AtNIGT1/HRS1 TARGET approach
Ontology highlight
ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
PROVIDER: GSE64352 | GEO | 2015/01/05
SECONDARY ACCESSION(S): PRJNA270809
REPOSITORIES: GEO
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Project description:This work studies the impact of AtNIGT1/HRS1-GR entrance in the nucleus upond DEX treatment in protoplasts. AtNIGT1/HRS1 TARGET. The whole procedure has been performed as previously described in Bargmann et al Mol Plant 2013. In brief, the protoplasts were transfected with the plasmid pBeaconRFP_GR-HRS1 that trigger the expression of HRS1 protein fused with the glucocorticoid receptor under control of CaMV35S promoter. Protoplasts were treated with 35µM cycloheximide (CHX) to inhibit translation and to select only direct target genes and 10µM dexamethasone (DEX) to induce HRS1-GR entry in the nucleus. Nitrate is maintained during the whole TARGET procedure. The Red Fluorescent Protein was used as marker selection for fluorescent-activated cell sorting (FACS) of successfully transformed protoplasts. RNA were extracted and amplified in order to be tested with ATH1 Affymetrix™ chips.
2015-01-05 | E-GEOD-64352 | biostudies-arrayexpress
Project description:The effects of nitrogen and cycloheximide treatments on nitrogen response and TF-target identification was tested in the cell-based TARGET assay. For protoplast experiments, Arabidopsis plants were grown on low nitrogen (1mM KNO3) and after 10 days protoplasts were generated from root tissue. Cells were transfected with a vector containing the GR-TGA4 fusion or GR-only empty vector and incubated overnight. In the morning, samples were pre-treated with nitrogen as specified for 2 hours, and +/-cycloheximide for 20 minutes, before TF entry into the nucleus was induced with dexamethasone. 3 hours after dex treatment samples were FACS sorted into RNA extraction buffer. For whole roots, Arabidopsis were grown in liquid media with low nitrogen (1 mM KNO3) in Phytatrays for 11 days. In the morning and at the same time as the protoplasts, the plants were transferred to fresh media containing either 20 mM KCL or 20mM KNO3 + 20mM NH4NO3 for 5 hours. Roots were harvested and immediately frozen in liquid N2.
2019-03-14 | GSE128209 | GEO
Project description:The direct targets of 16 TFs were identified using the TARGET system. Arabidopsis plants were grown on 1/2X MS media and after 10 days protoplasts were generated from root tissue, Cells were transfected with a vector containing the GR-TF fusion and incubated overnight. Each experiment also included a GR-only empty vector control. In the morning, samples were pre-treated with cycloheximide for 20 minutes, before TF entry into the nucleus was induced with dexamethasone. 3 hours after dex treatment samples were FACS sorted into RNA extraction buffer and RNA-seq libraries were generated. Transcriptional response to each TF compared to empty vector was assayed.
2020-06-13 | GSE152405 | GEO
Project description:Strigolactone (SL) signalling plays many roles in plants, however, the downstream responsive genes remain unclear. SUPPRESSOR OF MORE AXILLARY GROWTH2-LIKE 7 (SMXL7) is degraded upon SL signalling. Here, we generated Arabidopsis carrying a transgene SMXL7pro:SMXL7d53-GR, where a SL-insensitive version of SMXL7 is tagged with the ligand binding domain of the glucocorticoid receptor (GR). SMXL7d53-GR will be translocated to the nucleus upon dexamethasone (dex), and we profiled the gene expression in mock or dex treated plants and revealed the downstream responsive genes of SL signalling.
2024-07-01 | GSE270808 | GEO
Project description:The direct targets of 33 Nitrogen-early responsive TFs were identified using the TARGET system. Arabidopsis plants were grown on low nitrogen (1mM KNO3) and after 10 days protoplasts were generated from root tissue, Cells were transfected with a vector containing the GR-TF fusion and incubated overnight. Each experiment also included a GR-only empty vector control. In the morning, samples were pre-treated with 20 mM KNO3 and 20 mM NH4NO3 for 2 hours, and cycloheximide for 20 minutes, before TF entry into the nucleus was induced with dexamethasone. 3 hours after dex treatment samples were FACS sorted into RNA extraction buffer and RNA-seq libraries were generated. Transcriptional response to each TF compared to empty vector was assayed.
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Project description:Deciphering gene regulatory networks (GRNs) is a key for understanding gene expression regulations in living systems. Here, we describe the investigation of the ABSCISIC ACID INSENSITIVE 3 (ABI3) plant transcription factor GRN vicinity by a technique called Network Walking. The method involves transient transformation of protoplasts and inducible nuclear re-localization of transcription factors along with transcriptomic analysis. This genome-wide approach allowed the de novo recovery of i) direct and indirect ABI3 target genes, ii) cis-binding site preference, and iii) biological processes regulated by this canonical abscisic acid response factor. This work improves our knowledge of ABI3 action by inferring network motifs (such as Feed Forwar Loops) under its influence. The novel high-throughput-oriented technique will help accelerate GRN systems investigations in plants, as well as in other organisms.
2013-01-01 | GSE33344 | GEO
Project description:Direct target genes of VND7 were explored with inducible expression system using glucocorticoid receptor (GR). Transgenic plants expressing 35S:VND7-VP16-GR were treated with dexamethazone (DEX) and/or protein synthesis inhibitor cycloheximide (CHX). A number of genes related to the formation of vascular vessel was induced by DEX even in the presence of CHX. Total RNAs of the transgenic plants expressing 35S:VND7-VP16-GR treated with DEX plus CHX and those treated with CHX only were compared. As a control experiment, transgenic plants harboring empty vector were treated similarly and the total RNAs were compared similarly to identify genes merely induced by DEX treatment itself.
2011-03-31 | E-GEOD-24169 | biostudies-arrayexpress
Project description:In Arabidopsis thaliana, cytokinin responsive B-type ARR transcription factors and HD-ZIP III transcription factors such as REVOLUTA (REV), act cooperatively as master regulators of shoot regeneration. To identify the downstream targets of ARR-HD-ZIP III transcriptional complex, we used an inducible line of REV, 35S::FLAG-GR-rREV, in which FLAG-tagged miR165/6-non-targetable form of REV (rREV)-GR fusion protein was expressed from 35S promoter. DEX treatment induced activation of REV by translocation of FLAG-GR-rREV fusion protein from cytoplasm to the nucleus. We treated 35S::FLAG-GR-rREV seedlings with 6-benzylaminopurine (6-BA, a cytokinin), dexamethasone (DEX), or 6-BA+DEX for 2 hours. Total RNAs were extracted and subjected to Agilent Arabidopsis Gene Expression Microarray analyses. The differentially expressed genes (>1.5-fold, p<0.05) were identified. 10-day-old 35S::FLAG-GR-rREV plants were treated with 6-benzylaminopurine (6-BA), dexamethasone (DEX), or 6-BA+DEX for 2 hours. DEX treatment induced activation of REV by translocation of FLAG-GR-rREV fusion protein from cytoplasm to the nucleus. Total RNA was extracted with RNeasy Mini Kit and hybridized to Agilent Arabidopsis Gene Expression Microarray. Differentially expressed genes were defined by a 1.5-fold expression difference with a P value<0.05. Biological replicates were performed.
2014-01-29 | E-GEOD-54469 | biostudies-arrayexpress
Project description:We report changes in ER and GR binding profiles genome-wide upon co-treatment with Dex and E2 when compared to Dex or E2 treatments alone. We examine ER and GR binding under four different treatments (unt, Dex, E2, and Dex + E2).
2013-07-01 | E-GEOD-46123 | biostudies-arrayexpress
Project description:We report changes in ER and GR binding profiles genome-wide upon co-treatment with Dex and E2 when compared to Dex or E2 treatments alone.
2013-07-01 | GSE46123 | GEO