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DamID-seq: A Genome-Wide DNA Methylation Method that Captures Both Transient and Stable TF-DNA Interactions in Plant Cells
Indexado
Scopus SCOPUS_ID:85170170249
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


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.

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Disciplinas de Investigación



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Scopus
Molecular Biology
Genetics
SciELO
Sin Disciplinas

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Publicaciones WoS (Ediciones: ISSHP, ISTP, AHCI, SSCI, SCI), Scopus, SciELO Chile.

Colaboración Institucional



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Autores - Afiliación



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
3 Leonelli, Lauriebeth - University of Illinois Urbana-Champaign - Estados Unidos
4 Brooks, Matthew D. - USDA Agricultural Research Service - Estados Unidos
5 Coruzzi, Gloria M. - New York University - Estados Unidos

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Financiamiento



Fuente
National Science Foundation
NSF-PGRP
Agencia Nacional de Investigación y Desarrollo
Zegar Family FoundationA16-0051

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Agradecimientos



Agradecimiento
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.

Muestra la fuente de financiamiento declarada en la publicación.