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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\u2010Sequencing (ChIP\u2010Seq) method. Here, we present an optimized fungal Cleavage Under Targets and Tagmentation\u2010Sequencing (fCUT&amp;Tag\u2010Seq) 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\u2010DNA binding efficiency. We then successfully applied fCUT&amp;Tag\u2010Seq to accurately profile multiple histone modifications like H3K9me3, H3K27me3, H3K4me3, and H3K18ac, across different plant pathogenic or model fungal species, including\n                    <jats:italic>Verticillium dahliae<\/jats:italic>\n                    ,\n                    <jats:italic>Neurospora crassa, Fusarium graminearum<\/jats:italic>\n                    , and\n                    <jats:italic>Sporisorium scitamineum<\/jats:italic>\n                    , showing good signal\u2010to\u2010noise ratios, reproducibility, and detection sensitivity. Furthermore, we extended this method to profile chromatin\u2010binding proteins, such as the histone acetyltransferase Gcn5. This study establishes fCUT&amp;Tag\u2010Seq 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.\n                  <\/jats:p>","DOI":"10.1002\/mlf2.70060","type":"journal-article","created":{"date-parts":[[2026,3,24]],"date-time":"2026-03-24T04:47:03Z","timestamp":1774327623000},"page":"239-253","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["fCUT&amp;Tag\u2010Seq: An optimized CUT&amp;Tag\u2010based method for high\u2010resolution profiling of histone modifications and chromatin\u2010binding proteins\u00a0in fungi"],"prefix":"10.1002","volume":"5","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3477-0217","authenticated-orcid":false,"given":"Haiting","family":"Wang","sequence":"first","affiliation":[{"name":"State Key Laboratory of Microbial Diversity and 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