fCUT&Tag‐Seq: An optimized CUT&Tag‐based method for high‐resolution profiling of histone modifications and chromatin‐binding proteins in fungi

Abstract Histone modifications and chromatin‐binding proteins play crucial roles in regulating gene expression in eukaryotes, with significant implications for fungal pathogenicity and development. However, profiling these modifications or proteins across the genome in fungi remains challenging due to the technical limitations of the traditional, widely used Chromatin Immunoprecipitation‐Sequencing (ChIP‐Seq) method. Here, we present an optimized fungal Cleavage Under Targets and Tagmentation‐Sequencing (fCUT&Tag‐Seq) protocol 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, including Verticillium dahliae , Neurospora crassa, Fusarium graminearum , and Sporisorium scitamineum , showing good signal‐to‐noise ratios, reproducibility, and 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.