title: Chromatin analyses of Zymoseptoria tritici: Methods for chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq).
abstract: The presence or absence of specific transcription factors, chromatin remodeling machineries, chromatin modification enzymes, post-translational histone modifications and histone variants all play crucial roles in the regulation of pathogenicity genes. Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) provides an important tool to study genome-wide protein-DNA interactions to help understand gene regulation in the context of native chromatin. ChIP-seq is a convenient in vivo technique to identify, map and characterize occupancy of specific DNA fragments with proteins against which specific antibodies exist or which can be epitope-tagged in vivo. We optimized existing ChIP protocols for use in the wheat pathogen Zymoseptoria tritici and closely related sister species. Here, we provide a detailed method, underscoring which aspects of the technique are organism-specific. Library preparation for Illumina sequencing is described, as this is currently the most widely used ChIP-seq method. One approach for the analysis and visualization of representative sequence is described; improved tools for these analyses are constantly being developed. Using ChIP-seq with antibodies against H3K4me2, which is considered a mark for euchromatin or H3K9me3 and H3K27me3, which are considered marks for heterochromatin, the overall distribution of euchromatin and heterochromatin in the genome of Z. tritici can be determined. Our ChIP-seq protocol was also successfully applied to Z. tritici strains with high levels of melanization or aberrant colony morphology, and to different species of the genus (Z. ardabiliae and Z. pseudotritici), suggesting that our technique is robust. The methods described here provide a powerful framework to study new aspects of chromatin biology and gene regulation in this prominent wheat pathogen.
authors: INRA, UMR 1290 INRA-AgroParisTech BIOGER, Avenue Lucien Brétignières, F-78850 Thiverval-Grignon, France; Max Planck Institute for Evolutionary Biology, August-Thienemann-Str. 2, 24306 Plön, and Christian-Albrechts University of Kiel, Am Botanischen Garten 1-9, 24118 Kiel, Germany.
authors: Jessica L
authors: JL
authors: Soyer
authors: Max Planck Institute for Evolutionary Biology, August-Thienemann-Str. 2, 24306 Plön, and Christian-Albrechts University of Kiel, Am Botanischen Garten 1-9, 24118 Kiel, Germany.
authors: Mareike
authors: M
authors: Möller
authors: Max Planck Institute for Evolutionary Biology, August-Thienemann-Str. 2, 24306 Plön, and Christian-Albrechts University of Kiel, Am Botanischen Garten 1-9, 24118 Kiel, Germany.
authors: Klaas
authors: K
authors: Schotanus
authors: Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331, USA.
authors: Lanelle R
authors: LR
authors: Connolly
authors: Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331, USA.
authors: Jonathan M
authors: JM
authors: Galazka
authors: Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331, USA. Electronic address: freitagm@cgrb.oregonstate.edu.
authors: Michael
authors: M
authors: Freitag
authors: Max Planck Institute for Evolutionary Biology, August-Thienemann-Str. 2, 24306 Plön, and Christian-Albrechts University of Kiel, Am Botanischen Garten 1-9, 24118 Kiel, Germany. Electronic address: estukenbrock@bot.uni-kiel.de.
authors: Eva H
authors: EH
authors: Stukenbrock
mesh_terms: D001203:Ascomycota / Q000235:genetics*; D047369:Chromatin Immunoprecipitation; D004271:DNA, Fungal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D059014:High-Throughput Nucleotide Sequencing; D010935:Plant Diseases / Q000382:microbiology; D014908:Triticum / Q000382:microbiology
chemical_list: D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins
keywords: ChIP; Chromatin immunoprecipitation; Genome-wide histone modification maps; Histone modifications; Protein–DNA interactions