scholarly journals Molecular Dissection of TaLTP1 Promoter Reveals Functional Cis-Elements Regulating Epidermis-Specific Expression

2020 ◽  
Vol 21 (7) ◽  
pp. 2261
Author(s):  
Guiping Wang ◽  
Guanghui Yu ◽  
Yongchao Hao ◽  
Xinxin Cheng ◽  
Jinxiao Zhao ◽  
...  
Keyword(s):  
Lipid Transfer Protein ◽  
Brachypodium Distachyon ◽  
Lipid Transfer ◽  
Cis Elements ◽  
Mobility Shift ◽  
Specific Expression ◽  
Pavement Cell ◽  
Pavement Cells ◽  
Promoter Deletion Analysis ◽  
Mobility Shift Assay

Plant epidermis serves important functions in shoot growth, plant defense and lipid metabolism, though mechanisms of related transcriptional regulation are largely unknown. Here, we identified cis-elements specific to shoot epidermis expression by dissecting the promoter of Triticum aestivum lipid transfer protein 1 (TaLTP1). A preliminary promoter deletion analysis revealed that a truncated fragment within 400 bp upstream from the translation start site was sufficient to confer conserved epidermis-specific expression in transgenic Brachypodium distachyon and Arabidopsis thaliana. Further, deletion or mutation of a GC(N4)GGCC motif at position −380 bp caused a loss of expression in pavement cells. With an electrophoretic mobility shift assay (EMSA) and transgenic reporter assay, we found that a light-responsive CcATC motif at position −268 bp was also involved in regulating pavement cell-specific expression that is evolutionary conserved. Moreover, expression specific to leaf trichome cells was found to be independently regulated by a CCaacAt motif at position −303 bp.

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.

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 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 Tissue-Specific Expression of a Gene Encoding a Cell Wall-Localized Lipid Transfer Protein from Arabidopsis

1994 ◽  
Vol 105 (1) ◽  
pp. 35-45 ◽  
Author(s):  
S. Thoma ◽  
U. Hecht ◽  
A. Kippers ◽  
J. Botella ◽  
S. De Vries ◽  
...  
Keyword(s):  
Cell Wall ◽  
Lipid Transfer Protein ◽  
Lipid Transfer ◽  
Gene Encoding ◽  
Specific Expression ◽  
Tissue Specific ◽  
Tissue Specific Expression ◽  
Transfer Protein ◽  
A Cell

Identification of a pancreatic beta-cell insulin gene transcription factor that binds to and appears to activate cell-type-specific expression: its possible relationship to other cellular factors that bind to a common insulin gene sequence

1990 ◽  
Vol 10 (4) ◽  
pp. 1564-1572
Author(s):  
J Whelan ◽  
S R Cordle ◽  
E Henderson ◽  
P A Weil ◽  
R Stein
Keyword(s):  
Beta Cell ◽  
Pancreatic Beta Cell ◽  
Insulin Gene ◽  
Mobility Shift ◽  
Base Pairs ◽  
Specific Expression ◽  
Cellular Factors ◽  
Cell Type Specific ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift

The insulin gene is expressed almost exclusively in pancreatic beta-cells. Previous work in our laboratory has shown that pancreatic beta-cell-specific expression of the rat insulin II gene is controlled by a number of positive and negative cis-acting DNA elements within the enhancer. We have shown that one element within the enhancer, located between nucleotides -100 and -91 (GCCATCTGCT; referred to as the insulin control element [ICE]) relative to the transcription start site, is controlled by both positive- and negative-acting cellular transcription factors. The positive-acting factor appears to be uniquely active in beta-cells. To identify the nucleotides within the ICE that mediate positive cell-type-specific regulation, point mutations within this element were generated and assayed for their effects on expression. Base pairs -97, -94, -93, and -92 were found to be crucial for the activator function of this region, while mutations at base pairs -100, -96, and -91 had little or no effect on activity. The gel mobility shift assay was used to determine whether specific cellular factors associated directly with the ICE. Several specific protein-DNA complexes were detected in extracts prepared from insulin-producing and non-insulin-producing cells, including a complex unique to beta-cell extracts. The ability of unlabeled wild-type and point mutant versions of the ICE to compete for binding to these cellular factors demonstrated that the beta-cell-specific complex appears to contain the insulin gene activator protein(s). Interestingly, the adenovirus type 2 major late promoter upstream element (USE; GCCACGTGAC) also competed in the gel mobility shift assay for binding of cellular proteins to the ICE.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Identification of a pancreatic beta-cell insulin gene transcription factor that binds to and appears to activate cell-type-specific expression: its possible relationship to other cellular factors that bind to a common insulin gene sequence.

1990 ◽  
Vol 10 (4) ◽  
pp. 1564-1572 ◽  
Author(s):  
J Whelan ◽  
S R Cordle ◽  
E Henderson ◽  
P A Weil ◽  
R Stein
Keyword(s):  
Beta Cell ◽  
Pancreatic Beta Cell ◽  
Insulin Gene ◽  
Mobility Shift ◽  
Base Pairs ◽  
Specific Expression ◽  
Cellular Factors ◽  
Cell Type Specific ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift

The insulin gene is expressed almost exclusively in pancreatic beta-cells. Previous work in our laboratory has shown that pancreatic beta-cell-specific expression of the rat insulin II gene is controlled by a number of positive and negative cis-acting DNA elements within the enhancer. We have shown that one element within the enhancer, located between nucleotides -100 and -91 (GCCATCTGCT; referred to as the insulin control element [ICE]) relative to the transcription start site, is controlled by both positive- and negative-acting cellular transcription factors. The positive-acting factor appears to be uniquely active in beta-cells. To identify the nucleotides within the ICE that mediate positive cell-type-specific regulation, point mutations within this element were generated and assayed for their effects on expression. Base pairs -97, -94, -93, and -92 were found to be crucial for the activator function of this region, while mutations at base pairs -100, -96, and -91 had little or no effect on activity. The gel mobility shift assay was used to determine whether specific cellular factors associated directly with the ICE. Several specific protein-DNA complexes were detected in extracts prepared from insulin-producing and non-insulin-producing cells, including a complex unique to beta-cell extracts. The ability of unlabeled wild-type and point mutant versions of the ICE to compete for binding to these cellular factors demonstrated that the beta-cell-specific complex appears to contain the insulin gene activator protein(s). Interestingly, the adenovirus type 2 major late promoter upstream element (USE; GCCACGTGAC) also competed in the gel mobility shift assay for binding of cellular proteins to the ICE.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Activation of proglucagon gene transcription through a novel promoter element by the caudal-related homeodomain protein cdx-2/3.

1996 ◽  
Vol 16 (1) ◽  
pp. 19-28 ◽  
Author(s):  
T Jin ◽  
D J Drucker
Keyword(s):  
Cell Types ◽  
Intestinal Cell ◽  
Homeodomain Protein ◽  
Promoter Element ◽  
Mobility Shift ◽  
Specific Expression ◽  
Coordinate Regulation ◽  
Cell Extracts ◽  
Homeobox Protein ◽  
Mobility Shift Assay

The proglucagon gene is expressed in a highly restricted tissue-specific manner in the A cells of the pancreatic islet and the L cells of the small and large intestines. The results of previous experiments indicate that cell-specific expression of the proglucagon gene is mediated by proteins that interact with the proximal G1 promoter element. We show here that the G1 element contains several AT-rich subdomains that bind proteins present in islet and enteroendocrine cell extracts. Electrophoretic mobility shift assay experiments using specific antisera identified the homeobox protein cdx-2/3 (which designates the same homeobox protein called cdx-2 for mice and cdx-3 for hamsters) as a major component of the G1-Gc2 complex in islet and intestinal cells. Mutations of the Gc element that decreased cdx-2/3 binding also resulted in decreased proglucagon promoter activity in islet and intestinal cell lines. The finding that cdx-2/3 mediates activation of the proglucagon promoter in both islet and enteroendocrine cells is consistent with the common endodermal lineage of these tissues and provides new insight into the coordinate regulation of genes expressed in both pancreatic and intestinal endocrine cell types.

scholarly journals 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 TaWRKY13-A Serves as a Mediator of Jasmonic Acid-Related Leaf Senescence by Modulating Jasmonic Acid Biosynthesis

2021 ◽  
Vol 12 ◽  
Author(s):  
Hualiang Qiao ◽  
Yongwei Liu ◽  
Lingling Cheng ◽  
Xuelin Gu ◽  
Pengcheng Yin ◽  
...  
Keyword(s):  
Jasmonic Acid ◽  
Leaf Senescence ◽  
Brachypodium Distachyon ◽  
Wrky Transcription Factor ◽  
Luciferase Reporter ◽  
Virus Induced Gene Silencing ◽  
Reporter System ◽  
Mobility Shift ◽  
Yield And Quality ◽  
Mobility Shift Assay

Leaf senescence is crucial for crop yield and quality. Transcriptional regulation is a key step for integrating various senescence-related signals into the nucleus. However, few regulators of senescence implicating transcriptional events have been functionally characterized in wheat. Based on our RNA-seq data, we identified a WRKY transcription factor, TaWRKY13-A, that predominately expresses at senescent stages. By using the virus-induced gene silencing (VIGS) method, we manifested impaired transcription of TaWRKY13-A leading to a delayed leaf senescence phenotype in wheat. Moreover, the overexpression (OE) of TaWRKY13-A accelerated the onset of leaf senescence under both natural growth condition and darkness in Brachypodium distachyon and Arabidopsis thaliana. Furthermore, by physiological and molecular investigations, we verified that TaWRKY13-A participates in the regulation of leaf senescence via jasmonic acid (JA) pathway. The expression of JA biosynthetic genes, including AtLOX6, was altered in TaWRKY13-A-overexpressing Arabidopsis. We also demonstrated that TaWRKY13-A can interact with the promoter of AtLOX6 and TaLOX6 by using the electrophoretic mobility shift assay (EMSA) and luciferase reporter system. Consistently, we detected a higher JA level in TaWRKY13-A-overexpressing lines than that in Col-0. Moreover, our data suggested that TaWRKY13-A is partially functional conserved with AtWRKY53 in age-dependent leaf senescence. Collectively, this study manifests TaWRKY13-A as a positive regulator of JA-related leaf senescence, which could be a new clue for molecular breeding in wheat.

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