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| Indexado |
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| DOI | 10.1007/978-1-0716-3354-0_7 | ||
| Año | 2023 | ||
| Tipo |
Citas Totales
Autores Afiliación Chile
Instituciones Chile
% Participación
Internacional
Autores
Afiliación Extranjera
Instituciones
Extranjeras
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.
| Ord. | Autor | Género | Institución - País |
|---|---|---|---|
| 1 | Alvarez, José M. | - |
Universidad Nacional Andrés Bello - Chile
Instituto Milenio de Biología Integrativa - Chile Millennium Institute for Integrative Biology (iBio) - Chile |
| 2 | Hinckley, Will E. | - |
New York University - Estados Unidos
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| 3 | Leonelli, Lauriebeth | - |
University of Illinois Urbana-Champaign - Estados Unidos
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| 4 | Brooks, Matthew D. | - |
USDA Agricultural Research Service - Estados Unidos
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| 5 | Coruzzi, Gloria M. | - |
New York University - Estados Unidos
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| Fuente |
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| National Science Foundation |
| NSF-PGRP |
| Agencia Nacional de Investigación y Desarrollo |
| Zegar Family FoundationA16-0051 |
| Agradecimiento |
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| This work is funded by ANID FONDECYT1210389 and ANID— Millennium Science Initiative Program—Millennium Institute for Integrative Biology (iBio) ICN17_022 to J.M.A, NIH-NIGMSR01 GM121753 to G.M.C., NSF Plant Genome grant NSF-PGRP: IOS – 1840761 to G.M.C. and J.M.A., the Zegar Family FoundationA16-0051 to G.M.C. |