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

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 (Ccnd1) 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 also feature broad H3K4me3 and H3K27ac marks, DNA hypomethylation, and often CTCF binding. Hi-C analyses reveal robust promoter-promoter physical interactions, 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. Therefore, ZBTB16 ensures uSPG cell cycle progression and colocalizes with SALL4, SOX3 and often CTCF to establish a novel chromatin poising network. Spermatogonial stem cells (SSCs) balance self-renewal and differentiation to ensure continuous sperm production in the testis. The transcription factor Zbtb16 (PLZF) supports undifferentiated SSC maintenance through partly unknown mechanisms. We combined genomics (RNA-seq and ChIP-seq) and genetic approaches to reveal multiple functions of Zbtb16 in juvenile mouse SSCs. Zbtb16-bound loci show a striking correlation with active promoters bearing H3K4me3 and the activator Sall4. Zbtb16 activates genes that support SSC self-renewal and cell cycle progression (e.g., Ccnd1) that help maintain undifferentiated SSC pools, including both self-renewing SSCs and transit-amplifying progenitors. Zbtb16 also attenuates certain genes, including meiotic genes and specific retrotransposons that confer genome instability. Notably, Zbtb16 genome localization and its impact on the transcriptome are dynamic, displaying mesenchymal gene targets in vivo, which are not maintained in cultured SSCs. Our data reveal dynamic roles for Zbtb16 in ensuring SSC identity, amplification, and maintenance in vivo. To better understand Zbtb16 function in vivo, we isolated SSCs and then performed RNA-seq and histone modification ChIP-seq. We also performed ChIP-seq for the transcription factors Zbtb16 and Sall4 using whole testis to reveal directly regulated genes in postnatal mouse spermatogonia.精原干细胞（Spermatogonial stem cells, SSCs）通过平衡自我更新与分化/精子发生过程，以确保精子的持续生成。本研究揭示了转录因子ZBTB16/PLZF在幼年小鼠未分化精原细胞（undifferentiated spermatogonia, uSPG）中的多重调控功能。ZBTB16可激活未分化精原细胞中促进自我更新与细胞周期进程的基因（如Ccnd1），以维持未分化精原细胞及转运扩增状态。值得注意的是，在未分化精原细胞中，ZBTB16、SALL4与SOX3共定位结合于超过12000个调控未分化精原细胞及减数分裂过程的启动子区域。这些区域同时具有宽泛的H3K4me3和H3K27ac修饰标记、DNA低甲基化特征，且常结合CTCF。Hi-C分析显示存在显著的启动子-启动子物理互作，揭示了未分化精原细胞内存在一个转录因子与高阶活性染色质互作网络，该网络可使减数分裂启动子处于预激活状态。反之，这些因子并未结合驱动精子形成的生殖细胞特异性启动子，此类启动子既不存在启动子-启动子物理互作，同时伴随DNA高甲基化。因此，ZBTB16可确保未分化精原细胞的细胞周期进程，并与SALL4、SOX3及常结合的CTCF共同构建了一种新型的染色质预激活网络。 精原干细胞（SSCs）通过平衡自我更新与分化过程，以确保睾丸内精子的持续生成。转录因子Zbtb16（PLZF）通过尚不明确的部分机制维持未分化精原干细胞的稳态。我们结合基因组学技术（RNA测序（RNA-seq）与染色质免疫沉淀测序（ChIP-seq））及遗传学方法，揭示了Zbtb16在幼年小鼠精原干细胞中的多重功能。Zbtb16结合位点与携带H3K4me3修饰的活性启动子及激活因子Sall4的结合区域呈现显著相关性。Zbtb16可激活支持精原干细胞自我更新与细胞周期进程的基因（如Ccnd1），这些基因有助于维持未分化精原干细胞库，包括自我更新型精原干细胞及转运扩增祖细胞。Zbtb16同时可抑制部分基因的表达，包括减数分裂相关基因及可引发基因组不稳定性的特定逆转录转座子。值得注意的是，Zbtb16的基因组定位及其对转录组的影响具有动态性：在体内可靶向间充质基因靶点，但此类靶点在体外培养的精原干细胞中并未得到维持。我们的研究揭示了Zbtb16在体内维持精原干细胞特性、扩增及稳态中的动态调控功能。 为进一步阐明Zbtb16在体内的功能，我们分离得到精原干细胞并开展了RNA-seq及组蛋白修饰ChIP-seq实验。同时，我们利用完整睾丸组织对转录因子Zbtb16与Sall4开展了ChIP-seq分析，以揭示出生后小鼠精原细胞中的直接调控基因。