Olf-1-binding site: characterization of an olfactory neuron-specific promoter motif

1993 ◽  
Vol 13 (5) ◽  
pp. 3002-3014
Author(s):  
K Kudrycki ◽  
C Stein-Izsak ◽  
C Behn ◽  
M Grillo ◽  
R Akeson ◽  
...  
Keyword(s):  
Nuclear Protein ◽  
Consensus Sequence ◽  
Binding Motif ◽  
Sequence Motif ◽  
Olfactory Neuron ◽  
Sequence Motifs ◽  
Mobility Shift ◽  
Specific Expression ◽  
Mobility Shift Assay

We report characterization of several domains within the 5' flanking region of the olfactory marker protein (OMP) gene that may participate in regulating transcription of this and other olfactory neuron-specific genes. Analysis by electrophoretic mobility shift assay and DNase I footprinting identifies two regions that contain a novel sequence motif. Interactions between this motif and nuclear proteins were detected only with nuclear protein extracts derived from olfactory neuroepithelium, and this activity is more abundant in olfactory epithelium enriched in immature neurons. We have designated a factor(s) involved in this binding as Olf-1. The Olf-1-binding motif consensus sequence was defined as TCCCC(A/T)NGGAG. Studies with transgenic mice indicate that a 0.3-kb fragment of the OMP gene containing one Olf-1 motif is sufficient for olfactory tissue-specific expression of the reporter gene. Some of the other identified sequence motifs also interact specifically with olfactory nuclear protein extracts. We propose that Olf-1 is a novel, olfactory neuron-specific trans-acting factor involved in the cell-specific expression of OMP.

scholarly journals Olf-1-binding site: characterization of an olfactory neuron-specific promoter motif.

1993 ◽  
Vol 13 (5) ◽  
pp. 3002-3014 ◽  
Author(s):  
K Kudrycki ◽  
C Stein-Izsak ◽  
C Behn ◽  
M Grillo ◽  
R Akeson ◽  
...  
Keyword(s):  
Nuclear Protein ◽  
Consensus Sequence ◽  
Binding Motif ◽  
Sequence Motif ◽  
Olfactory Neuron ◽  
Sequence Motifs ◽  
Mobility Shift ◽  
Specific Expression ◽  
Mobility Shift Assay

We report characterization of several domains within the 5' flanking region of the olfactory marker protein (OMP) gene that may participate in regulating transcription of this and other olfactory neuron-specific genes. Analysis by electrophoretic mobility shift assay and DNase I footprinting identifies two regions that contain a novel sequence motif. Interactions between this motif and nuclear proteins were detected only with nuclear protein extracts derived from olfactory neuroepithelium, and this activity is more abundant in olfactory epithelium enriched in immature neurons. We have designated a factor(s) involved in this binding as Olf-1. The Olf-1-binding motif consensus sequence was defined as TCCCC(A/T)NGGAG. Studies with transgenic mice indicate that a 0.3-kb fragment of the OMP gene containing one Olf-1 motif is sufficient for olfactory tissue-specific expression of the reporter gene. Some of the other identified sequence motifs also interact specifically with olfactory nuclear protein extracts. We propose that Olf-1 is a novel, olfactory neuron-specific trans-acting factor involved in the cell-specific expression of OMP.

scholarly journals Nitrogen regulator GlnR directly controls transcription ofprpDBCoperon involved in methylcitrate cycle inMycobacterium smegmatis

2018 ◽  
Author(s):  
Xin-Xin Liu ◽  
Wei-Bing Liu ◽  
Meng-Jia Shen ◽  
Bang-Ce Ye
Keyword(s):  
Fatty Acids ◽  
Mycobacterium Tuberculosis ◽  
Nitrogen Metabolism ◽  
Potential Application ◽  
Transcriptional Activator ◽  
Affinity Constant ◽  
Binding Motif ◽  
Mobility Shift ◽  
Poor Growth ◽  
Mobility Shift Assay

AbstractMycobacterium tuberculosisutilizes the fatty acids of the host as the carbon source. While the metabolism of odd chain fatty acids produces propionyl-CoA. Methylcitrate cycle is essential for Mycobacteria to utilize the propionyl-CoA to persist and grow on these fatty acids. InM. smegmatis, methylcitrate synthase, methylcitrate dehydratase, and methylisocitrate lyase involved in methylcitrate cycle were respectively encoded byprpC,prpD,and prpBin operonprpDBC. In this study, we found that the nitrogen regulator GlnR directly binds to the promoter region ofprpDBCoperon and inhibits its transcription. The typical binding sequence of GlnR was identified by bioinformatics analysis and electrophoretic mobility shift assay. The GlnR-binding motif was seperated by 164 bp with the binding site of PrpR which was a pathway-specific transcriptional activator of methylcitrate cycle. Moreover, the affinity constant of GlnR was much stronger than that of PrpR toprpDBC. The deletion ofglnRresulted in poor growth in propionate or cholesterol medium comparing with wild-type strain. The ΔglnRmutant strain also showed a higher survival in macrophages. These results illustrated that the nitrogen regulator GlnR regulated methylcitrate cycle through directly repressing the transcription ofprpDBCoperon. The finding reveals an unprecedented link between nitrogen metabolism and methylcitrate pathway, and provides a potential application for controlling populations of pathogenic mycobacteria.Author SummaryNutrients are crucial for the survival and pathogenicity ofMycobacterium tuberculosis. The success of this pathogen survival in macrophage due to its ability to assimilate fatty acids and cholesterol from host. The cholesterol and fatty acids are catabolized via β-oxidation to generate propionyl-CoA, which is then mainly metabolized via the methylcitrate cycle. The assimilation of propionyl-CoA needs to be tightly regulated to prevent its accumulation and alleviate toxicity in cell. Here, we identified a new regulator GlnR (the nitrogen transcriptional regulator) that repressed the transcription ofprpoperon involved in methylcitrate cycle inM. smegmatis. In this study, we found a typical GlnR binding box inprpoperon, and the affinity is much stronger than that of PrpR which is known as a pathway-specific transcriptional activator of methylcitrate cycle. In addition, deletion ofglnRobviously affect the growth of mutant in propionate or cholesterol medium, and show a better viability in macrophage. The findings not only provide the insights into the regulatory mechanism underlying crosstalk of nitrogen metabolism and carbon metabolism, but also reveal a potential application for controlling populations of pathogenic mycobacteria.

Identification of a killer cell-specific regulatory element of the mouse perforin gene: an Ets-binding site-homologous motif that interacts with Ets-related proteins

1993 ◽  
Vol 13 (11) ◽  
pp. 6690-6701
Author(s):  
H Koizumi ◽  
M F Horta ◽  
B S Youn ◽  
K C Fu ◽  
B S Kwon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Binding Site ◽  
Protein Interactions ◽  
Regulatory Element ◽  
Killer Cell ◽  
Mobility Shift ◽  
Specific Expression ◽  
Native Molecular Mass ◽  
Mobility Shift Assay

The gene encoding the cytolytic protein perforin is selectively expressed by activated killer lymphocytes. To understand the mechanisms underlying the cell-type-specific expression of this gene, we have characterized the regulatory functions and the DNA-protein interactions of the 5'-flanking region of the mouse perforin gene (Pfp). A region extending from residues +62 through -141, which possesses the essential promoter activity, and regions further upstream, which are able to either enhance or suppress gene expression, were identified. The region between residues -411 and -566 was chosen for further characterization, since it contains an enhancer-like activity. We have identified a 32-mer sequence (residues -491 to -522) which appeared to be capable of enhancing gene expression in a killer cell-specific manner. Within this segment, a 9-mer motif (5'-ACAGGAAGT-3', residues -505 to -497; designated NF-P motif), which is highly homologous to the Ets proto-oncoprotein-binding site, was found to interact with two proteins, NF-P1 and NF-P2. NF-P2 appears to be induced by reagents known to up-regulate the perforin message level and is present exclusively in killer cells. Electrophoretic mobility shift assay and UV cross-linking experiments revealed that NF-P1 and NF-P2 may possess common DNA-binding subunits. However, the larger native molecular mass of NF-P1 suggests that NF-P1 contains an additional non-DNA-binding subunit(s). In view of the homology between the NF-P motif and other Ets proto-oncoprotein-binding sites, it is postulated that NF-P1 and NF-P2 belong to the Ets protein family. Results obtained from the binding competition assay, nevertheless, suggest that NF-P1 and NF-P2 are related to but distinct from Ets proteins, e.g., Ets-1, Ets-2, and NF-AT/Elf-1, known to be expressed in T cells.

scholarly journals Regulation of differential proton-coupled folate transporter gene expression in human tumors: transactivation by KLF15 with NRF-1 and the role of Sp1

2019 ◽  
Vol 476 (8) ◽  
pp. 1247-1266
Author(s):  
Zhanjun Hou ◽  
Carrie O'Connor ◽  
Josephine Frühauf ◽  
Steve Orr ◽  
Seongho Kim ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Transcriptional Control ◽  
Consensus Sequence ◽  
Minimal Promoter ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Ht1080 Cells ◽  
Promoter Construct ◽  
Mobility Shift Assay ◽  
Reporter Gene Assays

Abstract Tumors can be therapeutically targeted with novel antifolates (e.g. AGF94) that are selectively transported by the human proton-coupled folate transporter (hPCFT). Studies were performed to determine the transcription regulation of hPCFT in tumors and identify possible mechanisms that contribute to the highly disparate levels of hPCFT in HepG2 versus HT1080 tumor cells. Transfection of hPCFT-null HT1080 cells with hPCFT restored transport and sensitivity to AGF94. Progressive deletions of the hPCFT promoter construct (−2005 to +96) and reporter gene assays in HepG2 and HT1080 cells confirmed differences in hPCFT transactivation and localized a minimal promoter to between positions −50 and +96. The minimal promoter included KLF15, GC-Box and NRF-1 cis-binding elements whose functional importance was confirmed by promoter deletions and mutations of core consensus sequences and reporter gene assays. In HepG2 cells, NRF-1, KLF15 and Sp1 transcripts were increased over HT1080 cells by ∼5.1-, ∼44-, and ∼2.4-fold, respectively. In Drosophila SL2 cells, transfection with KLF15 and NRF-1 synergistically activated the hPCFT promoter; Sp1 was modestly activating or inhibitory. Chromatin immunoprecipitation and electrophoretic mobility shift assay (EMSA) and supershifts confirmed differential binding of KLF15, Sp1, and NRF-1 to the hPCFT promoter in HepG2 and HT1080 cells that paralleled hPCFT levels. Treatment of HT1080 nuclear extracts (NE) with protein kinase A increased Sp1 binding to its consensus sequence by EMSA, suggesting a role for Sp1 phosphorylation in regulating hPCFT transcription. A better understanding of determinants of hPCFT transcriptional control may identify new therapeutic strategies for cancer by modulating hPCFT levels in combination with hPCFT-targeted antifolates.

PDGF Up-regulates CSF-1 Gene Transcription in Ameloblast-like Cells

2008 ◽  
Vol 87 (1) ◽  
pp. 33-38 ◽  
Author(s):  
Y. Wittrant ◽  
B. Sriniketan Bhandari ◽  
H. Abboud ◽  
N. Benson ◽  
K. Woodruff ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Binding Motif ◽  
Rt Pcr ◽  
Mobility Shift ◽  
Protein Levels ◽  
Macrophage Colony Stimulating Factor ◽  
Cellular Pathways ◽  
Mobility Shift Assay ◽  
Macrophage Colony ◽  
Stimulating Factor

Macrophage colony-stimulating factor (CSF-1) is a key regulatory cytokine for amelogenesis, and ameloblasts synthesize CSF-1. We hypothesized that PDGF stimulates DNA synthesis and regulates CSF-1 in these cells. We determined the effect of PDGF on CSF-1 expression using MEOE-3M ameloblasts as a model. By RT-PCR, MEOE-3M expressed PDGFRs and PDGF A- and B-chain mRNAs. PDGF-BB increased DNA synthesis and up-regulated CSF-1 mRNA and protein in MEOE-3M. Cells transfected with CSF-1 promoter deletion constructs were analyzed. A PDGF-responsive region between −1.7 and −0.795 kb, containing a consensus Pea3 binding motif, was identified. Electrophoretic mobility shift assay (EMSA) showed that PDGF-BB stimulated protein binding to this motif that was inhibited in the presence of anti-Pea3 antibody. Analysis of these data provides the first evidence that PDGF-BB is a mitogen for MEOE-3M and increases CSF-1 protein levels, predominantly by transcription. Elucidation of the cellular pathways that control CSF-1 expression may provide novel strategies for the regulation of enamel matrix formation.

scholarly journals A novel cis-acting element controlling the rat CYP2D5 gene and requiring cooperativity between C/EBP beta and an Sp1 factor.

1994 ◽  
Vol 14 (2) ◽  
pp. 1383-1394 ◽  
Author(s):  
Y H Lee ◽  
M Yano ◽  
S Y Liu ◽  
E Matsunaga ◽  
P F Johnson ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Ternary Complex ◽  
Sequence Similarity ◽  
Consensus Sequence ◽  
Regulatory Region ◽  
Mrna Levels ◽  
Binding Studies ◽  
Mobility Shift ◽  
Specific Expression ◽  
Nuclear Extracts

The rat CYP2D5 gene encodes a cytochrome P450 and is expressed in liver cells. Its expression commences a few days after birth, and maximal mRNA levels are achieved when animals reach puberty. Transfection and DNA binding studies were performed to investigate the mechanism controlling developmentally programmed, liver-specific expression of CYP2D5. Transfection studies using a series of CYP2D5 upstream DNA chloramphenicol acetyltransferase gene fusion constructs identified a segment of DNA between nucleotides -55 and -156 that conferred transcriptional activity in HepG2 cells. Activity was markedly increased by cotransfection with a vector expressing C/EBP beta but was unaffected by vectors producing other liver-enriched transcription factors (C/EBP alpha, HNF-1 alpha, and DBP). DNase I footprinting revealed a region protected by both HepG2 and liver cell nuclear extracts between nucleotides -83 and -112. This region displayed some sequence similarity to the Sp1 consensus sequence and was able to bind the Sp1 protein, as assessed by a gel mobility shift assay. The role of Sp1 in CYP2D5 transcription was confirmed by trans activation of the 2D5-CAT construct in Drosophila melanogaster cells by using an Sp1 expression vector. C/EBP beta alone was unable to directly bind the -83 to -112 region of the promoter but was able to produce a ternary complex when combined with HepG2 nuclear extracts or recombinant human Sp1. C/EBP alpha was unable to substitute for C/EBP beta in forming this ternary complex. A poor C/EBP binding site is present adjacent to the Sp1 site, and mutagenesis of this site abolished formation of the ternary complex with the CYP2D5 regulatory region. These result establish that two transcription factors can work in conjunction, possibly by protein-protein interaction, to activate the CYP2D5 gene.

scholarly journals A Microfluidics-Based Mobility Shift Assay to Discover New Tyrosine Phosphatase Inhibitors

2006 ◽  
Vol 11 (8) ◽  
pp. 996-1004 ◽  
Author(s):  
Dominique Perrin ◽  
Christèle Frémaux ◽  
Dominique Besson ◽  
Wolfgang HB Sauer ◽  
Alexander Scheer
Keyword(s):  
Drug Target ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Mobility Shift ◽  
Phosphatase Inhibitors ◽  
Mobility Shift Assay ◽  
Diabetes And Obesity ◽  
Major Drug ◽  
Specific Peptide

Protein tyrosine phosphatases (PTPs) play key roles in regulating tyrosine phosphorylation levels in cells. Since the discovery of PTP1B as a major drug target for diabetes and obesity, PTPs have emerged as a new and promising class of signaling targets for drug development in a variety of therapeutic areas. The routine use of generic substrate 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) in our hands led to the discovery of very similar and often not very selective molecules. Therefore, to increase the chances to discover novel chemical scaffolds, a side-by-side comparison between the DiFMUP assay and a chip-based mobility shift assay with a specific phosphopeptide was performed, on 1 PTP, using a focused set of compounds. Assay robustness and sensitivity were comparable for both the DiFMUP and mobility shift assays. The off-chip mobility shift assay required a longer development time because of identification, synthesis, and characterization of a specific peptide, and its cost per point was higher. However, although most potent scaffolds found with the DiFMUP assay were confirmed in the mobility shift format, the off-chip mobility shift assay led to the identification of previously unidentified chemical scaffolds with improved druglike properties.

scholarly journals HrpS Is a Global Regulator on Type III Secretion System (T3SS) and Non-T3SS Genes in Pseudomonas savastanoi pv. phaseolicola

2018 ◽  
Vol 31 (12) ◽  
pp. 1232-1243 ◽  
Author(s):  
Jingru Wang ◽  
Xiaolong Shao ◽  
Yingchao Zhang ◽  
Yanan Zhu ◽  
Pan Yang ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Binding Motif ◽  
Global Regulator ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Type Iii ◽  
Mobility Shift Assay ◽  
The Individual

The type III secretion system (T3SS) is the main machinery for Pseudomonas savastanoi and other gram-negative bacteria to invade plant cells. HrpR and HrpS form a hetero-hexamer, which activates the expression of HrpL, which induces all T3SS genes by binding to a ‘hrp box’ in promoters. However, the individual molecular mechanism of HrpR or HrpS has not been fully understood. Through chromatin immunoprecipitation coupled to high-throughput DNA sequencing, we found that HrpR, HrpS, and HrpL had four, 47, and 31 targets on the genome, respectively. HrpS directly bound to the promoter regions of a group of T3SS genes and non-T3SS genes. HrpS independently regulated these genes in a hrpL deletion strain. Additionally, a HrpS-binding motif (GTGCCAAA) was identified, which was verified by electrophoretic mobility shift assay and lux-reporter assay. HrpS also regulated motility and biofilm formation in P. savastanoi. The present study strongly suggests that HrpS alone can work as a global regulator on both T3SS and non-T3SS genes in P. savastanoi. [Formula: see text] Copyright © 2018 The Author(s). This is an open-access article distributed under the CC BY-NC-ND 4.0 International license .

scholarly journals Molecular regulation of the human IL-3 gene: inducible T cell-restricted expression requires intact AP-1 and Elf-1 nuclear protein binding sites.

1993 ◽  
Vol 178 (5) ◽  
pp. 1681-1692 ◽  
Author(s):  
L R Gottschalk ◽  
D M Giannola ◽  
S G Emerson
Keyword(s):  
T Cells ◽  
Transcription Factors ◽  
T Cell ◽  
Protein Binding ◽  
Binding Site ◽  
Binding Sites ◽  
Nuclear Protein ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Mobility Shift Assay

Interleukin 3 (IL-3) is a hematopoietic stem-cell growth and differentiation factor that is expressed solely in activated T and NK cells. Studies to date have identified elements 5' to the IL-3 coding sequences that regulate its transcription, but the sequences that confer T cell-specific expression remain to be clearly defined. We have now identified DNA sequences that are required for T cell-restricted IL-3 gene transcription. A series of transient transfections performed with human IL-3-chloramphenicol acetyltransferase (CAT) reporter plasmids in T and non-T cells revealed that a plasmid containing 319 bp of 5' flanking sequences was active exclusively in T cells. Deletion analysis revealed that T cell specificity was conferred by a 49-bp fragment (bp -319 to -270) that included a potential binding site for AP-1 transcription factors 6 bp upstream of a binding site for Elf-1, a member of the Ets family of transcription factors. DNaseI footprint and electrophoretic mobility shift assay analyses performed with MLA-144 T cell nuclear extracts demonstrated that this 49-bp region contains a nuclear protein binding region that includes consensus AP-1 and Elf-1 binding sites. In addition, extracts prepared from purified human T cells contained proteins that bound to synthetic oligonucleotides corresponding to the AP-1 and Elf-1 binding sites. In vitro-transcribed and -translated Elf-1 protein bound specifically to the Elf-1 site, and Elf-1 antisera competed and super shifted nuclear protein complexes present in MLA-144 nuclear extracts. Moreover, addition of anti-Jun family antiserum in electrophoretic mobility shift assay reactions completely blocked formation of the AP-1-related complexes. Transient transfection studies in MLA-144 T cells revealed that constructs containing mutations in the AP-1 site almost completely abolished CAT activity while mutation of the Elf-1 site or the NF-IL-3 site, a previously described nuclear protein binding site (bp. -155 to -148) in the IL-3 promoter, reduced CAT activity to < 25% of the activity given by wild-type constructs. We conclude that expression of the human IL-3 gene requires the AP-1 and Elf-1 binding sites; however, unlike other previously characterized cytokine genes such as IL-2, the AP-1 and Elf-1 factors can bind independently in the IL-3 gene.(ABSTRACT TRUNCATED AT 400 WORDS)

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