scholarly journals Transcription Factor MaMsn2 Regulates Conidiation Pattern Shift under the Control of MaH1 through Homeobox Domain in Metarhizium acridum

2021 ◽  
Vol 7 (10) ◽  
pp. 840
Author(s):  
Dongxu Song ◽  
Yueqing Cao ◽  
Yuxian Xia
Keyword(s):  
Cell Growth ◽  
Filamentous Fungi ◽  
Promoter Region ◽  
Molecular Mechanisms ◽  
Homologous Gene ◽  
Regulation Mechanism ◽  
Polar Growth ◽  
Mobility Shift ◽  
Metarhizium Acridum ◽  
Mobility Shift Assay

The growth pattern of filamentous fungi can switch between hyphal radial polar growth and non-polar yeast-like cell growth depending on the environmental conditions. Asexual conidiation after radial polar growth is called normal conidiation (NC), while yeast-like cell growth is called microcycle conidiation (MC). Previous research found that the disruption of MaH1 in Metarhizium acridum led to a conidiation shift from NC to MC. However, the regulation mechanism is not clear. Here, we found MaMsn2, an Msn2 homologous gene in M. acridum, was greatly downregulated when MaH1 was disrupted (ΔMaH1). Loss of MaMsn2 also caused a conidiation shift from NC to MC on a nutrient-rich medium. Yeast one-hybrid (Y1H) and electrophoretic mobility shift assay (EMSA) showed that MaH1 could bind to the promoter region of the MaMsn2 gene. Disrupting the interaction between MaH1 and the promoter region of MaMsn2 significantly downregulated the transcription level of MaMsn2, and the overexpression of MaMsn2 in ΔMaH1 could restore NC from MC of ΔMaH1. Our findings demonstrated that MaMsn2 played a role in maintaining the NC pattern directly under the control of MaH1, which revealed the molecular mechanisms that regulated the conidiation pattern shift in filamentous fungi for the first time.

scholarly journals RbsR Activates Capsule but Represses therbsUDKOperon in Staphylococcus aureus

2015 ◽  
Vol 197 (23) ◽  
pp. 3666-3675 ◽  
Author(s):  
Mei G. Lei ◽  
Chia Y. Lee
Keyword(s):  
Staphylococcus Aureus ◽  
Virulence Factor ◽  
Transcriptional Activation ◽  
Promoter Region ◽  
Mobility Shift ◽  
Content Type ◽  
Virulence Regulation ◽  
Important Virulence Factor ◽  
Dna Fragment ◽  
Mobility Shift Assay

ABSTRACTStaphylococcus aureuscapsule is an important virulence factor that is regulated by a large number of regulators. Capsule genes are expressed from a major promoter upstream of thecapoperon. A 10-bp inverted repeat (IR) located 13 bp upstream of the −35 region of the promoter was previously shown to affect capsule gene transcription. However, little is known about transcriptional activation of thecappromoter. To search for potential proteins which directly interact with thecappromoter region (Pcap), we directly analyzed the proteins interacting with the PcapDNA fragment from shifted gel bands identified by electrophoretic mobility shift assay. One of these regulators, RbsR, was further characterized and found to positively regulatecapgene expression by specifically binding to thecappromoter region. Footprinting analyses showed that RbsR protected a DNA region encompassing the 10-bp IR. Our results further showed thatrbsRwas directly controlled by SigB and that RbsR was a repressor of therbsUDKoperon, involved in ribose uptake and phosphorylation. The repression ofrbsUDKby RbsR could be derepressed byd-ribose. However,d-ribose did not affect RbsR activation of capsule.IMPORTANCEStaphylococcus aureusis an important human pathogen which produces a large number of virulence factors. We have been using capsule as a model virulence factor to study virulence regulation. Although many capsule regulators have been identified, the mechanism of regulation of most of these regulators is unknown. We show here that RbsR activates capsule by direct promoter binding and that SigB is required for the expression ofrbsR. These results define a new pathway wherein SigB activates capsule through RbsR. Our results further demonstrate that RbsR inhibits therbsoperon involved in ribose utilization, thereby providing an example of coregulation of metabolism and virulence inS. aureus. Thus, this study further advances our understanding of staphylococcal virulence regulation.

scholarly journals A Sequence of the CIS Gene Promoter Interacts Preferentially with Two Associated STAT5A Dimers: a Distinct Biochemical Difference between STAT5A and STAT5B

1998 ◽  
Vol 18 (10) ◽  
pp. 5852-5860 ◽  
Author(s):  
Frédérique Verdier ◽  
Raquel Rabionet ◽  
Fabrice Gouilleux ◽  
Christian Beisenherz-Huss ◽  
Paule Varlet ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Target Genes ◽  
Prolactin Receptor ◽  
Gene Promoter ◽  
Expression Vectors ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Mobility Shift Assay ◽  
Binding Sequence ◽  
Biochemical Difference

ABSTRACT Two distinct genes encode the closely related signal transducer and activator of transcription proteins STAT5A and STAT5B. The molecular mechanisms of gene regulation by STAT5 and, particularly, the requirement for both STAT5 isoforms are still undetermined. Only a few STAT5 target genes, among them the CIS (cytokine-inducible SH2-containing protein) gene, have been identified. We cloned the human CIS gene and studied the human CIS gene promoter. This promoter contains four STAT binding elements organized in two pairs. By electrophoretic mobility shift assay studies using nuclear extracts of UT7 cells stimulated with erythropoietin, we showed that these four sequences bound to STAT5-containing complexes that exhibited different patterns and affinities: the three upstream STAT binding sequences bound to two distinct STAT5-containing complexes (C0 and C1) and the downstream STAT box bound only to the slower-migrating C1 band. Using nuclear extracts from COS-7 cells transfected with expression vectors for the prolactin receptor, STAT5A, and/or STAT5B, we showed that the C1 complex was composed of a STAT5 tetramer and was dependent on the presence of STAT5A. STAT5B lacked this property and bound with a stronger affinity than did STAT5A to the four STAT sequences as a homodimer (C0 complex). This distinct biochemical difference between STAT5A and STAT5B was confirmed with purified activated STAT5 recombinant proteins. Moreover, we showed that the presence on the same side of the DNA helix of a second STAT sequence increased STAT5 binding and that only half of the palindromic STAT binding sequence was sufficient for the formation of a STAT5 tetramer. Again, STAT5A was essential for this cooperative tetrameric association. This property distinguishes STAT5A from STAT5B and could be essential to explain the transcriptional regulation diversity of STAT5.

scholarly journals Pituitary homeobox 1 (Pitx1) stimulates rat LHβ gene expression via two functional DNA-regulatory regions

2005 ◽  
Vol 35 (1) ◽  
pp. 145-158 ◽  
Author(s):  
Qiaorong Jiang ◽  
Kyeong-Hoon Jeong ◽  
Cheryl D Horton ◽  
Lisa M Halvorson
Keyword(s):  
Gene Expression ◽  
Molecular Mechanisms ◽  
Gene Promoter ◽  
Orphan Nuclear Receptor ◽  
Mobility Shift ◽  
Regulatory Regions ◽  
Steroidogenic Factor 1 ◽  
Reproductive Axis ◽  
Functional Dna ◽  
Mobility Shift Assay

Luteinizing hormone (LH) plays a central role in the reproductive axis, stimulating both gonadal steroid biosynthesis and the development of mature gametes. Over the past decade, significant progress has been made in characterizing the transcription factors and associated DNA-regulatory sites which mediate expression of the LH β-subunit gene (LHβ). One of these factors, pituitary homeobox 1 (Pitx1), has been shown to stimulate LHβ gene promoter activity, both alone and in synergy with the orphan nuclear receptor, steroidogenic factor-1 (SF-1), and the early growth response gene 1 (Egr-1). Prior reports have attributed the Pitx1 response to a cis-element located at position -101 in the rat LHβ gene promoter. While investigating the role of Pitx1 in regulating rat LHβ gene expression, we observed a small, but significant, residual Pitx1 response despite mutation or deletion of this site. In the studies presented here, we identify the presence of a second functional Pitx1 region spanning positions −73 to −52 in the rat LHβ gene promoter. Based on electrophoretic mobility shift assay, Pitx1 binds to both the initially described 5′Pitx1 site as well as this putative 3′Pitx1 region. In transient transfection analysis, mutation of the LHβ-3′Pitx1 site significantly blunted Pitx1 responsiveness, with elimination of the Pitx1 response in a construct containing mutations in both Pitx1 cis-elements. We also analyzed the importance of each of these Pitx1 sites for providing functional synergy with SF-1 and with Egr-1. We observed a markedly decreased synergistic response with mutation of the 5′Pitx1 site with further loss following mutation of the 3′Pitx1 site. In contrast, functional interaction between Pitx1 and Egr-1 persisted with mutation of both Pitx1 regions. We conclude that Pitx1 stimulates the rat LHβ gene promoter via two Pitx1 DNA-regulatory regions. These results further our understanding of the molecular mechanisms that regulate expression of this critical reproductive gene promoter.

Characterization of the human E2F4 promoter region and its response to 12-O-tetradecanoylphorbol-13-acetate

2019 ◽  
Vol 166 (4) ◽  
pp. 363-373 ◽  
Author(s):  
Hiroshi Hamada ◽  
Yuta Goto ◽  
Jun Arakawa ◽  
Erisa Murayama ◽  
Yui Ogawa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Promoter Region ◽  
Expression Vectors ◽  
Reporter Assay ◽  
Rt Pcr ◽  
Mobility Shift ◽  
A Cell ◽  
Mobility Shift Assay ◽  
E2f Transcription Factors

Abstract The E2F transcription factors (TFs), which control the progression of the cell cycle in response to DNA-damage and various stresses, are known to interact with a tumour suppressor, Retinoblastoma 1 (RB1). We previously showed that the response of the human RB1 promoter to a 12-O-tetradecanoylphorbol-13-acetate (TPA) in HL-60 cells is mediated by a duplicated GGAA motif, which is also present in the 5′-upstream of the E2F family genes. The motifs are especially rich in the 5′-upstream of the E2F4 gene. In the present study, we constructed luciferase (Luc) expression vectors containing a 466 bp of the 5′-upstream of the human E2F4 gene. The transfection of this plasmid and deletion/mutation-introduced derivatives into HL-60 cells and a Luc reporter assay showed that duplicated and triplicated GGAA (TTCC) motifs in the E2F4 promoter respond to TPA. As expected, electrophoretic mobility shift assay indicated that SPI1 (PU.1) binds to the GGAA motif-containing element. A quantitative RT-PCR and western blotting showed that the E2F4 transcripts and its encoding proteins accumulate during the differentiation of HL-60 into macrophage-like cells. In contrast, the expression of the E2F1 gene and the protein, which possibly acts as a cell cycle accelerator, was greatly diminished.

scholarly journals Role of MexZ and PA5471 in transcriptional regulation of mexXY in Pseudomonas aeruginosa

Microbiology ◽  
2009 ◽  
Vol 155 (10) ◽  
pp. 3312-3321 ◽  
Author(s):  
Masaki Yamamoto ◽  
Atsuhisa Ueda ◽  
Makoto Kudo ◽  
Yasuhiro Matsuo ◽  
Jun Fukushima ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Transcriptional Regulation ◽  
Molecular Mechanisms ◽  
Efflux Pump ◽  
Mobility Shift ◽  
Drug Induced ◽  
Drug Efflux Pump ◽  
Mobility Shift Assay

MexXY, a drug efflux pump in Pseudomonas aeruginosa, confers resistance to aminoglycoside antibiotics. We recently reported that MexZ binds to the promoter region of the mexXY operon. Electrophoretic mobility shift assay (EMSA) using recombinant MexZ and oligonucleotide probes prepared from the intergenic region between mexZ and mexX revealed that MexZ binds to a 20 bp palindromic sequence. Culture of P. aeruginosa in the presence of tetracycline induced higher levels of MexX and MexZ, as measured by immunoblotting and EMSA, than in the absence of antibiotics. When MexZ was expressed by a mexZ expression plasmid, the plasmid-borne MexZ repressed drug-induced MexX production, further confirming that MexZ acts as a repressor of the mexXY operon. PA5471 protein has been reported to be essential for drug-induced MexXY production. Similarly to that report, we observed that plasmid-borne PA5471 induced both MexX and MexZ production in PAO1 cells. Interestingly, interaction between MexZ and PA5471 was observed in a yeast two-hybrid assay. Furthermore, EMSA and in vitro transcription assays revealed that interaction between PA5471 and MexZ reduced MexZ DNA-binding ability, leading to mexXY transcription. These findings contribute to the understanding of the molecular mechanisms underlying the transcriptional regulation of mexZ and mexXY by drug-induced PA5471 expression.

scholarly journals Interaction of Transcriptional Repressor ArgR with Transcriptional Regulator FarR at theargBPromoter Region inCorynebacterium glutamicum

2010 ◽  
Vol 77 (3) ◽  
pp. 711-718 ◽  
Author(s):  
Soo Youn Lee ◽  
Jae-Min Park ◽  
Jin Hyung Lee ◽  
Suk-Tai Chang ◽  
Jin-Soo Park ◽  
...  
Keyword(s):  
Dna Binding ◽  
Promoter Region ◽  
Mrna Level ◽  
Protein A ◽  
Transcriptional Repressor ◽  
Binding Domain ◽  
Mobility Shift ◽  
Mobility Shift Assay ◽  
Do So

ABSTRACTInCorynebacterium glutamicum, the ArgR protein, a transcriptional repressor, affects the expression level of theargBgene through binding to its promoter region. TheargBpromoter region (positions −77 to −25) has been found byin vitroelectrophoretic mobility shift assay (EMSA) results andin silicoanalysis to be important for the DNA binding of ArgR. Proline supplementation prevented the DNA binding of ArgR to theargBpromoter region and triggered an increase of theargBmRNA level. Additional mutational analyses of theargBpromoter region found nucleotides critical for ArgR binding (G located at position −58, C at position −55, and A at position −41 of theargBpromoter) in that region. Another transcriptional repressor, FarR, was also demonstrated to bind to theargBpromoter region. This binding was delimited to positions −57 to −77 on theargBpromoter. FarR has only one putative binding domain located at positions −57 to −77, but this region exactly overlapped with the binding region located from positions −55 to −77 for the binding of ArgR within theargBpromoter; thus, if ArgR bound with theargBpromoter first, the binding of FarR was not observed in this region. However, if FarR bound to the binding domain located at positions −57 to −77 first, ArgR could bind other binding sites located at positions −49 to −25 within theargBpromoter. Finally, this study suggests that ArgR can affect FarR binding to theargBpromoter region, as protein binding is dominated by the protein most able to do so.

scholarly journals CodY Is a Nutritional Repressor of Flagellar Gene Expression in Bacillus subtilis

2003 ◽  
Vol 185 (10) ◽  
pp. 3118-3126 ◽  
Author(s):  
F. Bergara ◽  
C. Ibarra ◽  
J. Iwamasa ◽  
J. C. Patarroyo ◽  
R. Aguilera ◽  
...  
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Bacillus Subtilis ◽  
Promoter Region ◽  
Stringent Response ◽  
Mobility Shift ◽  
Flagellin Gene ◽  
New Members ◽  
Environment Effects ◽  
Mobility Shift Assay

ABSTRACT Expression of the σD-dependent flagellin gene, hag, is repressed by the CodY protein in nutrient-rich environments. Analysis of a codY mutant bearing a hag-lacZ reporter suggests that the availability of amino acids in the environment is the specific signal that triggers this repression. Further, hag-lacZ expression appears to be sensitive to intracellular GTP levels, as demonstrated by increased expression upon addition of decoyinine. This result is consistent with the postulate that the availability of amino acids in the environment effects intracellular GTP levels through the stringent response. However, the levels of hag-lacZ measured upon the addition of subsets of amino acids suggest an additional mechanism(s). CodY is a DNA binding protein that could repress flagellin expression directly by binding to the hag promoter region, or indirectly by binding to the fla/che promoter region that governs expression of the σD transcriptional activator required for hag gene expression. Using an electrophoretic mobility shift assay, we have demonstrated that purified CodY protein binds specifically to both the hag and fla/che promoter fragments. Additionally, CodY acts as a nutritional repressor of transcription from the fla/che promoter region that contains two functional promoters. CodY binds to both the σD- and σA-dependent promoters in this region, as demonstrated by DNase I footprint analyses. Footprint analyses of the hag gene demonstrated that CodY binds downstream of its σD-dependent promoter. Taken together, these results identify new members of the CodY regulon that encode motility functions in Bacillus subtilis and are controlled by the σD alternate sigma factor.

Wilms tumor suppressor, Wt1, is a transcriptional activator of the erythropoietin gene

Blood ◽  
2006 ◽  
Vol 107 (11) ◽  
pp. 4282-4290 ◽  
Author(s):  
Christof Dame ◽  
Karin M. Kirschner ◽  
Katharina V. Bartz ◽  
Thomas Wallach ◽  
Christiane S. Hussels ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Molecular Mechanisms ◽  
Wilms Tumor ◽  
Transcriptional Activator ◽  
Mobility Shift ◽  
Specific Expression ◽  
Tissue Specific ◽  
Hek 293 ◽  
Mobility Shift Assay ◽  
Specific Regulation

AbstractMolecular mechanisms for the developmental stage and tissue-specific regulation of the erythropoietin (EPO) gene are poorly understood. Recent findings indicate a role of the Wilms tumor suppressor, Wt1, in the formation of the hematopoietic system. Herein, we tested the hypothesis that Wt1 is a transcriptional regulator of the EPO gene. Binding of the transcriptionally competent Wt1(–KTS) isoform to the minimal EPO promoter was demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation. Under normoxia, EPO expression was significantly increased in HEK 293 and HepG2 cells with forced expression of Wt1(–KTS). A reporter construct harboring the 117-bp minimal human EPO promoter was activated up to 20-fold by transient cotransfection of Wt1(–KTS) in different cell lines. Mutation of the Wt1 binding site in the EPO promoter abrogated this stimulatory effect of the Wt1(–KTS) protein. Hepatic Epo mRNA expression was significantly reduced in embryonic mice with homozygous Wt1 deletion. Furthermore, Wt1 and EPO were colocalized in hepatocytes of the liver and in neuronal cells of the dorsal root ganglia in developing mice. Both proteins were also detected in Sertoli cells of the adult murine testis. In conclusion, we identified Wt1(–KTS) as a novel transcriptional activator for the tissue-specific expression of the EPO gene.

Role of NF-κB in the Rescue of Multiple Myeloma Cells From Glucocorticoid-Induced Apoptosis by Bcl-2

Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 3044-3052 ◽  
Author(s):  
Rena Feinman ◽  
Jadd Koury ◽  
Michael Thames ◽  
Bart Barlogie ◽  
Joshua Epstein ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Dna Binding ◽  
Molecular Mechanisms ◽  
Annexin V ◽  
Early Event ◽  
Protective Effects ◽  
Mobility Shift ◽  
Myeloma Cells ◽  
Mobility Shift Assay ◽  
Induced Apoptosis

Abstract The molecular mechanisms by which multiple myeloma (MM) cells evade glucocorticoid-induced apoptosis have not been delineated. Using a human IgAκ MM cell line (ARP-1), we found that dexamethasone (Dex)-induced apoptosis is associated with decreased NF-κB DNA binding and κB-dependent transcription. Both nuclear p50:p50 and p50:p65 NF-κB complexes are detected in ARP-1 cells by supershift electrophoretic mobility shift assay (EMSA). Dex-mediated inhibition of NF-κB DNA binding precedes a notable increase in annexin V binding, thereby indicating that diminished NF-κB activity is an early event in Dex-induced apoptosis. Overexpression of bcl-2 in ARP-1 cells prevents Dex-mediated repression of NF-κB activity and apoptosis. Sustained NF-κB DNA binding is also observed in two previously characterized Dex-resistant MM cell lines (RPMI8226 and ARH-77) that express moderate levels of endogenous bcl-2 and IκB proteins. In addition, enforced bcl-2 expression in ARP-1 cells did not prevent the augmentation of IκB protein by Dex. We also noted a possible association between Dex-mediated downregulation of NF-κB in freshly obtained primary myeloma cells and the patients’ responsiveness to glucocorticoid-based chemotherapy. Collectively, our data suggest that the protective effects of bcl-2 in MM cells act upstream in the NF-κB activation–signaling pathway and the potential use of NF-κB as a biomarker in progressive MM.

scholarly journals Association between polymorphism in the promoter region of lncRNA GAS5 and the risk of colorectal cancer

2019 ◽  
Vol 39 (4) ◽  
Author(s):  
Yajie Wang ◽  
Shenshen Wu ◽  
Xi Yang ◽  
Xiaobo Li ◽  
Rui Chen
Keyword(s):  
Colorectal Cancer ◽  
Flow Cytometry ◽  
Promoter Region ◽  
Expression Level ◽  
Luciferase Assay ◽  
Migration And Invasion ◽  
Mobility Shift ◽  
Dual Luciferase Assay ◽  
Invasion And Migration ◽  
Mobility Shift Assay

AbstractThe growth arrest special 5 (GAS5), as a research hotspot of long noncoding RNAs (lncRNAs), has been reported to be associated with colorectal cancer (CRC). However, the association between polymorphisms in GAS5 and the risk of CRC was not clear. In the present study, a case–control study in 1078 CRC patients and 1175 matched healthy controls was performed to evaluate the association between the potential functional genetic variants in GAS5 and the risk of CRC. PCR-TaqMan, qPCR, dual-luciferase assay, electrophoretic mobility shift assay (EMSA), flow cytometry, migration and invasion assays were performed to evaluate the function of polymorphism. Results showed that subjects carrying the rs55829688 CT/TT genotypes had a significantly higher risk of CRC when compared with the CC genotype. Further qPCR assay confirmed that the CRC tissues with rs55829688 CT/TT genotypes had a higher GAS5 mRNA expression level. The dual-luciferase assay, qPCR and EMSA assay revealed that rs55829688 T>C polymorphism could decrease the expression level of GAS5 by impacting the binding ability of the transcription factor Yin Yang-1 (YY1) to the GAS5 promoter region. The expression of apoptosis-related proteins were detected by Western blot. Further, flow cytometry, migration, and invasion experiments showed that GAS5 repressed apoptosis and increased invasion and migration capability of CRC cells. Taken together, our findings provided evidence that the rs55829688 variant in the GAS5 promoter was associated with the risk of CRC and decreased expression of GAS5 by affecting the binding affinity of the transcription factors YY1 to GAS5.

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