fCUT&Tag-seq: An Optimized CUT&Tag-based Method for High-Resolution Histone Modification and Chromatin-Binding Protein Profiling in both Model and Plant Pathogenic Fungi

Histone modifications and chromatin-binding proteins are important regulators of gene expression in eukaryotes and have pivotal roles in fungal pathogenicity and development. However, profiling these modifications or proteins across the genome in fungi is still challenging, due to the technical limitations of the traditional widely-used ChIP-Seq method. Here, we present an optimized CUT&Tag-Seq protocol (fCUT&Tag-Seq) specifically designed for filamentous fungi and dimorphic fungi. Our approach involves the preparation of protoplasts and nuclear extraction to enhance antibody accessibility, along with formaldehyde crosslinking to improve protein-DNA binding efficiency. We then successfully applied fCUT&Tag-Seq to accurately profile multiple histone modifications like H3K9me3, H3K27me3, H3K4me3, and H3K18ac, across different plant pathogenic or model fungal species includingVerticillium dahliae,Neurospora crassa,Fusarium graminearum, andSporisorium scitamineum. Compared to the traditional ChIP-Seq, our method showed superior signal-to-noise ratios, higher reproducibility, and enhanced detection sensitivity. Furthermore, we extended this method to profile chromatin-binding proteins, such as the histone acetyltransferase Gcn5. This study establishes fCUT&Tag-Seq as a robust and useful tool for fungal epigenetic research, enabling detailed exploration of chromatin dynamics and advancing our understanding of fungal gene regulation, development, and pathogenicity.