ABSTRACT: The cellular origins and transcriptional regulation of allergens from house dust mites (HDMs), a predominant source of indoor aeroallergens, remain poorly understood. To address this, we constructed the first single-nucleus multi-omic atlas of Dermatophagoides farinae to delineate its cellular composition, map allergen expression, and identify transcriptional regulators. We performed single-nucleus RNA-seq (snRNA-seq) and ATAC-seq (snATAC-seq) on whole mites, using clustering, functional enrichment, and integrative analyses to define cell types and chromatin landscapes. Focusing on the allergen Der f 9, candidate regulatory transcription factors (TFs) were validated by qPCR and dual-luciferase assays. snRNA-seq of 16,335 high-quality nuclei resolved six major cell types: Stromal_cell, Proliferative, Neuron1, Neuron2, Metabolism1, and Metabolism_lipid. Stromal_cells were identified as the dominant allergen source, with 28 of 40 allergen groups enriched, including major (Der f 1, Der f 2) and mid-tier (Der f 5, Der f 7, Der f 21) allergens. Integrated snATAC-seq confirmed distinct epigenomic states for each cell type and revealed a highly accessible promoter region for Der f 9. Motif analysis nominated Br-family TFs as putative regulators. Among six candidate br homologs identified, qPCR confirmed their expression in D. farinae. Dual-luciferase assays demonstrated that Def04G00825 and Def10G00316 significantly transactivated the Der f 9 promoter. In conclusion, this first single-nucleus atlas of D. farinae defines cellular heterogeneity, identifies Stromal_cells as the primary allergen reservoir, and validates TFs regulating Der f 9, thereby providing a foundational resource for understanding allergen biology and advancing targeted diagnostic and therapeutic strategies.
