Dynamic Landscape of H3K4me3 and H3K27me3 Modification During Postnatal Leydig Cell Fate Determination

ABSTRACT Background Histone methylation plays a crucial role in regulating chromatin architecture and gene expression during spermatogenesis. H3K4me3 is enriched at the transcription start sites of active genes, whereas H3K27me3 is deposited on chromatin to confer transcriptional repression. However, the dynamics of H3K4me3 and H3K27me3 modifications in the differentiation of postnatal Leydig cells remain poorly characterized. Objectives The purpose of this study was to elucidate the dynamics of H3K4me3 and H3K27me3 modifications in the differentiation of postnatal Leydig cells. Methods Through an integrated analysis of single‐cell RNA‐seq (scRNA‐seq), cleavage under targets and tagmentation sequencing (CUT&Tag‐seq), bulk RNA‐seq, and immunohistochemistry across different developmental stages of mouse Leydig cells, we mapped the stage‐specific landscapes of H3K4me3 and H3K27me3. Results Our results demonstrate that during progenitor to immature Leydig cell differentiation, H3K4me3 levels exhibited a diphasic fluctuation pattern (first decreasing and then increasing), contrasting with the progressive accumulation of H3K27me3. In progenitor Leydig cells, the resolution of bivalent chromatin domains (accounting for 24.5% of promoters) helps balance cell proliferation and differentiation. Strikingly, steroidogenic genes such as Star , Cyp17a1 , and Hsd17b3 are exclusively regulated by H3K4me3 and lack H3K27me3 marking. In terminally differentiated adult Leydig cells, H3K4me3 sustains the steroidogenic capacity by activating transcription factors (such as Gata6 , Cebpb , Nr1d1 ) and maturation markers (including Hsd17b3 , Insl3 , Sult1e1 ), while H3K27me3 permanently silences proliferation‐related networks (such as Ccna2 , Mki67 , Ccnd1 ) to eliminate the self‐renewal potential of these cells. Discussion and Conclusion Our work identifies an H3K4me3/H3K27me3‐transcription factors (TFs)‐target gene axis as a central regulatory mechanism governing postnatal Leydig cell differentiation. This discovery clarifies how this axis endows Leydig cells with steroidogenic potential while suppressing stemness properties.