Abstract
Dynamic reprogramming of gene regulatory networks (GRNs) enables organisms to rapidly respond to environmental perturbation. However, the underlying transient interactions between transcription factors (TFs) and genome-wide targets typically elude biochemical detection. Here, we capture both stable and transient TF-target interactions genome-wide within minutes after controlled TF nuclear import using time-series chromatin immunoprecipitation (ChIP-seq) and/or DNA adenine methyltransferase identification (DamID-seq). The transient TF-target interactions captured uncover the early mode-of-action of NIN-LIKE PROTEIN 7 (NLP7), a master regulator of the nitrogen signaling pathway in plants. These transient NLP7 targets captured in root cells using temporal TF perturbation account for 50% of NLP7-regulated genes not detectably bound by NLP7 in planta. Rapid and transient NLP7 binding activates early nitrogen response TFs, which we validate to amplify the NLP7-initiated transcriptional cascade. Our approaches to capture transient TF-target interactions genome-wide can be applied to validate dynamic GRN models for any pathway or organism of interest.
Highlights
Dynamic reprogramming of gene regulatory networks (GRNs) enables organisms to rapidly respond to environmental perturbation
The very large set of transcription factors (TFs)-regulated, but unbound genes are typically dismissed as indirect targets, because standard approaches can only identify direct TF targets based on TF binding[7,8]
We set out to address the paradox that NIN-LIKE PROTEIN 7 (NLP7) binds to hundreds of genes, yet only 10% of NLP7-regulated targets are NLP7-bound based on chromatin immunoprecipitation (ChIP)-chip assay in planta[11]
Summary
Dynamic reprogramming of gene regulatory networks (GRNs) enables organisms to rapidly respond to environmental perturbation. The underlying transient interactions between transcription factors (TFs) and genome-wide targets typically elude biochemical detection We capture both stable and transient TF-target interactions genome-wide within minutes after controlled TF nuclear import using time-series chromatin immunoprecipitation (ChIP-seq) and/or DNA adenine methyltransferase identification (DamID-seq). To capture potential transient TF–target interactions of NLP7, we exploited the cell-based TARGET system, which can identify direct TF-mediated gene regulation: (i) in the absence of stable TF binding, and (ii) within minutes of controlled TF nuclear entry, using time-series ChIP14,15. We previously used this system to capture early and transient TF–target interactions for bZIP1, which support a Hit-and-Run model of transcription, in which transient TF binding initiates transcription that persists long after the TF has dissociated from its targets[14,16]. The stable adenine methylation signature left on the DNA, allows one to capture even the briefest TF–DNA interaction, a major advantage of DamID over ChIP (reviewed in Aughey and Southall[17])
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