ZBTB16/PLZF regulates self-renewal and differentiation of juvenile spermatogonial stem cells through an extensive transcription factor-chromatin poising network

Spermatogonial stem cells (SSCs) balance self-renewal versus differentiation/spermatogenesis to ensure continuous sperm production. Here, we uncover multiple roles for the transcription factor ZBTB16/PLZF in juvenile mouse undifferentiated spermatogonia (uSPG). ZBTB16 activates genes in uSPG promoting self-renewal and cell cycle progression to maintain uSPG and transit-amplifying states. Remarkably, in uSPG, ZBTB16, SALL4, SOX3 all co-localize at over 12,000 promoters regulating uSPG and meiosis. These regions feature broad H3K4me3 and H3K27ac, DNA hypomethylation, RNAPol2 and often CTCF. Hi-C analyses reveal robust 3D physical interactions among these co-bound promoters, revealing a transcription factor and higher-order active chromatin interaction network within uSPG that poises meiotic promoters for subsequent activation. Conversely, these factors do not 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 establish a novel and extensive chromatin poising network. Overall design: To investigate the function of ZBTB16 in vivo, we isolated undifferentiated spermatogonia (uSPG) from the testis and conducted RNA-seq and histone modification ChIP-seq. We also performed ChIP-seq for ZBTB16, SALL4, SOX3, TBP, and RNAPol2 (Pan, CTD Ser5P, and Ser2P) using whole testis samples. Given that ZBTB16 expression is restricted to uSPG at postnatal day 7 (P7), these experiments aimed to identify genes directly regulated in postnatal mouse spermatogonia.