Precise Identification of DNA-Binding Proteins Genomic Location by Exonuclease Coupled Chromatin Immunoprecipitation (ChIP-exo)
DNA-binding proteins play a crucial role in all living organisms by interacting with various DNA sequences across the genome. While several methods have been used to study the interaction between DNA and proteins in vitro, chromatin immunoprecipitation followed by sequencing (ChIP-seq) has become the standard technique for identifying the genome-wide location of DNA-binding proteins in vivo. However, the resolution of standard ChIP-seq methodology is limited by the DNA fragmentation process and presence of contaminating DNA. A significant improvement of the ChIP-seq technique results from the addition of an exonuclease treatment during the immunoprecipitation step (ChIP-exo) that lowers background noise and more importantly increases the identification of binding sites to a level near to single-base resolution by effectively footprinting DNA-bound proteins. By doing so, ChIP-exo offers new opportunities for a better characterization of the complex and fascinating architecture that resides in DNA-proteins interactions and provides new insights for the comprehension of important molecular mechanisms.
Key wordsChromatin immunoprecipitation ChIP-exo Protein-DNA interaction DNA-binding protein Next-generation sequencing DNA footprinting Formaldehyde cross-linking Genomics Exonuclease degradation Gene regulation
We are grateful to Nicolas Carraro and Vincent Burrus for the gift of the E. coli pVCR94ΔX strain. We thank Pierre-Étienne Jacques and Stéphanie Bianco for technical assistance, the Centre de calcul scientifique of Université de Sherbrooke for computational resources and technical support, as well as Vincent Baby for his precious comments on the manuscript. This work was supported by the Fonds québécois de la recherche sur la nature et les technologies through an MSc scholarship awarded to D.M. and a Projet de recherche en équipe grant awarded to S.R., and Vincent Burrus. S.R. holds a Chercheur boursier Junior 1 award from the Fonds de recherche Québec-Santé.
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