RUNX proteins and CBFβ create an interconnected network of transcriptional regulation in the male germline

The foundation for lifelong spermatogenesis depends on a highly coordinated program of prepubertal germline development, during which a precise balance between spermatogonial proliferation and differentiation is established to ensure sustained spermatogenesis. Any disruptions to this balance can impair germ cell maturation and overall fertility. However, factors critical in maintaining this balance remain incompletely understood. Our previous studies revealed that core-binding factor subunit-β (CBFβ) regulates both proliferation and differentiation during the onset of spermatogenesis. Canonically, CBFβ functions as a co-factor for the Runt-related transcription factor (RUNX) family by forming heterodimeric complexes that can act either as transcriptional activators or repressors. Here, we reveal interactions between CBFβ and RUNX proteins within the male germline and highlight distinct expression patterns of RUNX1 and RUNX3, particularly differential temporal expression during discrete cell cycle phases within spermatogonia. Moreover, Cleavage Under Targets & Release Using Nuclease (CUT&RUN) analyses revealed both overlapping and distinct genomic localization of RUNX1 and RUNX3. Surprisingly, knockdown studies determined that RUNX1 and RUNX3 act in opposition as either transcriptional activators or repressors within overlapping genomic targets. By contrast, genomic regions with differential RUNX1 or RUNX3 localization suggest distinct regulation of proliferation or differentiation, respectively. Furthermore, motif analysis revealed enrichment of disparate transcription factor motifs, including canonical regulators of the germline. Collectively, our findings suggest that CBFβ, RUNX1, and RUNX3 participate in a network to precisely coordinate proliferation and differentiation during prepubertal germline development, thus ensuring continuous spermatogenesis and male fertility.