fCUT&Tag-Seq:An optimized CUT&Tag-based method for high-resolution profiling of histone modifications and chromatin-binding proteins in fungiOA
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.
Haiting Wang;Yongjunlin Tan;Jiayue Ma;Jie Yang;Mengran Liu;Peng Jiang;Shan Lu;Haoxue Xia;Guangfei Tang;Wende Liu;Hui-Shan Guo;Chun-Min Shan
State Key Laboratory of Microbial Diversity and Innovative Utilization,Department of Agri-microbiomics and Biotechnology,Institute of Microbiology,Chinese Academy of Sciences,Beijing,ChinaShanxi Agricultural University,Jinzhong,ChinaState Key Laboratory of Microbial Diversity and Innovative Utilization,Department of Agri-microbiomics and Biotechnology,Institute of Microbiology,Chinese Academy of Sciences,Beijing,China University of Chinese Academy of Sciences,Beijing,ChinaState Key Laboratory of Microbial Diversity and Innovative Utilization,Department of Agri-microbiomics and Biotechnology,Institute of Microbiology,Chinese Academy of Sciences,Beijing,China University of Chinese Academy of Sciences,Beijing,ChinaState Key Laboratory of Microbial Diversity and Innovative Utilization,Department of Agri-microbiomics and Biotechnology,Institute of Microbiology,Chinese Academy of Sciences,Beijing,China University of Chinese Academy of Sciences,Beijing,ChinaState Key Laboratory of Microbial Diversity and Innovative Utilization,Department of Agri-microbiomics and Biotechnology,Institute of Microbiology,Chinese Academy of Sciences,Beijing,China University of Chinese Academy of Sciences,Beijing,ChinaCollege of Agriculture,Guangxi Key Laboratory of Sugarcane Biology,Guangxi University,Nanning,ChinaState Key Laboratory for Biology of Plant Diseases and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing,ChinaState Key Laboratory for Biology of Plant Diseases and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing,ChinaState Key Laboratory for Biology of Plant Diseases and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing,ChinaState Key Laboratory of Microbial Diversity and Innovative Utilization,Department of Agri-microbiomics and Biotechnology,Institute of Microbiology,Chinese Academy of Sciences,Beijing,ChinaState Key Laboratory of Microbial Diversity and Innovative Utilization,Department of Agri-microbiomics and Biotechnology,Institute of Microbiology,Chinese Academy of Sciences,Beijing,China
生物科学
chromatin-binding proteinsfCUT&Tag-Seqfungihistone modificationsplant pathogen
《mLife》 2026 (2)
P.239-253,15
supported by the China National Key Research and Development Program(Grant No.2024YFC3406000)the Chinese Academy of Sciences(CAS)Projects for Young Scientists in Basic Research(Grant No.YSBR-080)the Strategic Priority Research Program of Chinese Academy of Sciences(Grant No.XDA0450000)to C-M.S.the Young Scientists Fund of the National Natural Science Foundation of China(Grant No.32402338)to H.W.
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