DamID-seq: A Genome-Wide DNA Methylation Method that Captures Both Transient and Stable TF-DNA Interactions in Plant Cells

José M. Alvarez, Will E. Hinckley, Lauriebeth Leonelli, Matthew D. Brooks, Gloria M. Coruzzi

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages87-107
Number of pages21
DOIs
StatePublished - 2023

Publication series

NameMethods in Molecular Biology
Volume2698
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • DNA adenine methyltransferase identification (DamID)
  • Plant cell protoplasts
  • Transcription factors (TFs)
  • Transient/stable DNA-binding sites

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology

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