ZBTB16/PLZF regulates juvenile spermatogonial stem cell development via an extensive transcription factor poising network

Spermatogonial stem cells balance self-renewal and differentiation/spermatogenesis to ensure continuous sperm production. Here, we identify roles for the transcription factor ZBTB16/PLZF in juvenile mouse undifferentiated spermatogonia (uSPG) in promoting self-renewal and cell cycle progression to maintain uSPG and transit-amplifying states. Notably, ZBTB16, SALL4, SOX3 co-localize at over 12,000 promoters regulating uSPG and meiosis. These regions largely share broad H3K4me3 and H3K27ac, DNA hypomethylation, RNAPol2 and often CTCF. Hi-C analyses show robust 3D physical interactions among these co-bound promoters, suggesting the existence of a transcription factor and higher-order active chromatin interaction network within uSPG that poises meiotic promoters for subsequent activation. Conversely, these factors do not notably occupy germline-specific promoters driving spermiogenesis, which instead lack promoter-promoter physical interactions and bear DNA hypermethylation, even when active. Overall, ZBTB16 promotes uSPG cell cycle progression and colocalizes with SALL4, SOX3, CTCF and RNAPol2 to help establish an extensive and interactive chromatin poising network.