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Identification of Plant Transcription Factor DNA-Binding Sites Using seq-DAP-seq.

Stephanie Hutin¹, Romain Blanc-Mathieu¹, Philippe Rieu¹

¹Laboratoire de Physiologie Cellulaire et Végétale, CNRS, CEA, Université Grenoble Alpes, INRAE, IRIG, CEA Grenoble, Grenoble, France.

Methods in Molecular Biology (Clifton, N.J.)

|September 8, 2023

Summary

This summary is machine-generated.

DNA affinity purification sequencing (DAP-seq) and its modification, sequential DAP-seq (seq-DAP-seq), map genome-wide transcription factor binding sites (TFBS). These robust in vitro methods identify TF binding sites for deciphering regulatory networks.

Keywords:

DNA affinity purification sequencing (DAP-seq)DNA methylation Recombinant protein expression Sequential DNA affinity purification sequencing (seq-DAP-seq)Sequential pull-down Transcription factor binding sites (TFBS)Transcription factors (TF)

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Area of Science:

Molecular Biology
Genomics
Bioinformatics

Background:

Identifying genome-wide transcription factor binding sites (TFBS) is crucial for understanding gene regulation.
Current methods for mapping TFBS, especially for TF complexes, face technical challenges.

Purpose of the Study:

To present a detailed protocol for sequential DNA affinity purification sequencing (seq-DAP-seq) for heterooligomeric TF complexes.
To describe the downstream bioinformatics pipeline for developing TFBS models from DAP-seq data.

Main Methods:

DNA affinity purification sequencing (DAP-seq) and sequential DAP-seq (seq-DAP-seq) are in vitro methods using fragmented genomic DNA libraries.
Libraries are incubated with affinity-tagged TFs or TF complexes, followed by elution, PCR amplification, and next-generation sequencing.
Bioinformatics pipelines analyze sequencing reads to identify TFBS and generate predictive models.

Main Results:

The study outlines a comprehensive protocol for seq-DAP-seq, applicable to various organisms.
The described bioinformatics pipeline enables the robust modeling of TFBS from DAP-seq experimental data.
This approach allows for the determination of TF binding on naked or endogenously modified DNA.

Conclusions:

Seq-DAP-seq provides a robust method for genome-wide mapping of TF and TF complex binding sites.
The protocol and associated bioinformatics pipeline facilitate the deciphering of transcriptional regulatory networks.
This work advances the technical capabilities for studying gene regulation at a genome-wide scale.