Chromatin analyses of Zymoseptoria tritici: Methods for chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq)

• Published in: Fungal genetics and biology Vol. 79; pp. 63 - 70
• Main Authors: Soyer, Jessica L., Möller, Mareike, Schotanus, Klaas, Connolly, Lanelle R., Galazka, Jonathan M., Freitag, Michael, Stukenbrock, Eva H.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.06.2015; Elsevier
• Subjects: Ascomycota - genetics; ChIP; Chromatin Immunoprecipitation; DNA, Fungal - metabolism; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Fungal Proteins - genetics; Fungal Proteins - metabolism; Gene Expression Regulation, Fungal; Genome-wide histone modification maps; High-Throughput Nucleotide Sequencing; Histone modifications; Life Sciences; Plant Diseases - microbiology; Protein–DNA interactions; Triticum - microbiology; Triticum aestivum; Chromatin immunoprecipitation; Genome-wide histone modification maps; Histone modifications; ChIP; Protein–DNA interactions; protein–DNA interactions
• DOI: 10.1016/j.fgb.2015.03.006
• ISSN: 1087-1845

•Chromatin structures and modifications important for gene regulation.•Chromatin immunoprecipitation and NGS sequencing (ChIP-seq) to study histone modifications.•Optimized ChIPseq based methods for the wheat pathogen Zymoseptoria tritici.•Genome-wide maps of chromatin modifications.•Heterochromatin and euchromatin distribution in Z. tritici. 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.