Unknown,Transcriptomics,Genomics,Proteomics

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Hit-and-run transcriptional control by bZIP1 mediates rapid nutrient signaling in Arabidopsis

ABSTRACT: To identify potential transient interactions between a TF and its targets, we developed an approach that can identify primary targets based either on TF-induced regulation or TF-binding, assayed in the same samples. Our studies focused on the TF bZIP1 (BASIC LEUCINE ZIPPER 1), a central integrator of cellular and metabolic signaling. To discern the mechanisms by which bZIP1 regulates a gene netowork in response to a nitrogen (N)-signal perceived in vivo, we perturbed both bZIP1 and the N-signal that it transduces in a cell-based transient expression system called TARGET (Transient Assay Reporting Genome-wide Effects of Transcription factors). To identify the genome-wide targets of bZIP1 in response to a N-signal, we transiently perturbed both the TF and the signal in the TARGET cell-based system. Specifically, bZIP1 was transiently overexpressed in root protoplasts following transfection with a 35S::GR::bZIP1 construct containing an RFP (red fluorescent protein) selectable marker gene. bZIP1 transfected cells were sequentially treated with: i) an inorganic nitrogen signal (+/-N), ii) cycloheximide (CHX) to block the synthesis of proteins, and iii) dexamethasone (DEX) to induce nuclear import of the GR::bZIP1 protein. To identify both the TF-regulated and the TF-bound genes, transcriptome analysis of transfected cells and micro-ChIP data (using anti-GR antibodies), were carried out in parallel . This submission represents transcriptome component of study.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Ying Li

PROVIDER: E-GEOD-54049 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Hit-and-run transcriptional control by bZIP1 mediates rapid nutrient signaling in Arabidopsis.

Para Alessia A Li Ying Y Marshall-Colón Amy A Varala Kranthi K Francoeur Nancy J NJ Moran Tara M TM Edwards Molly B MB Hackley Christopher C Bargmann Bastiaan O R BO Birnbaum Kenneth D KD McCombie W Richard WR Krouk Gabriel G Coruzzi Gloria M GM

Proceedings of the National Academy of Sciences of the United States of America 20140623 28

The dynamic nature of gene regulatory networks allows cells to rapidly respond to environmental change. However, the underlying temporal connections are missed, even in kinetic studies, as transcription factor (TF) binding within at least one time point is required to identify primary targets. The TF-regulated but unbound genes are dismissed as secondary targets. Instead, we report that these genes comprise transient TF-target interactions most relevant to rapid signal transduction. We temporall ...[more]

PMID: 24958886

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Project description:BACKGROUND:Dynamic transcriptional regulation is critical for an organism's response to environmental signals and yet remains elusive to capture. Such transcriptional regulation is mediated by master transcription factors (TF) that control large gene regulatory networks. Recently, we described a dynamic mode of TF regulation named "hit-and-run". This model proposes that master TF can interact transiently with a set of targets, but the transcription of these transient targets continues after the TF dissociation from the target promoter. However, experimental evidence validating active transcription of the transient TF-targets is still lacking. RESULTS:Here, we show that active transcription continues after transient TF-target interactions by tracking de novo synthesis of RNAs made in response to TF nuclear import. To do this, we introduced an affinity-labeled 4-thiouracil (4tU) nucleobase to specifically isolate newly synthesized transcripts following conditional TF nuclear import. Thus, we extended the TARGET system (Transient Assay Reporting Genome-wide Effects of Transcription factors) to include 4tU-labeling and named this new technology TARGET-tU. Our proof-of-principle example is the master TF Basic Leucine Zipper 1 (bZIP1), a central integrator of metabolic signaling in plants. Using TARGET-tU, we captured newly synthesized mRNAs made in response to bZIP1 nuclear import at a time when bZIP1 is no longer detectably bound to its target. Thus, the analysis of de novo transcripomics demonstrates that bZIP1 may act as a catalyst TF to initiate a transcriptional complex ("hit"), after which active transcription by RNA polymerase continues without the TF being bound to the gene promoter ("run"). CONCLUSION:Our findings provide experimental proof for active transcription of transient TF-targets supporting a "hit-and-run" mode of action. This dynamic regulatory model allows a master TF to catalytically propagate rapid and broad transcriptional responses to changes in environment. Thus, the functional read-out of de novo transcripts produced by transient TF-target interactions allowed us to capture new models for genome-wide transcriptional control. Root protoplasts were transfected with 35S::GR::bZIP1 or Empty vector (EV=pJD385_35S::GR) constructs and cells were sequentially treated with: i) the nitrogen signal (N), ii) cycloheximide (CHX), iii) dexamethasone (DEX; for review see Bargmann et. al 2013). Cells were exposed to 4tU to affinity-label newly synthesized transcripts prior RNA extraction followed by specific pull down of 4tu-fractions and hybridization on Arabidopsis ATH1 microarray chips.

Dataset Information

Hit-and-run transcriptional control by bZIP1 mediates rapid nutrient signaling in Arabidopsis

Publications

Hit-and-run transcriptional control by bZIP1 mediates rapid nutrient signaling in Arabidopsis.

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