Genome-Wide Analysis of Chromatin Accessibility in Arabidopsis Infected with Pseudomonas syringae.

Abstract

Changes in chromatin accessibility are an important aspect of the molecular changes that occur in eukaryotic cells responding to stress, and they appear to play a critical role in stress-induced transcriptional activation/reprogramming and epigenetic changes. In plants, pathogen infection has been shown to induce rapid and drastic transcriptional reprogramming; growing evidence suggests that chromatin remodeling plays an essential role in this phenomenon. The recent development of genomic tools to assess chromatin accessibility presents a significant opportunity to investigate the relationship between chromatin dynamicity and gene expression. In this protocol, we have adopted a popular chromatin accessibility assay, DNase-seq, to measure chromatin accessibility in Arabidopsis infected with the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). DNase-seq provides information on chromatin accessibility through the sequencing of DNA fragments generated by DNase I digestion of open chromatin, followed by mapping these sequences on a reference genome. Of the two popular DNase-seq approaches, we based our method on the Stamatoyannopoulos protocol, which involves two DNase cleavages rather than a single cleavage, followed by size fractionation. Please note that this two-cleavage approach is widely accepted and has been used extensively by ENCODE (Encyclopedia of DNA Elements) project, a public research consortium investigating cis- and trans-elements in the transcriptional regulation in animal cells. To enhance the quality of the chromatin accessibility assay, we modified this protocol by including two centrifugation steps for nuclear enrichment and size fractionation and an extra washing step for removal of chloroplasts and Pst. The outcomes obtained by this approach are also discussed.