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Using single-nucleus multi-omics, we jointly profile gene expression and chromatin accessibility in 10,335 cells from the <jats:italic>Drosophila<\/jats:italic> testis apical tip. This enables inference of 147 cell type-specific enhancer-gene regulons using SCENIC\u2009+\u2009. We functionally validate key transcription factors, including <jats:italic>ovo<\/jats:italic> and <jats:italic>klumpfuss<\/jats:italic>, known from other stem cell systems but not previously linked to spermatogenesis. CRISPR-mediated knockout reveals their essential roles in germline stem cell regulation, and we provide evidence that they co-regulate shared targets through overlapping enhancer elements. We further uncover a critical role for canonical Wnt signaling, with Pangolin\/Tcf activating lineage-specific targets in the germline, soma, and niche. The Pan eRegulon links Wnt activity to cell adhesion, intercellular signaling and germline stem cell maintenance. Together, our study defines the enhancer-driven regulatory landscape of early spermatogenesis and reveals conserved, combinatorial mechanisms of niche-dependent stem cell control.<\/jats:p>","DOI":"10.1038\/s41467-025-62046-9","type":"journal-article","created":{"date-parts":[[2025,7,22]],"date-time":"2025-07-22T22:50:37Z","timestamp":1753224637000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Dissecting the enhancer gene regulatory network in early Drosophila spermatogenesis"],"prefix":"10.1038","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8182-7063","authenticated-orcid":false,"given":"Patrick","family":"van Nierop y Sanchez","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-2781-1404","authenticated-orcid":false,"given":"Pallavi 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