YAP/TEAD complex restricts H3K27ac deposition to suppress testosterone synthesis in adult Leydig cells

The Hippo signaling pathway regulates the development of multiple organs, yet its role in the maturation of adult Leydig cells and testosterone synthesis is unclear. In this study, by integrating bulk RNA sequencing and immunohistochemistry, we characterized the spatiotemporal expression patterns of YAP and TEAD family members during postnatal Leydig cell development. Functional assays combining pharmacological inhibition, genetic knockdown, and CUT&Tag-seq analysis were employed to elucidate the underlying mechanisms, and single-cell transcriptomic data from patients with idiopathic non-obstructive azoospermia (iNOA) were analyzed to assess clinical relevance. Results of single-cell transcriptomic analysis revealed that YAP, TEAD1, and TEAD3 are significantly elevated in Leydig cells from patients with iNOA compared with normal controls. In contrast, under normal physiological conditions, YAP protein levels progressively declined during Leydig cell maturation into adulthood, accompanied by increased phosphorylation (p-YAP). Correspondingly, Tead1 and Tead2 are markedly downregulated as immature Leydig cells differentiate into adult Leydig cells. Functional assays further revealed that knockdown of either YAP or TEAD and K975 treatment significantly increased testosterone synthesis and upregulated the expression of Hsd3b6, Cyp17a1, and Hsd17b3. Mechanistically, the YAP/TEAD complex may transcriptionally repress these steroidogenic genes by restricting the deposition of the active histone mark H3K27ac at their promoter regions. Our findings demonstrate that YAP/TEAD orchestrates postnatal Leydig cell maturation by epigenetically repressing the steroidogenic program, revealing a novel role for the Hippo pathway in male reproductive development and providing insights into the pathogenesis of iNOA.