TY - CHAP
T1 - DamID-seq
T2 - A Genome-Wide DNA Methylation Method that Captures Both Transient and Stable TF-DNA Interactions in Plant Cells
AU - Alvarez, José M.
AU - Hinckley, Will E.
AU - Leonelli, Lauriebeth
AU - Brooks, Matthew D.
AU - Coruzzi, Gloria M.
N1 - Publisher Copyright:
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023.
PY - 2023
Y1 - 2023
N2 - Capturing the dynamic and transient interactions of a transcription factor (TF) with its genome-wide targets whose regulation leads to plants’ adaptation to their changing environment is a major technical challenge. This is a widespread problem with biochemical methods such as chromatin immunoprecipitation-sequencing (ChIP-seq) which are biased towards capturing stable TF-target gene interactions. Herein, we describe how DNA adenine methyltransferase identification and sequencing (DamID-seq) can be used to capture both transient and stable TF-target interactions by DNA methylation. The DamID technique uses a TF protein fused to a DNA adenine methyltransferase (Dam) from E. coli. When expressed in a plant cell, the Dam-TF fusion protein will methylate adenine (A) bases near the sites of TF-DNA interactions. In this way, DamID results in a permanent, stable DNA methylation mark on TF-target gene promoters, even if the target gene is only transiently “touched” by the Dam-TF fusion protein. Here we provide a step-by-step protocol to perform DamID-seq experiments in isolated plant cells for any Dam-TF fusion protein of interest. We also provide information that will enable researchers to analyze DamID-seq data to identify TF-binding sites in the genome. Our protocol includes instructions for vector cloning of the Dam-TF fusion proteins, plant cell protoplast transfections, DamID preps, library preparation, and sequencing data analysis. The protocol outlined in this chapter is performed in Arabidopsis thaliana, however, the DamID-seq workflow developed in this guide is broadly applicable to other plants and organisms.
AB - Capturing the dynamic and transient interactions of a transcription factor (TF) with its genome-wide targets whose regulation leads to plants’ adaptation to their changing environment is a major technical challenge. This is a widespread problem with biochemical methods such as chromatin immunoprecipitation-sequencing (ChIP-seq) which are biased towards capturing stable TF-target gene interactions. Herein, we describe how DNA adenine methyltransferase identification and sequencing (DamID-seq) can be used to capture both transient and stable TF-target interactions by DNA methylation. The DamID technique uses a TF protein fused to a DNA adenine methyltransferase (Dam) from E. coli. When expressed in a plant cell, the Dam-TF fusion protein will methylate adenine (A) bases near the sites of TF-DNA interactions. In this way, DamID results in a permanent, stable DNA methylation mark on TF-target gene promoters, even if the target gene is only transiently “touched” by the Dam-TF fusion protein. Here we provide a step-by-step protocol to perform DamID-seq experiments in isolated plant cells for any Dam-TF fusion protein of interest. We also provide information that will enable researchers to analyze DamID-seq data to identify TF-binding sites in the genome. Our protocol includes instructions for vector cloning of the Dam-TF fusion proteins, plant cell protoplast transfections, DamID preps, library preparation, and sequencing data analysis. The protocol outlined in this chapter is performed in Arabidopsis thaliana, however, the DamID-seq workflow developed in this guide is broadly applicable to other plants and organisms.
KW - DNA adenine methyltransferase identification (DamID)
KW - Plant cell protoplasts
KW - Transcription factors (TFs)
KW - Transient/stable DNA-binding sites
UR - http://www.scopus.com/inward/record.url?scp=85170170249&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85170170249&partnerID=8YFLogxK
U2 - 10.1007/978-1-0716-3354-0_7
DO - 10.1007/978-1-0716-3354-0_7
M3 - Chapter
C2 - 37682471
AN - SCOPUS:85170170249
T3 - Methods in Molecular Biology
SP - 87
EP - 107
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -