  
Cell Membrane Integrity ◽  
Effective Strategies ◽  
Photosynthesis Efficiency

Na+ compartmentalization into vacuoles is one of the effective strategies for adaptation of halophytes to saline environments. The tonoplast Na+/H+ antiporter (NHX) has been proved to be involved in the compartmentalization of Na+ into vacuoles to alleviate Na+ toxicity in cytoplasm. However, the function of NHX in halophyte Iris lactea is still unclear under salt stress. In this study, a significant positive correlation was observed between Na+ accumulations and IlNHX expression levels in shoots and roots under different concentrations of NaCl (0-200 mM), indicating IlNHX might be involved in Na+ accumulation of I. lactea in response to salt stress. More important, IlNHX was specifically localized to the tonoplast. Transgenic tobacco plants expressing IlNHX grew better and showed higher salinity tolerance under salt (200 mM NaCl) stress than those of wild type (WT) plants. Compared to WT plants, transgenic tobacco plants accumulated more Na+ and K+ and maintained higher K+/Na+ ratios in tissues by salt stress, accompanied by the reduction of chlorophyll loss and lipid peroxidation in the presence of salt. Interestingly, we found that transgenic tobacco plants exhibited markedly higher tonoplast H+-ATPase activity relative to WT plants subjected to salt. Overall, overexpression of IlNHX in tobacco could compartmentalize excessive Na+ into vacuoles to keep the cytosolic K+/Na+ balance by enhanced tonoplast proton pumps activity, which would be contributed to maintain K+ and Na+ homeostasis, to improve photosynthesis efficiency and to protect cell membrane integrity under salt stress.

35 Characterization of the promoter region of ZNFO, an oocyte-specific gene in cattle

2021 ◽  
Vol 33 (2) ◽  
pp. 125
Author(s):  
M. Zhang ◽  
H. Baldwin ◽  
J. Current ◽  
J. Yao
Keyword(s):  
Transcription Factor ◽  
Embryonic Development ◽  
Promoter Region ◽  
Core Promoter ◽  
Regulatory Region ◽  
Early Embryonic Development ◽  
Luciferase Reporter ◽  
Transcription Start ◽  
Core Promoter Region ◽  
The Core

Maternal factors are essential aspects of oocyte competence, which orchestrate early embryonic development. ZNFO is a Krüppel-associated box domain (KRAB) containing zinc finger transcription factor, which is exclusively expressed in bovine oocyte. Previous studies have demonstrated that ZNFO is essential for early embryonic development. However, the mechanisms regulating ZNFO transcription remain elusive. The objective of present study is to elucidate regulatory mechanisms of ZNFO transcription invitro, and specifically to identify putative core promoter and transcription factor(s) regulating ZNFO expression. 5′ Random amplification of cDNA ends (RACE) was performed using RNA isolated from 100 germinal vesicle (GV) stage oocytes to identify the transcription start site (TSS) of ZNFO. To elucidate the molecular mechanisms of ZNFO transcription, a 1.7-kb fragment of the 5′ regulatory region was obtained and cloned into pGL4.14 promoterless vector. The luciferase reporter assay was performed to confirm the promoter activity of the regulatory region. To further identify the core promoter region of the putative ZNFO promoter, a series of 5′ deletions in the ZNFO promoter followed by luciferase reporter assay was performed. The luciferase results indicated that the core promoter region of ZNFO was located within a region 57 to 31bp upstream of the transcription start site. Bioinformatics analysis indicated that a putative USF1/USF2 binding site (GGTCTCGTGACC) is located within the core promoter region. USF1 is a basic helix–loop–helix leucine zipper transcription factor, which regulates the expression of various maternal genes, which are essential for oocyte maturation and early embryonic development in cattle. To confirm that USF1 regulated ZNFO expression, bovine USF1 open reading frame (ORF) was cloned into pcDNA3.1A-HA vector, generating a USF1 overexpression construct. Overexpression of USF1 by transfecting USF1 plasmid enhanced ZNFO promoter activity within HEK293 cells, confirming that ZNFO expression is regulated by USF1. From these results, we conclude that USF1 activates the ZNFO promoter by binding to its target site, GGTCTCGTGACC.

PeSTZ1 confers salt stress tolerance by scavenging the accumulation of ROS through regulating the expression of PeZAT12 and PeAPX2 in Populus

2020 ◽  
Vol 40 (9) ◽  
pp. 1292-1311
Author(s):  
Fang He ◽  
Meng-Xue Niu ◽  
Cong-Hua Feng ◽  
Hui-Guang Li ◽  
Yanyan Su ◽  
...  
Keyword(s):  
Salt Stress ◽  
Stress Tolerance ◽  
Stress Responses ◽  
Populus Euphratica ◽  
Biomass Accumulation ◽  
Luciferase Reporter ◽  
Transgenic Poplar ◽  
Salt Stress Tolerance ◽  
Mobility Shift ◽  
Mobility Shift Assay

Abstract ZINC FINGER OF ARABIDOPSIS THALIANA12 (ZAT12) plays an important role in stress responses, but the transcriptional regulation of ZAT12 in response to abiotic stress remains unclear. In this study, we confirmed that a SALT TOLERANCE ZINC FINGER1 transcription factor from Populus euphratica (PeSTZ1) could regulate the expression of PeZAT12 by dual-luciferase reporter (DLR) assay and electrophoretic mobility shift assay. The expression of PeSTZ1 was rapidly induced by NaCl and hydrogen peroxide (H2O2) treatments. Overexpressing PeSTZ1 in poplar 84K (Populus alba × Populus glandulosa) plant was endowed with a strong tolerance to salt stress. Under salt stress, transgenic poplar exhibited higher expression levels of PeZAT12 and accumulated a larger amount of antioxidant than the wild-type plants. Meanwhile, ASCORBATE PEROXIDASE2 (PeAPX2) can be activated by PeZAT12 and PeSTZ1, promoting the accumulation of cytosolic ascorbate peroxidase (APX) to scavenge reactive oxygen species (ROS) under salt stress. This new regulatory model (PeSTZ1–PeZAT12–PeAPX2) was found in poplar, providing a new idea and insight for the interpretation of poplar resistance. Transgenic poplar reduced the accumulation of ROS, restrained the degradation of chlorophyll and guaranteed the photosynthesis and electron transport system. On the other hand, transgenic poplar slickly adjusted K+/Na+ homeostasis to alleviate salt toxicity in photosynthetic organs of plants under salt stress and then increased biomass accumulation. In summary, PeSTZ1 confers salt stress tolerance by scavenging the accumulation of ROS through regulating the expression of PeZAT12 and PeAPX2 in poplar.

scholarly journals A mitogen-responsive promoter region that is synergistically activated through multiple signalling pathways.

1993 ◽  
Vol 13 (3) ◽  
pp. 1796-1804 ◽  
Author(s):  
Q Ouyang ◽  
M Bommakanti ◽  
W K Miskimins
Keyword(s):  
Promoter Region ◽  
Elevated Level ◽  
Receptor Gene ◽  
Regulatory Region ◽  
Gene Promoter ◽  
Signalling Pathways ◽  
Sodium Orthovanadate ◽  
Dibutyryl Cyclic Amp ◽  
Human Transferrin Receptor ◽  
Promoter Construct

A regulatory region of the human transferrin receptor gene promoter was found to be required for increased expression in response to serum or growth factors. This region contains two elements that appear to cooperate for full responsiveness. We found that sodium orthovanadate treatment of cells significantly activated expression of promoter constructs containing these elements. 12-O-Tetradecanoylphorbol-13-acetate alone induced a twofold increase in expression but acted synergistically with vanadate to generate a highly elevated level of expression. Dibutyryl cyclic AMP alone had no effect on expression, but when added together with vanadate and 12-O-tetradecanoylphorbol-13-acetate, led to superinduction of the promoter construct. Induction of expression by these reagents was delayed several hours, and the kinetics were identical to those observed for serum induction.

scholarly journals The transcription factor USF1 promotes glioma cell invasion and migration by activating lncRNA HAS2-AS1

2020 ◽  
Vol 40 (8) ◽  
Author(s):  
Juntong Wang ◽  
Jingshun Gu ◽  
Aiwu You ◽  
Jun Li ◽  
Yuyan Zhang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cell Invasion ◽  
Promoter Region ◽  
Glioma Cell ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Mesenchymal Transition ◽  
Invasion And Migration ◽  
Starting Point ◽  
And Migration

Abstract Objective: The role of lncRNAs in tumor has been widely concerned. The present study took HAS2-AS1 (the antisense RNA 1 of HAS2) as a starting point to explore its expression in glioma and its role in the process of migration and invasion, providing a strong theoretical basis for mining potential therapeutic targets of glioma. Methods: Clinical data of glioma were obtained from The Cancer Genome Atlas (TCGA) database and differentially expressed lncRNAs were analyzed by edgeR. The hTFtarget database was used to predict the upstream transcription factors of HAS2-AS1 and the JASPAR website was used to predict the binding sites of human upstream transcription factor 1 (USF1) and HAS2-AS1. qRT-PCR was used to detect the expressions of HAS2-AS1 and USF1 in glioma tissues and cell lines. The effects of silencing HAS2-AS1 on the migration and invasion of cancer cells were verified by wound healing and Transwell invasion assays. The chromatin immunoprecipitation (ChIP) and dual luciferase reporter assays were applied to demonstrate the binding of USF1 and HAS2-AS1 promoter region. Western blot was used to detect the expressions of epithelial–mesenchymal transition (EMT)-related proteins. Results: HAS2-AS1 was highly expressed in glioma tissues and cells, and was significantly associated with poor prognosis. Silencing HAS2-AS1 expression inhibited glioma cell migration, invasion and EMT. USF1 was highly expressed in glioma and positively correlated with HAS2-AS1. The transcription of HAS2-AS1 was activated by USF1 via binding to HAS2-AS1 promoter region, consequently potentiating the invasion and migration abilities of glioma cells. Conclusion: These results suggested that the transcription factor USF1 induced up-regulation of lncRNA HAS2-AS1 and promoted glioma cell invasion and migration.

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