scholarly journals MAPK-activated transcription factor PxJun suppresses PxABCB1 expression and confers resistance to Bacillus thuringiensis Cry1Ac toxin in Plutella xylostella (L.)

2021 ◽  
Author(s):  
Jianying Qin ◽  
Le Guo ◽  
Fan Ye ◽  
Shi Kang ◽  
Dan Sun ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Regulation ◽  
Bacillus Thuringiensis ◽  
Insect Resistance ◽  
Plutella Xylostella ◽  
Molecular Basis ◽  
Luciferase Reporter ◽  
Regulation Mechanism ◽  
Cry Toxins ◽  
Regulation Mechanisms

Deciphering the molecular mechanisms underlying insect resistance to Cry toxins produced by Bacillus thuringiensis (Bt) is pivotal for the sustainable utilization of Bt biopesticides and transgenic Bt crops. Previously, we identified that MAPK-mediated reduced expression of the PxABCB1 gene is associated with Bt Cry1Ac resistance in the diamondback moth, Plutella xylostella (L.). However, the underlying transcriptional regulation mechanism remains enigmatic. Herein, the PxABCB1 promoter in Cry1Ac-susceptible and Cry1Ac-resistant P. xylostella strains was cloned and analyzed and found to contain a putative Jun binding site (JBS). A dual-luciferase reporter assay and yeast one-hybrid assay (Y1H) demonstrated that the transcription factor PxJun repressed PxABCB1 expression by interacting with this JBS. The expression levels of PxJun were increased in the midguts of all resistant strains compared to the susceptible strain. Silencing of PxJun expression significantly elevated PxABCB1 expression and Cry1Ac susceptibility in the resistant NIL-R strain, and silencing of PxMAP4K4 expression decreased PxJun expression and also increased PxABCB1 expression. These results indicate that MAPK-activated PxJun suppresses PxABCB1 expression to confer Cry1Ac resistance in P. xylostella, deepening our understanding of the transcriptional regulation of midgut Cry receptor genes and the molecular basis of insect resistance to Bt Cry toxins. Importance The transcriptional regulation mechanisms underlying reduced expression of Bt toxin receptor genes in Bt-resistant insects remain elusive. This study unveils that a transcription factor PxJun activated by the MAPK signaling pathway represses PxABCB1 expression and confers Cry1Ac resistance in P. xylostella. Our results provide new insights into the transcriptional regulation mechanisms of midgut Cry receptor genes and deepen our understanding of the molecular basis of insect resistance to Bt Cry toxins. To our knowledge, this study identified the first transcription factor that can be involved in the transcriptional regulation mechanisms of midgut Cry receptor genes in Bt-resistant insects.

scholarly journals A cis-Acting Mutation in the PxABCG1 Promoter Is Associated with Cry1Ac Resistance in Plutella xylostella (L.)

2021 ◽  
Vol 22 (11) ◽  
pp. 6106
Author(s):  
Jianying Qin ◽  
Fan Ye ◽  
Linzheng Xu ◽  
Xuguo Zhou ◽  
Neil Crickmore ◽  
...  
Keyword(s):  
Insect Resistance ◽  
Plutella Xylostella ◽  
Molecular Mechanisms ◽  
Resistance Mechanism ◽  
Luciferase Reporter ◽  
Cry Toxins ◽  
Cis Acting ◽  
Cry1ac Toxin ◽  
Bt Resistance ◽  
Bacterium Bacillus

The molecular mechanisms of insect resistance to Cry toxins generated from the bacterium Bacillus thuringiensis (Bt) urgently need to be elucidated to enable the improvement and sustainability of Bt-based products. Although downregulation of the expression of midgut receptor genes is a pivotal mechanism of insect resistance to Bt Cry toxins, the underlying transcriptional regulation of these genes remains elusive. Herein, we unraveled the regulatory mechanism of the downregulation of the ABC transporter gene PxABCG1 (also called Pxwhite), a functional midgut receptor of the Bt Cry1Ac toxin in Plutella xylostella. The PxABCG1 promoters of Cry1Ac-susceptible and Cry1Ac-resistant strains were cloned and analyzed, and they showed clear differences in activity. Subsequently, a dual-luciferase reporter assay, a yeast one-hybrid (Y1H) assay, and RNA interference (RNAi) experiments demonstrated that a cis-mutation in a binding site of the Hox transcription factor Antennapedia (Antp) decreased the promoter activity of the resistant strain and eliminated the binding and regulation of Antp, thereby enhancing the resistance of P. xylostella to the Cry1Ac toxin. These results advance our knowledge of the roles of cis- and trans-regulatory variations in the regulation of midgut Cry receptor genes and the evolution of Bt resistance, contributing to a more complete understanding of the Bt resistance mechanism.

scholarly journals Klotho is regulated by transcription factor Sp1 in renal tubular epithelial cells

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Yan Li ◽  
Yong Liu ◽  
Kailong Wang ◽  
Yinghui Huang ◽  
Wenhao Han ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Regulation ◽  
Epithelial Cells ◽  
Renal Disease ◽  
Luciferase Reporter ◽  
Tubular Epithelial Cells ◽  
Renal Tubular Epithelial Cells ◽  
Transcription Factor Sp1 ◽  
Renal Tubular ◽  
Tgf Β1

Abstract Background Klotho is a multifunctional protein, which exists both in a membrane bound and a soluble form. In renal tubules, Klotho is involved in cell senescence, anti-oxidant response, and renal fibrosis, thus regulation of its expression is critical to understand its roles in renal diseases. Indeed, reduced expression was observed in various renal disease. However, the mechanisms underlying transcriptional regulation of the human klotho gene (KL) largely remain unknown. Results Here we demonstrated that the Klotho expression in human renal tubular epithelial cells (RTECs) was enhanced by overexpression of the transcription factor Sp1. On the contrary, Klotho expression was decreased by Sp1 knockdown. Besides, increased expression of Sp1 alleviated TGF-β1-induced fibrosis in HK-2 cells by inducing Klotho expression. Luciferase reporter assays and chromatin immunoprecipitation assays further identified the binding site of Sp1 was located in − 394 to − 289 nt of the KL promoter, which was further confirmed by mutation analysis. Conclusions These data demonstrate that KL is a transcriptional target of Sp1 and TGF-β1-induced fibrosis was alleviated by Sp1 in human RTECs by directly modulating Klotho expression, which help to further understand the transcriptional regulation of Klotho in renal disease models.

Transcriptional Regulation of the Bach2 B-Cell Differentiation and Apoptotic Factor by the Bcr-Abl Oncoprotein.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2991-2991
Author(s):  
Maria Makri ◽  
Chikashi Yoshida ◽  
Akihiko Muto ◽  
Kazuhiko Igarashi ◽  
Junia V Melo
Keyword(s):  
Transcription Factor ◽  
Transcriptional Regulation ◽  
Cell Differentiation ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Promoter Activity ◽  
Lymphoid Cells ◽  
Luciferase Reporter ◽  
B Cell Differentiation ◽  
B Lymphoid

Abstract Transformation by the Bcr-Abl oncoprotein of CML is mediated by the activation of a variety of signalling pathways, leading to transcriptional regulation of genes conferring the malignant phenotype of increased proliferation, altered adhesion and inhibition of apoptosis. We previously reported that expression of the BACH2 gene is downregulated by Bcr-Abl. Bach2 is a B-lymphoid specific transcription factor, which regulates somatic hypermutation and class switch recombination of Ig genes. It is also a pro-apoptotic factor, coupling oxidative stress to transcription repression. It is possible that in an environment of increased genomic instability, Bcr-Abl transformed cells may repress pro-apoptotic signals by suppressing BACH2 transcription. To determine the direct association between Bcr-Abl and decreased BACH2 transcription, we infected human B-lymphoid cells with a retroviral vector expressing both p210Bcr-Abl and eGFP genes. Infected cells were treated with imatinib, an Abl tyrosine kinase inhibitor, prior to quantification of BACH2 transcripts by Real Time RT/PCR. Ectopic expression of BCR-ABL significantly decreased BACH2 mRNA levels, and this effect was completely abolished by imatinib. To investigate whether this regulation was exerted at the transcriptional level, we identified the BACH2 transcription initiation site (TIS), and then cloned and characterised a 3.9 Kb genomic DNA fragment including the BACH2 promoter region. By generating luciferase reporter constructs of various lengths of the BACH2 promoter we found that a region of 725 bp upstream the TIS conferred maximum promoter activity in human B-lymphoid cells. The effect of Bcr-Abl on promoter activity was demonstrated by co-transfection of the reporter and p210Bcr-Abl constructs. BACH2-promoter activity was reduced up to 60% in the presence of Bcr-Abl. Furthermore, when co-transfected cells were incubated with different concentrations of imatinib, the Bcr-Abl-mediated promoter repression was abrogated in a dose dependent manner, confirming the dependence of the effect on the tyrosine kinase activity of the oncoprotein. In support of these data, no effect on promoter activity was seen when the BACH2 promoter was co-transfected with a kinase-dead BCR-ABL construct. Moreover, treatment with imatinib of the BCR-ABL+ cell line BV173 transfected with the reporter induced a nearly 2-fold upregulation in its activity. Bioinformatics inspection of the promoter sequence revealed potential sites for the Pax5 B-cell differentiation factor and the Foxo3a transcription factor, a regulator of pro-apoptotic genes. In co-transfection experiments of either factor with the BACH2 promoter, both demonstrated a significant inducing effect on its activity. Gel shift and chromatin immunoprecipitation showed direct binding of Pax5 within the BACH2 promoter in vitro and in vivo. Moreover, Western analysis showed elevated Pax5 levels in BCR-ABL+ cell lines after imatinib treatment, indicating that inhibition of Bach2 expression by Bcr-Abl is mediated at least in part by Pax5. As to Foxo3a, it has been reported to be constitutively phosphorylated and inactivated in BCR-ABL+ cells, processes which prevent its translocation to the cell nucleus. Altogether, our data suggest that Bcr-Abl transcriptional repression of Bach2 via Pax5 could lead to a differentiation arrest in transformed B-cells, and that Foxo3a may induce imatinib-mediated apoptosis through up-regulation of the Bach2 apoptotic function.

NF-κB Is Active in Mcl-1 Promoter Regulation in Human CLL.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2265-2265
Author(s):  
Erin K Hertlein ◽  
Derek A. West ◽  
Ruth W Craig ◽  
David M Lucas ◽  
John C. Byrd
Keyword(s):  
Transcription Factor ◽  
Transcriptional Regulation ◽  
Dna Binding ◽  
Cell Survival ◽  
Binding Site ◽  
Nuclear Localization ◽  
Lymphocytic Leukemia ◽  
Luciferase Reporter ◽  
Critical Event ◽  
Direct Link

Abstract The NF-κB family of transcription factors is linked to neoplasia due to its role in increasing cell proliferation as well as inhibiting apoptosis. NF-κB has been reported to be constitutively active in chronic lymphocytic leukemia (CLL), but the mechanism underlying this this activation is not fully understood and the critical target genes involved have not been identified. To further define the mechanism(s) by which NF-κB promotes survival in CLL, we investigated the role of this transcription factor in the regulation of the myeloid cell leukemia 1 (Mcl-1) gene. The promoter region of Mcl-1 contains a putative NF-κB binding site, and increased expression of the Mcl-1 protein has been related to rapid disease progression and resistance to apoptosis in CLL. However, a direct link between NF-κB and Mcl-1 transcriptional regulation has not yet been established in B-cells or human CLL. We demonstrate here that NF-κB binding is a critical event in the transcriptional regulation of Mcl-1, as deletion of the NF-κB binding site in the promoter results in decreased activity of an Mcl-1-luciferase reporter construct. In addition, pharmacological inhibition of NF-κB reduced p65 nuclear localization and binding of NF-κB to the Mcl-1 promoter. Interestingly, when CLL patient samples were exposed to an IKK inhibitor, Bay-11, the extent of Mcl-1 inhibition varied in patients. We therefore examined whether the effect on Mcl-1 correlated with in vitro cell survival, a result that might be expected given that NF-κB expression has been previously reported to correlate with CLL cell survival. We found that CLL patient cells more sensitive to Bay-11-induced apoptosis also showed a larger decrease in Mcl-1 mRNA, suggesting that Mcl-1 message level may be a useful diagnostic to predict patients that will respond to NF-κB targeted therapy. Furthermore, different inducers of the NF-κB signaling pathway (CD40L, immune stimulatory CpG-ODN and TNF-α), promote differential effects on Mcl-1 regulation in CLL patient cells. Although all three treatments increase NF-κB nuclear localization and DNA binding, Mcl-1 RNA and protein increased with CpG and CD40L treatment, but not TNFα. This result provides evidence of stimulus-specific regulation of Mcl-1 by NF-κB. Based on these observations, we hypothesize that there is a direct link between NF-κB DNA binding and transcriptional control of Mcl-1, and that this gene could serve as a pharmacodynamic endpoint to monitor the efficacy of NF-κB inhibitors in CLL cells. Additionally, a direct link of NF-κB activity to Mcl-1 expression and cell survival provide further justification for targeting this transcription factor for treatment in CLL.

scholarly journals Cyt1A from Bacillus thuringiensis Lacks Toxicity to Susceptible and Resistant Larvae of Diamondback Moth (Plutella xylostella) and Pink Bollworm (Pectinophora gossypiella)

2001 ◽  
Vol 67 (1) ◽  
pp. 462-463 ◽  
Author(s):  
Susan K. Meyer ◽  
Bruce E. Tabashnik ◽  
Yong-Biao Liu ◽  
Margaret C. Wirth ◽  
Brian A. Federici
Keyword(s):  
Bacillus Thuringiensis ◽  
Plutella Xylostella ◽  
Diamondback Moth ◽  
Pink Bollworm ◽  
Pectinophora Gossypiella ◽  
Cry Toxins ◽  
Lepidopteran Pests ◽  
Lepidopteran Larvae

ABSTRACT We tested Cyt1Aa, a cytolytic endotoxin of Bacillus thuringiensis, against susceptible and Cry1A-resistant larvae of two lepidopteran pests, diamondback moth (Plutella xylostella) and pink bollworm (Pectinophora gossypiella). Unlike previous results obtained with mosquito and beetle larvae, Cyt1Aa alone or in combination with Cry toxins was not highly toxic to the lepidopteran larvae that we examined.

scholarly journals STAT3 Inhibits Cell Proliferation at Least in Part Via Directly Negatively Regulating FST Gene Expression

2021 ◽  
Author(s):  
Haidong Xu ◽  
Guangwei Ma ◽  
Fang Mu ◽  
Bolin Ning ◽  
Hui Li ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Transcription Factor ◽  
Transcriptional Regulation ◽  
Binding Site ◽  
Hair Follicle ◽  
Luciferase Reporter ◽  
Functional Study ◽  
Follicle Development ◽  
Hacat Cells ◽  
Hair Follicle Development

Abstract Background: Follistatin (FST) is a secretory glycoprotein and belongs to the TGF-β superfamily. Previously, we found that two single nucleotide polymorphisms (SNPs) of sheep FST gene were significantly associated with wool quality traits in Chinese Merino sheep (Junken type), indicating that FST is involved in the regulation of hair follicle development and hair trait formation. The transcription regulation of human and mouse FST genes has been widely investigated, and many transcription factors have been identified to regulate FST gene, such as erythroid 2-related factor 2 (Nrf2), Estrogen-related receptor-β (ERRβ), β-catenin/transcription factor 4 (TCF4) and transcription factor Sp1. However, to date, the transcriptional regulation of sheep FST is largely unknown. The objective of this study was to investigate the transcriptional regulation of sheep FST gene in hair follicles. Results: Genome walking analysis revealed that the gap region upstream of sheep genomic FST gene was 775 bp long. Transcription factor binding site analysis showed sheep FST promoter region contained a conserved putative binding site for signal transducer and activator of transcription 3 (STAT3), located at nucleotides -423 to -416 relative to the first nucleotide (A, +1) of the initiation codon (ATG). The dual-luciferase reporter assays showed that STAT3 inhibited the activity of the FST promoter reporter, and the mutation of the putative STAT3 binding site attenuated the inhibitory effect of STAT3 on the FST promoter activity. Furthermore, chromatin immunoprecipitation assay (ChIP) indicated that STAT3 directly binds to the FST promoter. The further functional study displayed that FST and STAT3 played opposite roles in cell proliferation. Overexpression of FST significantly promoted the proliferation of sheep fetal fibroblasts (SFFs) and human keratinocyte (HaCaT) cells, and overexpression of STAT3 significantly inhibited the proliferation of SFFs and HaCaT cells, which was accompanied by a significantly reduced expression of FST gene (P < 0.05). Conclusions: STAT3 directly negatively regulates sheep FST gene and inhibits cell proliferation. The findings will contribute to understanding molecular mechanisms that underlie hair follicle development and wool trait formation.

scholarly journals Profiling of MicroRNAs in Midguts of Plutella xylostella Provides Novel Insights Into the Bacillus thuringiensis Resistance

2021 ◽  
Vol 12 ◽  
Author(s):  
Jie Yang ◽  
Xuejiao Xu ◽  
Sujie Lin ◽  
Shiyao Chen ◽  
Guifang Lin ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Plutella Xylostella ◽  
Target Genes ◽  
Expression Patterns ◽  
Mapk Signaling ◽  
Differentially Expressed ◽  
Luciferase Reporter ◽  
Sequencing Analysis ◽  
Bt Resistance ◽  
Differentially Expressed Mirnas

The diamondback moth (DBM), Plutella xylostella, one of the most destructive lepidopteran pests worldwide, has developed field resistance to Bacillus thuringiensis (Bt) Cry toxins. Although miRNAs have been reported to be involved in insect resistance to multiple insecticides, our understanding of their roles in mediating Bt resistance is limited. In this study, we constructed small RNA libraries from midguts of the Cry1Ac-resistant (Cry1S1000) strain and the Cry1Ac-susceptible strain (G88) using a high-throughput sequencing analysis. A total of 437 (76 known and 361 novel miRNAs) were identified, among which 178 miRNAs were classified into 91 miRNA families. Transcripts per million analysis revealed 12 differentially expressed miRNAs between the Cry1S1000 and G88 strains. Specifically, nine miRNAs were down-regulated and three up-regulated in the Cry1S1000 strain compared to the G88 strain. Next, we predicted the potential target genes of these differentially expressed miRNAs and carried out GO and KEGG pathway analyses. We found that the cellular process, metabolism process, membrane and the catalytic activity were the most enriched GO terms and the Hippo, MAPK signaling pathway might be involved in Bt resistance of DBM. In addition, the expression patterns of these miRNAs and their target genes were determined by RT-qPCR, showing that partial miRNAs negatively while others positively correlate with their corresponding target genes. Subsequently, novel-miR-240, one of the differentially expressed miRNAs with inverse correlation with its target genes, was confirmed to interact with Px017590 and Px007885 using dual luciferase reporter assays. Our study highlights the characteristics of differentially expressed miRNAs in midguts of the Cry1S1000 and G88 strains, paving the way for further investigation of miRNA roles in mediating Bt resistance.

scholarly journals Repressor Element 1 Silencing Transcription Factor (REST) Governs Microglia-Like BV2 Cell Migration via Progranulin (PGRN)

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Tongya Yu ◽  
Yingying Lin ◽  
Yuzhen Xu ◽  
Yunxiao Dou ◽  
Feihong Wang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cell Migration ◽  
Molecular Basis ◽  
Promoter Sequence ◽  
Microglia Activation ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Bv2 Cells ◽  
Potential Binding Site ◽  
Repressor Element

Microglia activation contributes to Alzheimer’s disease (AD) etiology, and microglia migration is a fundamental function during microglia activation. The repressor element-1 silencing transcription factor (REST), a powerful transcriptional factor, was found to play a neuroprotective role in AD. Despite its possible role in disease progression, little is known about whether REST participates in microglia migration. In this study, we aimed to explore the function of REST and its molecular basis during microglia migration under Aβ1-42-treated pathological conditions. When treated by Aβ1-42 REST was upregulated through JAK2/STAT3 signal pathway in BV2 cells. And transwell coculture system was used to evaluate cell migration function of microglia-like BV2. Small interfering RNA (siRNA) targeting progranulin (PGRN) were delivered into BV2 cells, and results showed that PGRN functions to promote BV2 migration. REST expression was inhibited by sh-RNA, which induced BV2 cell migration obviously. On the contrary, REST was overexpressed by REST recombinant plasmid transfection, which repressed BV2 cell migration, indicating that REST may act as a repressor of cell migration. To more comprehensively examine the molecular basis, we analyzed the promoter sequence of PGRN and found that it has the potential binding site of REST. Moreover, knocking-down of REST can increase the expression of PGRN, which confirms the inhibiting effect of REST on PGRN expression. Further detection of double luciferase reporter gene also confirmed the inhibition of REST on the activity of PGRN promoter, indicating that REST may be an inhibitory transcription factor of PGRN which governs microglia-like BV2 cell migration. In conclusion, the present study demonstrates that transcription factor REST may act as a repressor of microglia migration through PGRN.

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