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. Overall design: 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/早幼粒细胞白血病锌指蛋白（PLZF）在幼年小鼠未分化精原细胞（undifferentiated spermatogonia, uSPG）中的多重功能。ZBTB16可在未分化精原细胞中激活促进自我更新与细胞周期进程的基因（如细胞周期蛋白D1（Ccnd1）），以维持未分化精原细胞及过渡扩增状态。值得注意的是，在未分化精原细胞中，ZBTB16、SALL4与SOX3可在超过12000个调控未分化精原细胞及减数分裂的启动子区域共定位。这些区域同时具备宽泛的组蛋白H3赖氨酸4三甲基化（H3K4me3）与组蛋白H3赖氨酸27乙酰化（H3K27ac）修饰特征、DNA低甲基化特征，且常存在CCCTC结合因子（CTCF）结合。Hi-C染色质构象捕获分析显示存在显著的启动子-启动子物理互作，揭示未分化精原细胞内存在由转录因子构成的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。相反，上述因子并不结合驱动精子形成的生殖细胞特异性启动子，这类启动子既缺乏启动子-启动子物理互作，同时呈现DNA高甲基化特征。因此，ZBTB16可保障未分化精原细胞的细胞周期进程，并与SALL4、SOX3及CTCF共同构建了一种全新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，确保睾丸内精子的持续生成。转录因子Zbtb16（PLZF）可通过部分尚不明确的机制维持未分化精原干细胞的稳态。本研究结合基因组学技术（RNA测序（RNA-seq）与染色质免疫共沉淀测序（ChIP-seq））与遗传学方法，揭示了Zbtb16在幼年小鼠精原干细胞中的多重功能。Zbtb16结合的位点与携带H3K4me3修饰的活性启动子及激活因子Sall4存在显著相关性。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与过渡扩增祖细胞。此外，Zbtb16还可抑制部分基因，包括减数分裂相关基因及可引发基因组不稳定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，但这类靶向关系在培养的精原干细胞中并不存在。本研究数据揭示了Zbtb16在体内保障精原干细胞身份维持、扩增与稳态的动态功能。实验整体设计：为深入解析Zbtb16在体内的功能，我们分离了精原干细胞，随后开展了RNA测序及组蛋白修饰染色质免疫共沉淀测序。同时，我们利用全睾丸样本对转录因子Zbtb16与Sall4进行了染色质免疫共沉淀测序，以揭示出生后小鼠精原细胞中直接受其调控的基因。