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.

Distinct functional contributions of 2 GABP–NRF-2 recognition sites within the context of the human TOMM70 promoter

2006 ◽  
Vol 84 (5) ◽  
pp. 813-822 ◽  
Author(s):  
José R. Blesa ◽  
José Hernández-Yago
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Site Directed Mutagenesis ◽  
Outer Mitochondrial Membrane ◽  
Mobility Shift ◽  
A Subunit ◽  
Expression Evidence ◽  
Electrophoretic Mobility Shift Assays

TOMM70 is a subunit of the outer mitochondrial membrane translocase that plays a major role as a receptor of hydrophobic preproteins targeted to mitochondria. We have previously reported 2 binding sites for the transcription factor GABP–NRF-2 in the promoter region of the human TOMM70 gene that are important in activating transcription. To assess the functionality and actual role of these sites, chromatin immunoprecipitation, site-directed mutagenesis, and electrophoretic mobility shift assays were carried out. We conclude that GABP–NRF-2 binds in vivo to the TOMM70 promoter, and that the 2 GABP–NRF-2 binding sites of the promoter have different functional contributions in promoting TOMM70 expression. Evidence is provided that they work in an additive manner as single sites.

C-MYB and C/EBP Family Transcription Factors Regulate the Gfi-1 Promoter.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4214-4214
Author(s):  
Richard Dahl ◽  
Kristin S. Owens
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Myeloid Cells ◽  
Transient Transfection ◽  
Mobility Shift ◽  
Reporter Assays ◽  
Immature Myeloid Cells ◽  
Factor C

Abstract Gfi-1 −/− mice generate abnormal immature myeloid cells exhibiting characteristics of both monocytes and granulocytes. One of Gfi-1’s critical functions is to downregulate monocyte specific genes in order for granulocytes to develop properly. Since the transcription factors C/EBP alpha and C/EBP epsilon are needed for granulocyte development we hypothesized that these factors may regulate Gfi-1 expression. The Gfi-1 promoter contains several putative C/EBP binding sites and we show by electrophoretic mobility shift and chromatin immunoprecipitation that C/EBP family members can bind to some of these sites. However we were unable to see activation of the Gfi-1 promoter by C/EBP proteins in transient transfection reporter assays. Other groups have shown that C/EBP proteins can synergize with the transcription factor c-myb. We observed that the Gfi-1 promoter contains sites for the hematopoietic transcription factor c-myb. Sevral of these c-myb binding sites are adjacent to C/EBP binding sites. In reporter assays in non-hematopoietic cells c-myb activated the Gfi-1 promoter by itself and this activity was enhanced when we included either C/EBP alpha or epsilon in the transfection. Our data suggests that C/EBP proteins and c-myb regulate the transcription of Gfi-1 in myeloid cells.

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 Pseudomonas aeruginosa LysR PA4203 Regulator NmoR Acts as a Repressor of the PA4202nmoAGene, Encoding a Nitronate Monooxygenase

2014 ◽  
Vol 197 (6) ◽  
pp. 1026-1039 ◽  
Author(s):  
Ken Vercammen ◽  
Qing Wei ◽  
Daniel Charlier ◽  
Andreas Dötsch ◽  
Susanne Haüssler ◽  
...  
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Mobility Shift ◽  
Quinone Oxidoreductase ◽  
Gene Encoding ◽  
Nitropropionic Acid ◽  
Gene Coding ◽  
Intergenic Regions ◽  
Increased Sensitivity ◽  
Electrophoretic Mobility Shift Assays

The PA4203 gene encodes a LysR regulator and lies between theppgLgene (PA4204), which encodes a periplasmic gluconolactonase, and, in the opposite orientation, the PA4202 (nmoA) gene, coding for a nitronate monooxygenase, andddlA(PA4201), encoding ad-alanine alanine ligase. The intergenic regions between PA4203 andppgLand between PA4203 andnmoAare very short (79 and 107 nucleotides, respectively). Here we show that PA4203 (nmoR) represses its own transcription and the expression ofnmoA. A chromatin immunoprecipitation analysis showed the presence of a single NmoR binding site betweennmoAandnmoR, which was confirmed by electrophoretic mobility shift assays (EMSAs) with the purified NmoR protein. Despite this observation, a transcriptome analysis revealed more genes to be affected in annmoRmutant, including genes known to be part of the MexT LysR activator regulon. The PA1225 gene, encoding a quinone oxidoreductase, was the most highly upregulated gene in thenmoRdeletion mutant, independently of MexT. Finally, deletion of thenmoAgene resulted in an increased sensitivity of the cells to 3-nitropropionic acid (3-NPA), confirming the role of the nitronate monooxygenase protein in the detoxification of nitronate.

NRF-1 is the major transcription factor regulating the expression of the human TOMM34 gene

2008 ◽  
Vol 86 (1) ◽  
pp. 46-56 ◽  
Author(s):  
José R. Blesa ◽  
Jesús A. Prieto-Ruiz ◽  
Beth A. Abraham ◽  
Bridget L. Harrison ◽  
Anita A. Hegde ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Colorectal Tumors ◽  
Methylation Analysis ◽  
Mobility Shift ◽  
Nuclear Respiratory Factors ◽  
Hela S3 ◽  
Electrophoretic Mobility Shift Assays

The human TOMM34 gene encodes a cytosolic protein with chaperone-like activity that helps import some preproteins to the mitochondria by keeping them in an unfolded, import-compatible state. TOMM34 was found to be upregulated frequently in colorectal tumors, suggesting that it also has a role in the growth of cancer cells. In this context, TOMM34 is a potential target for novel anticancer drugs, and it might also be used in the diagnosis of colorectal cancer. Nuclear respiratory factors (NRFs) play an important role in governing the nuclear–mitochondrial interactions implicated in mitochondrial biogenesis. Our previous studies revealed that NRFs promote the expression of the major members of the mitochondrial transport machinery, TOMM70 and TOMM20. Here we report the existence of binding sites for NRF-1, Sp1, and NRF-2 in the 5′ region of the human TOMM34 gene. We determined the effects of mutations at these sites on promoter activity in HeLa S3 and A204 cells, in conjunction with chromatin immunoprecipitation experiments, electrophoretic mobility shift assays, and in vivo methylation analysis of the promoter region. We conclude that NRF-1 is the main transcription factor regulating the expression of TOMM34. Sp1 interacts with NRF-1 to stimulate the promoter's full activity.

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 Structural and Functional Dissection of Mif2p, a Conserved DNA-binding Kinetochore Protein

2008 ◽  
Vol 19 (10) ◽  
pp. 4480-4491 ◽  
Author(s):  
R. L. Cohen ◽  
C. W. Espelin ◽  
P. De Wulf ◽  
P. K. Sorger ◽  
S. C. Harrison ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Chromatin Immunoprecipitation ◽  
Polypeptide Chain ◽  
Budding Yeast ◽  
Spatial Association ◽  
Dimerization Domain ◽  
Mobility Shift ◽  
Bacterial Transcription ◽  
Kinetochore Assembly ◽  
Electrophoretic Mobility Shift Assays

Mif2p is the budding-yeast orthologue of the mammalian centromere-binding protein CENP-C. We have mapped domains of Saccharomyces cerevisiae Mif2p and studied the phenotyptic consequences of their deletion. Using chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays, we have further shown that Mif2p binds in the CDEIII region of the budding-yeast centromere, probably in close spatial association with Ndc10p. Moreover, ChIP experiments show that Mif2p recruits to yeast kinetochores a substantial subset of inner and outer kinetochore proteins, but not the Ndc80 or Spc105 complexes. We have determined the crystal structure of the C-terminal, dimerization domain of Mif2p. It has a “cupin” fold, extremely similar both in polypeptide chain conformation and in dimer geometry to the dimerization domain of a bacterial transcription factor. The Mif2p dimer seems to be part of an enhanceosome-like structure that nucleates kinetochore assembly in budding yeast.

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.

β-Arm flexibility of HU fromStaphylococcus aureusdictates the DNA-binding and recognition mechanism

2014 ◽  
Vol 70 (12) ◽  
pp. 3273-3289 ◽  
Author(s):  
Do-Hee Kim ◽  
Hookang Im ◽  
Jun-Goo Jee ◽  
Sun-Bok Jang ◽  
Hye-Jin Yoon ◽  
...  
Keyword(s):  
Dna Binding ◽  
Md Simulations ◽  
Titration Calorimetry ◽  
Salt Bridges ◽  
Mobility Shift ◽  
Bent Dna ◽  
Recognition Mechanism ◽  
Local Shape ◽  
Associated Proteins ◽  
Electrophoretic Mobility Shift Assays

HU, one of the major nucleoid-associated proteins, interacts with the minor groove of DNA in a nonspecific manner to induce DNA bending or to stabilize bent DNA. In this study, crystal structures are reported for both free HU fromStaphylococcus aureusMu50 (SHU) and SHU bound to 21-mer dsDNA. The structures, in combination with electrophoretic mobility shift assays (EMSAs), isothermal titration calorimetry (ITC) measurements and molecular-dynamics (MD) simulations, elucidate the overall and residue-specific changes in SHU upon recognizing and binding to DNA. Firstly, structural comparison showed the flexible nature of the β-sheets of the DNA-binding domain and that the β-arms bend inwards upon complex formation, whereas the other portions are nearly unaltered. Secondly, it was found that the disruption and formation of salt bridges accompanies DNA binding. Thirdly, residue-specific free-energy analyses using the MM-PBSA method with MD simulation data suggested that the successive basic residues in the β-arms play a central role in recognizing and binding to DNA, which was confirmed by the EMSA and ITC analyses. Moreover, residue Arg55 resides in the hinge region of the flexible β-arms, exhibiting a remarkable role in their flexible nature. Fourthly, EMSAs with various DNAs revealed that SHU prefers deformable DNA. Taken together, these data suggest residue-specific roles in local shape and base readouts, which are primarily mediated by the flexible β-arms consisting of residues 50–80.

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