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Network Walking charts transcriptional dynamics of nitrogen signaling by integrating validated and predicted genome-wide interactions.

ABSTRACT: Charting a temporal path in gene networks requires linking early transcription factor (TF)-triggered events to downstream effects. We scale-up a cell-based TF-perturbation assay to identify direct regulated targets of 33 nitrogen (N)-early response TFs encompassing 88% of N-responsive Arabidopsis genes. We uncover a duality where each TF is an inducer and repressor, and in vitro cis-motifs are typically specific to regulation directionality. Validated TF-targets (71,836) are used to refine precision of a time-inferred root network, connecting 145 N-responsive TFs and 311 targets. These data are used to chart network paths from direct TF₁-regulated targets identified in cells to indirect targets responding only in planta via Network Walking. We uncover network paths from TGA1 and CRF4 to direct TF₂ targets, which in turn regulate 76% and 87% of TF₁ indirect targets in planta, respectively. These results have implications for N-use and the approach can reveal temporal networks for any biological system.

SUBMITTER: Brooks MD

PROVIDER: S-EPMC6451032 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Network Walking charts transcriptional dynamics of nitrogen signaling by integrating validated and predicted genome-wide interactions.

Brooks Matthew D MD Cirrone Jacopo J Pasquino Angelo V AV Alvarez Jose M JM Swift Joseph J Mittal Shipra S Juang Che-Lun CL Varala Kranthi K Gutiérrez Rodrigo A RA Krouk Gabriel G Shasha Dennis D Coruzzi Gloria M GM

Nature communications 20190405 1

Charting a temporal path in gene networks requires linking early transcription factor (TF)-triggered events to downstream effects. We scale-up a cell-based TF-perturbation assay to identify direct regulated targets of 33 nitrogen (N)-early response TFs encompassing 88% of N-responsive Arabidopsis genes. We uncover a duality where each TF is an inducer and repressor, and in vitro cis-motifs are typically specific to regulation directionality. Validated TF-targets (71,836) are used to refine preci ...[more]

PMID: 30952851

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Project description:BackgroundInteractions between transcription factors and DNA lie at the centre of many biological processes including DNA recombination, replication, repair and transcription. Most bacteria encode diverse proteins that act as transcription factors to regulate various traits. Several technologies for identifying protein-DNA interactions at the genomic level have been developed. Bind-n-seq is a high-throughput in vitro method first deployed to analyse DNA interactions associated with eukaryotic zinc-finger proteins. The method has three steps (i) binding protein to a randomised oligonucleotide DNA target library, (ii) deep sequencing of bound oligonucleotides, and (iii) a computational algorithm to define motifs among the sequences. The classical Bind-n-seq strategy suffers from several limitations including a lengthy wet laboratory protocol and a computational algorithm that is difficult to use. We introduce here an improved, rapid, and simplified Bind-n-seq protocol coupled with a user-friendly downstream data analysis and handling algorithm, which has been optimized for bacterial target proteins. We validate this new protocol by showing the successful characterisation of the DNA-binding specificities of YipR (YajQ interacting protein regulator), a well-known transcriptional regulator of virulence genes in the bacterial phytopathogen Xanthomonas campestris pv. campestris (Xcc).ResultsThe improved Bind-n-seq approach identified several DNA binding motif sequences for YipR, in particular the CCCTCTC motif, which were located in the promoter regions of 1320 Xcc genes. Informatics analysis revealed that many of these genes regulate functions associated with virulence, motility, and biofilm formation and included genes previously found involved in virulence. Additionally, electromobility shift assays show that YipR binds to the promoter region of XC_2633 in a CCCTCTC motif-dependent manner.ConclusionWe present a new and rapid Bind-n-seq protocol that should be useful to investigate DNA-binding proteins in bacteria. The analysis of YipR DNA binding using this protocol identifies a novel DNA sequence motif in the promoter regions of target genes that define the YipR regulon.

Dataset Information

Network Walking charts transcriptional dynamics of nitrogen signaling by integrating validated and predicted genome-wide interactions.

Publications

Network Walking charts transcriptional dynamics of nitrogen signaling by integrating validated and predicted genome-wide interactions.

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