Involvement of HD-ZIP I transcription factors LcHB2 and LcHB3 in fruitlet abscission by promoting transcription of genes related to the biosynthesis of ethylene and ABA in litchi

2019 ◽  
Vol 39 (9) ◽  
pp. 1600-1613 ◽  
Author(s):  
Xingshuai Ma ◽  
Caiqin Li ◽  
Xuming Huang ◽  
Huicong Wang ◽  
Hong Wu ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Leucine Zipper ◽  
Ethylene Biosynthesis ◽  
Limiting Factor ◽  
Mobility Shift ◽  
Plant Organ ◽  
Key Genes ◽  
Electrophoretic Mobility Shift Assays ◽  
Aba Biosynthesis ◽  
Fruitlet Abscission

Abstract Abnormal fruitlet abscission is a limiting factor in the production of litchi, an economically important fruit in Southern Asia. Both ethylene and abscisic acid (ABA) induce organ abscission in plants. Although ACS/ACO and NCED genes are known to encode key enzymes required for ethylene and ABA biosynthesis, respectively, the transcriptional regulation of these genes is unclear in the process of plant organ shedding. Here, two polygalacturonase (PG) genes (LcPG1 and LcPG2) and two novel homeodomain-leucine zipper I transcription factors genes (LcHB2 and LcHB3) were identified as key genes associated with the fruitlet abscission in litchi. The expression of LcPG1 and LcPG2 was strongly associated with litchi fruitlet abscission, consistent with enhanced PG activity and reduced homogalacturonan content in fruitlet abscission zones (FAZs). The promoter activities of LcPG1/2 were enhanced by ethephon and ABA. In addition, the production of ethylene and ABA in fruitlets was significantly increased during fruit abscission. Consistently, expression of five genes (LcACO2, LcACO3, LcACS1, LcACS4 and LcACS7) related to ethylene biosynthesis and one gene (LcNCED3) related to ABA biosynthesis in FAZs were activated. Further, electrophoretic mobility shift assays and transient expression experiments demonstrated that both LcHB2 and LcHB3 could directly bind to the promoter of LcACO2/3, LcACS1/4/7 and LcNCED3 genes and activate their expression. Collectively, we propose that LcHB2/3 are involved in the litchi fruitlet abscission through positive regulation of ethylene and ABA biosynthesis.

Transcriptional Regulation of Hepcidin by Iron.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2664-2664
Author(s):  
Pauline Lee ◽  
Truksa Jaroslav ◽  
Hongfan Peng ◽  
Ernest Beutler
Keyword(s):  
Transcriptional Regulation ◽  
Transcription Factors ◽  
Iron Homeostasis ◽  
Mobility Shift ◽  
Consensus Sequences ◽  
High Iron ◽  
Amino Acid Peptide ◽  
Nuclear Extracts ◽  
Iron Deficient ◽  
Electrophoretic Mobility Shift Assays

Abstract Transcriptional regulation by iron in mammalian systems is poorly understood. Hepcidin, a 25 amino acid peptide that plays a central role in iron homeostasis, is transcriptionally regulated by iron. A region of the murine hepcidin promoter 1.6 to 1.8 kb upstream from the start of translation was recently identified to be important in transcriptional regulation by iron (Truksa J, et al. The distal location of the iron responsive region of the hepcidin promoter. Blood DOI 10.1182/blood-2007-05-091108, 2007). In order to identify transcription factors that might be important in regulation by iron, transcription factor microarray analyses (Panomics TranSignal Protein/DNA Array) were performed with nuclear extracts from livers of mice made iron deficient or iron loaded for 4 weeks. The analyses revealed 43 transcription factors that were upregulated in iron loaded liver nuclear extracts and 39 transcription factors that were upregulated in iron deficient nuclear extracts. In the region of the promoter we had found essential for transcriptional regulation by iron, −1.6 to −1.8 kb, consensus motifs were identified by Genomatix MatInspector for 10 transcription factors that corresponded to transcription factors upregulated in high iron nuclear extracts by array analyses. Similarly, the consensus sequences for 5 transcription factors corresponded to transcription factors identified in iron deficient nuclear extracts. Electrophoretic mobility shift assays were performed with probes across this region of the murine hepcidin promoter. Several probes exhibited differential binding between deficient and high iron nuclear extracts. These include the probe encompassing the CCAAT box and MEL1 motif, a probe containing a HLH motif, and a probe containing a bZIP and COUP motif. The probe containing the CCAAT motif was supershifted with antibodies against CBF, but was not supershifted with antibodies against SMAD4, CEBPα, and COUP. The probe containing a bZIP and COUP motif can be supershifted with antibodies against COUP-Tf and HNF4α, but not with antibodies against SMAD4, CEBPα, and COUP. Our data suggest that CBFA, COUP, and HNF4α are involved in transcriptional regulation of hepcidin by iron.

scholarly journals Binding of the RING Polycomb Proteins to Specific Target Genes in Complex with the grainyhead-Like Family of Developmental Transcription Factors

2002 ◽  
Vol 22 (6) ◽  
pp. 1936-1946 ◽  
Author(s):  
Annabel Tuckfield ◽  
David R. Clouston ◽  
Tomasz M. Wilanowski ◽  
Lin-Lin Zhao ◽  
John M. Cunningham ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Target Genes ◽  
Regulatory Elements ◽  
Polycomb Group ◽  
Mobility Shift ◽  
Polycomb Proteins ◽  
Complex Forms ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT The Polycomb group (PcG) of proteins represses homeotic gene expression through the assembly of multiprotein complexes on key regulatory elements. The mechanisms mediating complex assembly have remained enigmatic since most PcG proteins fail to bind DNA. We now demonstrate that the human PcG protein dinG interacts with CP2, a mammalian member of the grainyhead-like family of transcription factors, in vitro and in vivo. The functional consequence of this interaction is repression of CP2-dependent transcription. The CP2-dinG interaction is conserved in evolution with the Drosophila factor grainyhead binding to dring, the fly homologue of dinG. Electrophoretic mobility shift assays demonstrate that the grh-dring complex forms on regulatory elements of genes whose expression is repressed by grh but not on elements where grh plays an activator role. These observations reveal a novel mechanism by which PcG proteins may be anchored to specific regulatory elements in developmental genes.

scholarly journals A widespread family of WYL-domain transcriptional regulators co-localises with diverse phage defence systems and islands

2021 ◽  
Author(s):  
David M Picton ◽  
Joshua D Harling-Lee ◽  
Samuel J Duffner ◽  
Sam C Went ◽  
Richard D Morgan ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Transcriptional Regulator ◽  
Transcriptional Regulators ◽  
Multidrug Resistant ◽  
Common Mechanism ◽  
Defence Mechanisms ◽  
Emerging Pathogen ◽  
Mobility Shift ◽  
Genes Encoding ◽  
Electrophoretic Mobility Shift Assays

Bacteria are under constant assault by bacteriophages and other mobile genetic elements. As a result, bacteria have evolved a multitude of systems that protect from attack. Genes encoding bacterial defence mechanisms can be clustered into 'defence islands', providing a potentially synergistic level of protection against a wider range of assailants. However, there is a comparative paucity of information on how expression of these defence systems is controlled. Here, we functionally characterise a transcriptional regulator, BrxR, encoded within a recently described phage defence island from a multidrug resistant plasmid of the emerging pathogen Escherichia fergusonii. Using a combination of reporters and electrophoretic mobility shift assays, we discovered that BrxR acts as a repressor. We present the structure of BrxR to 2.15 Å, the first structure of this family of transcription factors, and pinpoint a likely binding site for ligands within the WYL-domain. Bioinformatic analyses demonstrated that BrxR homologues are widespread amongst bacteria. About half (48%) of identified BrxR homologues were co-localised with a diverse array of known phage defence systems, either alone or clustered into defence islands. BrxR is a novel regulator that reveals a common mechanism for controlling the expression of the bacterial phage defence arsenal.

scholarly journals Reverse Chromatin Immunoprecipitation (R-ChIP) enables investigation of the upstream regulators of plant genes

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Xuejing Wen ◽  
Jingxin Wang ◽  
Daoyuan Zhang ◽  
Yu Ding ◽  
Xiaoyu Ji ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Chromatin Immunoprecipitation ◽  
Specific Gene ◽  
Mobility Shift ◽  
Dna Integration ◽  
Protein Capture ◽  
Associated Proteins ◽  
Plant Genes ◽  
Electrophoretic Mobility Shift Assays ◽  
Upstream Regulators

AbstractDNA binding proteins carry out important and diverse functions in the cell, including gene regulation, but identifying these proteins is technically challenging. In the present study, we developed a technique to capture DNA-associated proteins called reverse chromatin immunoprecipitation (R-ChIP). This technology uses a set of specific DNA probes labeled with biotin to isolate chromatin, and the DNA-associated proteins are then identified using mass spectrometry. Using R-ChIP, we identified 439 proteins that potentially bind to the promoter of the Arabidopsis thaliana gene AtCAT3 (AT1G20620). According to functional annotation, we randomly selected 5 transcription factors from these candidates, including bZIP1664, TEM1, bHLH106, BTF3, and HAT1, to verify whether they in fact bind to the AtCAT3 promoter. The binding of these 5 transcription factors was confirmed using chromatin immunoprecipitation quantitative real-time PCR and electrophoretic mobility shift assays. In addition, we improved the R-ChIP method using plants in which the DNA of interest had been transiently introduced, which does not require the T-DNA integration, and showed that this substantially improved the protein capture efficiency. These results together demonstrate that R-ChIP has a wide application to characterize chromatin composition and isolate upstream regulators of a specific gene.

scholarly journals A NAC transcription factor and its interaction protein hinder abscisic acid biosynthesis by synergistically repressing NCED5 in Citrus reticulata

2020 ◽  
Vol 71 (12) ◽  
pp. 3613-3625
Author(s):  
Feng Zhu ◽  
Tao Luo ◽  
Chaoyang Liu ◽  
Yang Wang ◽  
Li Zheng ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Abscisic Acid ◽  
Citrus Fruit ◽  
Bimolecular Fluorescence Complementation ◽  
Citrus Reticulata ◽  
Mobility Shift ◽  
Abscisic Acid Biosynthesis ◽  
Multiple Transcription Factor ◽  
Electrophoretic Mobility Shift Assays ◽  
Aba Biosynthesis

Abstract Although abscisic acid (ABA) is a vital regulator of fruit ripening and several transcription factors have been reported to regulate ABA biosynthesis, reports of the effect of ABA on citrus ripening and the regulation of its biosynthesis by a multiple-transcription-factor complex are scarce. In the present study, a systematic metabolic, cytological, and transcriptome analysis of an ABA-deficient mutant (MT) of Citrus reticulata cv. Suavissima confirmed the positive effect of ABA on the citrus ripening process. The analysis of transcriptome profiles indicated that CrNAC036 played an important role in the ABA deficiency of the mutant, most likely due to an effect on the expression of 9-cis-epoxycarotenoid dioxygenase 5 (CrNCED5). Electrophoretic mobility shift assays and dual luciferase assays demonstrated that CrNAC036 can directly bind and negatively regulate CrNCED5 expression. Furthermore, yeast two-hybrid, bimolecular fluorescence complementation, and dual luciferase assays demonstrated that CrNAC036 interacted with CrMYB68, also down-regulating the expression of CrNCED5. Taken together, our results suggest that CrNAC036 and CrMYB68 synergistically inhibit ABA biosynthesis in citrus fruit by regulating the expression of CrNCED5.

scholarly journals Mechanisms of Transcription in Eosinophils: GATA-1, but not GATA-2, Transactivates the Promoter of the Eosinophil Granule Major Basic Protein Gene

Blood ◽  
1998 ◽  
Vol 91 (9) ◽  
pp. 3447-3458 ◽  
Author(s):  
Yuji Yamaguchi ◽  
Steven J. Ackerman ◽  
Naoko Minegishi ◽  
Masaki Takiguchi ◽  
Masayuki Yamamoto ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Promoter Activity ◽  
Basic Protein ◽  
Transcriptional Start Site ◽  
Luciferase Reporter ◽  
Major Basic Protein ◽  
Mobility Shift ◽  
P2 Promoter ◽  
Eosinophil Granule ◽  
Electrophoretic Mobility Shift Assays

Granule major basic protein (MBP) is expressed exclusively in eosinophils, basophils, and placental trophoblasts. To identify thecis-elements and transcription factors involved in regulating MBP expression, we subcloned 3.2 kb of sequence upstream of the exon 9 transcriptional start site (P2 promoter) and serial 5′ deletions into the pXP2 luciferase reporter vector. An 80% decrement in promoter activity was obtained when MBP sequences between bp −117 to −67 were deleted. To identify transcription factors that bind to and transactivate through the bp −117 to −67 region, we first compared the upstream genomic sequences of human and murine MBP; a potential GATA binding consensus site was conserved in the 50-bp region between the two genes. To determine which GATA proteins bind this consensus site, we performed electrophoretic mobility shift assays (EMSAs), which showed that both GATA-1 and GATA-2 can bind to this consensus site. To determine the functionality of this site, we tested whether GATA-1 and GATA-2, either individually or in combination, can transactivate the MBP promoter in the Jurkat T cell line. Cotransfection with a GATA-1 expression vector produced 20-fold augmentation of MBP promoter activity, whereas GATA-2 had no activity. In contrast, combined cotransfection of GATA-1 and GATA-2 decreased the ability of GATA-1 to transactivate the MBP promoter by approximately 50%. Our results provide the first evidence for a GATA-1 target gene in eosinophils, a negative regulatory role for GATA-2 in MBP expression, and possibly eosinophil gene transcription in general during myelopoiesis.

scholarly journals Synergistic binding of bHLH transcription factors to the promoter of the maize NADP-ME gene used in C4 photosynthesis is based on an ancient code found in the ancestral C3 state

2018 ◽  
Author(s):  
Ana Rita Borba ◽  
Tânia S. Serra ◽  
Alicja Górska ◽  
Paulo Gouveia ◽  
André M. Cordeiro ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Regulatory Networks ◽  
Molecular Mechanisms ◽  
Mobility Shift ◽  
Helix Loop Helix ◽  
Specific Accumulation ◽  
Cell Type Specific ◽  
Bhlh Transcription Factors ◽  
Electrophoretic Mobility Shift Assays ◽  
Element Pair

AbstractC4 photosynthesis has evolved repeatedly from the ancestral C3 state to generate a carbon concentrating mechanism that increases photosynthetic efficiency. This specialised form of photosynthesis is particularly common in the PACMAD clade of grasses, and is used by many of the world’s most productive crops. The C4 cycle is accomplished through cell-type specific accumulation of enzymes but cis-elements and transcription factors controlling C4 photosynthesis remain largely unknown. Using the NADP-Malic Enzyme (NADP-ME) gene as a model we aimed to better understand molecular mechanisms associated with the evolution of C4 photosynthesis. Two basic Helix-Loop-Helix (bHLH) transcription factors, ZmbHLH128 and ZmbHLH129, were shown to bind the C4NADP-ME promoter from maize. These proteins form heterodimers and ZmbHLH129 impairs trans-activation by ZmbHLH128. Electrophoretic mobility shift assays indicate that a pair of cis-elements separated by a seven base pair spacer synergistically bind either ZmbHLH128 or ZmbHLH129. This pair of cis-elements is found in both C3 and C4 species of the PACMAD clade. Our analysis is consistent with this cis-element pair originating from a single motif present in the ancestral C3 state. We conclude that C4 photosynthesis has co-opted an ancient C3 regulatory code built on G-box recognition by bHLH to regulate the NADP-ME gene. More broadly, our findings also contribute to the understanding of gene regulatory networks controlling C4 photosynthesis.

scholarly journals ATF2 Interacts with β-Cell-enriched Transcription Factors, MafA, Pdx1, and Beta2, and Activates Insulin Gene Transcription

2011 ◽  
Vol 286 (12) ◽  
pp. 10449-10456 ◽  
Author(s):  
Song-iee Han ◽  
Kunio Yasuda ◽  
Kohsuke Kataoka
Keyword(s):  
Transcription Factors ◽  
Leucine Zipper ◽  
Binding Capacity ◽  
Immunohistochemical Analysis ◽  
Regulatory Elements ◽  
Insulin Gene ◽  
Β Cell ◽  
Mobility Shift ◽  
Basic Leucine Zipper ◽  
Insulin Promoter

Pancreatic β-cell-restricted expression of insulin is established through several critical cis-regulatory elements located in the insulin gene promoter region. The principal cis elements are A-boxes, E1, and C1/RIPE3b. The β-cell-enriched transcription factors Pdx1 and Beta2 bind to the A-boxes and E1 element, respectively. A β-cell-specific trans-acting factor binding to C1/RIPE3b (termed RIPE3b1 activator) was detected by electrophoretic mobility shift assay and has been identified as MafA, a member of the Maf family of basic leucine zipper (bZip) proteins. Here, ATF2, a member of the ATF/CREB family of basic leucine zipper proteins, was identified as a component of the RIPE3b1 activator. ATF2 alone was unable to bind to the C1/RIPE3b element but acquired binding capacity upon complex formation with MafA. ATF2 also interacted with Pdx1 and Beta2, and co-expression of ATF2, MafA, Pdx1, and Beta2 resulted in a synergistic activation of the insulin promoter. Immunohistochemical analysis of mouse pancreas tissue sections showed that ATF2 is enriched in islet endocrine cells, including β-cells. RNAi-mediated knockdown of MafA or ATF2 in the MIN6 β-cell line resulted in a significant decrease in endogenous levels of insulin mRNA. These data indicate that ATF2 is an essential component of the positive regulators of the insulin gene expression.

scholarly journals A mutant cell line defective in response to double-stranded RNA and in regulating basal expression of interferon-stimulated genes

1998 ◽  
Vol 95 (16) ◽  
pp. 9442-9447 ◽  
Author(s):  
Douglas W. Leaman ◽  
Anupama Salvekar ◽  
Rekha Patel ◽  
Ganes C. Sen ◽  
George R. Stark
Keyword(s):  
Transcription Factors ◽  
Mutant Cell ◽  
Mobility Shift ◽  
Serine Threonine Kinase ◽  
Double Stranded Rna ◽  
Interferon Stimulated Genes ◽  
Basal Expression ◽  
Recessive Mutant ◽  
Electrophoretic Mobility Shift Assays ◽  
Mutant Cells

Although much progress has been made in identifying the signaling pathways that mediate the initial responses to interferons (IFNs), much less is known about how IFN-stimulated genes (ISGs) are kept quiescent in untreated cells, how the response is sustained after the initial induction, and how ISG expression is down-regulated, even in the continued presence of IFN. We have used the cell sorter to isolate mutant cells with constitutively high ISG expression. A recessive mutant, P2.1, has higher constitutive ISG levels than the parental U4C cells, which do not respond to any IFN. Unexpectedly, P2.1 cells also are deficient in the expression of ISGs in response to double-stranded RNA (dsRNA). Electrophoretic mobility-shift assays revealed that the defect is upstream of the activation of the transcription factors NFκB and IFN regulatory factor 1. Analysis of the pivotal dsRNA-dependent serine/threonine kinase PKR revealed that the wild-type kinase is present and is activated normally in response to dsRNA in P2.1 cells. Together, these data suggest that the defect in P2.1 cells is either downstream of PKR or in a component of a distinct pathway that is involved both in activating multiple transcription factors in response to dsRNA and in regulating the basal expression of ISGs.

scholarly journals Mechanisms of Transcription in Eosinophils: GATA-1, but not GATA-2, Transactivates the Promoter of the Eosinophil Granule Major Basic Protein Gene

Blood ◽  
1998 ◽  
Vol 91 (9) ◽  
pp. 3447-3458 ◽  
Author(s):  
Yuji Yamaguchi ◽  
Steven J. Ackerman ◽  
Naoko Minegishi ◽  
Masaki Takiguchi ◽  
Masayuki Yamamoto ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Promoter Activity ◽  
Basic Protein ◽  
Transcriptional Start Site ◽  
Luciferase Reporter ◽  
Major Basic Protein ◽  
Mobility Shift ◽  
P2 Promoter ◽  
Eosinophil Granule ◽  
Electrophoretic Mobility Shift Assays

Abstract Granule major basic protein (MBP) is expressed exclusively in eosinophils, basophils, and placental trophoblasts. To identify thecis-elements and transcription factors involved in regulating MBP expression, we subcloned 3.2 kb of sequence upstream of the exon 9 transcriptional start site (P2 promoter) and serial 5′ deletions into the pXP2 luciferase reporter vector. An 80% decrement in promoter activity was obtained when MBP sequences between bp −117 to −67 were deleted. To identify transcription factors that bind to and transactivate through the bp −117 to −67 region, we first compared the upstream genomic sequences of human and murine MBP; a potential GATA binding consensus site was conserved in the 50-bp region between the two genes. To determine which GATA proteins bind this consensus site, we performed electrophoretic mobility shift assays (EMSAs), which showed that both GATA-1 and GATA-2 can bind to this consensus site. To determine the functionality of this site, we tested whether GATA-1 and GATA-2, either individually or in combination, can transactivate the MBP promoter in the Jurkat T cell line. Cotransfection with a GATA-1 expression vector produced 20-fold augmentation of MBP promoter activity, whereas GATA-2 had no activity. In contrast, combined cotransfection of GATA-1 and GATA-2 decreased the ability of GATA-1 to transactivate the MBP promoter by approximately 50%. Our results provide the first evidence for a GATA-1 target gene in eosinophils, a negative regulatory role for GATA-2 in MBP expression, and possibly eosinophil gene transcription in general during myelopoiesis.

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