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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Tao Y et al (2003) Quantitative nature of Arabidopsis responses during compatible and incompatible interactions with the bacterial pathogen Pseudomonas syringae. Plant Cell 15(2):317–330
Rivas S (2011) Nuclear dynamics during plant innate immunity. Plant Physiol 158(1):87–94
Gross DS, Garrard WT (1988) Nuclease hypersensitive sites in chromatin. Annu Rev Biochem 57:159–197
Galas DJ (2001) The invention of footprinting. Trends Biochem Sci 26(11):690–693
Metzker ML (2010) Sequencing technologies—the next generation. Nat Rev Genet 11(1):31–46
Tsompana M, Buck MJ (2014) Chromatin accessibility: a window into the genome. Epigenetics Chromatin 7(1):33
Jiang J (2015) The ‘dark matter’ in the plant genomes: non-coding and unannotated DNA sequences associated with open chromatin. Curr Opin Plant Biol 24:17–23
Encode Project Consortium (2012) An integrated encyclopedia of DNA elements in the human genome. Nature 489(7414):57–74
Hesselberth JR et al (2009) Global mapping of protein-DNA interactions in vivo by digital genomic footprinting. Nat Methods 6(4):283–289
Boyle AP et al (2008) High-resolution mapping and characterization of open chromatin across the genome. Cell 132(2):311–322
Zhang W et al (2012) High-resolution mapping of open chromatin in the rice genome. Genome Res 22(1):151–162
Zhang W et al (2012) Genome-wide identification of regulatory DNA elements and protein-binding footprints using signatures of open chromatin in Arabidopsis. Plant Cell 24(7):2719–2731
Sullivan AM et al (2014) Mapping and dynamics of regulatory DNA and transcription factor networks in A. thaliana. Cell Rep 8(6):2015–2030
Pajoro A et al (2014) Dynamics of chromatin accessibility and gene regulation by MADS-domain transcription factors in flower development. Genome Biol 15(3):R41
Kang HG et al (2008) CRT1, an Arabidopsis ATPase that interacts with diverse resistance proteins and modulates disease resistance to Turnip Crinkle Virus. Cell Host Microbe 3(1):48–57
Zhang W, Jiang J (2015) Genome-wide mapping of DNase I hypersensitive sites in plants. Methods Mol Biol 1284:71–89
Langmead B et al (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10(3):R25
Boyle AP et al (2008) F-Seq: a feature density estimator for high-throughput sequence tags. Bioinformatics 24(21):2537–2538
Anders S, Huber W (2010) Differential expression analysis for sequence count data. Genome Biol 11(10):R106
Clarke JD (2009) Cetyltrimethyl ammonium bromide (CTAB) DNA miniprep for plant DNA isolation. Cold Spring Harb Protoc 2009(3):pdb.prot5177
Hiroshi Nikaido MV (1985) Molecular basis of bacterial outer membrane permeability. Microbiol Rev 49(1):1–32
Ellison RT, Giehl TJ (1991) Killing of Gram-negative bacteria by lactofernn and lysozyme. J Clin Invest 88(4):1080–1091
Acknowledgments
We thank D’Maris Dempsey and Angela H. Kang for critical comments on the manuscript. This work is supported by Texas State University-Faculty Startup Program, Multi-disciplinary Internal Research Grant and National Science Foundation Grant (IOS-1553613) to H.G.K.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Bordiya, Y., Kang, HG. (2017). Genome-Wide Analysis of Chromatin Accessibility in Arabidopsis Infected with Pseudomonas syringae . In: Shan, L., He, P. (eds) Plant Pattern Recognition Receptors. Methods in Molecular Biology, vol 1578. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6859-6_22
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6859-6_22
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6858-9
Online ISBN: 978-1-4939-6859-6
eBook Packages: Springer Protocols