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The identification of genome-wide transcription factor binding sites (TFBS) is a critical step in deciphering gene and transcriptional regulatory networks. However, determining the genome-wide binding of specific TFs or TF complexes remains a technical challenge. DNA affinity purification sequencing (DAP-seq) and modifications such as sequential DAP-seq (seq-DAP-seq) are robust in vitro methods for mapping individual TF or TF complex binding sites in a genome-wide manner. DAP-seq protocols use a genomic DNA (gDNA) library from any target organism with or without amplification, allowing the determination of TF binding on naked or endogenously modified DNA, respectively. As a first step, the gDNA is fragmented to ~200 bp, end-repaired, and sequencing adaptors are added. This gDNA library can be used directly or an amplification step may be performed to remove DNA modifications such as cytosine methylation. DNA libraries are then incubated with an affinity-tagged TF or TF- complex immobilized on magnetic beads. The TF or TF complex of interest is generally produced using recombinant protein expression and purified prior to DNA affinity purification. After incubation of the DNA library with the immobilized TF of interest, multiple wash steps are performed to reduce non-specific DNA binding and the TF-DNA complexes eluted. The eluted DNA is PCR-amplified and sequenced using next-generation sequencing. The resulting sequence reads are mapped to the corresponding reference genome, identifying direct potential bound regions and binding sites of the TF or TF complex of interest. Predictive TFBS models are generated from the bound regions using downstream bioinformatics analysis pipelines. Here, we present a detailed protocol outlining the steps required for seq-DAP-seq of a heterooligomeric TF complex (Fig. 1) and briefly describe the downstream bioinformatics pipeline used to develop a robust TFBS model from sequencing data generated from a DAP-seq experiment.

Titel:	Identification of Plant Transcription Factor DNA-Binding Sites Using seq-DAP-seq.
Autor/in / Beteiligte Person:	Hutin, S ; Blanc-Mathieu, R ; Rieu, P ; Parcy, F ; Lai, X ; Zubieta, C
Zeitschrift:	Methods in molecular biology (Clifton, N.J.), Jg. 2698 (2023), S. 119-145
Veröffentlichung:	Totowa, NJ : Humana Press ; <i>Original Publication</i>: Clifton, N.J. : Humana Press,, 2023
Medientyp:	academicJournal
ISSN:	1940-6029 (electronic)
DOI:	10.1007/978-1-0716-3354-0_9
Schlagwort:	DNA, Plant genetics Binding Sites Sequence Analysis, DNA Transcription Factors genetics Gene Expression Regulation
Sonstiges:	Nachgewiesen in: MEDLINE Sprachen: English Publication Type: Journal Article Language: English [Methods Mol Biol] 2023; Vol. 2698, pp. 119-145. MeSH Terms: Transcription Factors* / genetics ; Gene Expression Regulation* ; DNA, Plant / genetics ; Binding Sites ; Sequence Analysis, DNA References: Babu MM, Luscombe NM, Aravind L et al (2004) Structure and evolution of transcriptional regulatory networks. Curr Opin Struct Biol 14:283–291. (PMID: 10.1016/j.sbi.2004.05.00415193307) ; Robertson G, Hirst M, Bainbridge M et al (2007) Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing. Nat Methods 4:651–657. (PMID: 10.1038/nmeth106817558387) ; Mukhopadhyay A, Deplancke B, Walhout AJM et al (2008) Chromatin immunoprecipitation (ChIP) coupled to detection by quantitative real-time PCR to study transcription factor binding to DNA in Caenorhabditis elegans. Nat Protoc 3:698–709. 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(PMID: 10.1186/s13007-018-0336-1301817646114050) Contributed Indexing: 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) Substance Nomenclature: 0 (DNA, Plant) ; 0 (Transcription Factors) Entry Date(s): Date Created: 20230908 Date Completed: 20230911 Latest Revision: 20230916 Update Code: 20240514

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