Cell type-specific role of CBX2-cPRC1 at the onset of spermatogonial differentiation [scRNA-Seq]

Polycomb group (PcG) proteins maintain the repressed state of lineage-inappropriate genes and are therefore essential for embryonic development and adult tissue homeostasis. One critical function of PcG complexes is modulating chromatin structure. Canonical Polycomb repressive complex 1 (cPRC1), particularly its component CBX2, can compact chromatin and phase separate in vitro. These activities are hypothesized to be critical for forming a repressed physical environment in cells. While much has been learned by studying these PcG activities in cell culture models, it is largely unexplored how cPRC1 regulates adult stem cells and their subsequent differentiation during tissue homeostasis in living animals. Here we show that CBX2 is upregulated as spermatogonial stem cells differentiate and is required in the differentiating spermatogonia of the male germ line. CBX2 forms condensates, similar to previously described Polycomb-bodies, that co-localize with target genes that are repressed in differentiating spermatogonia. Single cell analyses of mosaic Cbx2 mutant testes show CBX2 is specifically required to produce differentiating, A1 spermatogonia. Furthermore, the domain of CBX2 responsible for compaction and phase separation is needed for the long-term maintenance of male germ cells in the animal. These results emphasize that the regulation of chromatin structure by CBX2 at a specific stage of spermatogenesis is critical for the continued production of sperm and, ultimately, the transmission of genetic material to the next generation. Overall design: Goal#1) Obtain single cell gene expression profile of adult testes, Goal#2) Induce Cbx2 deletion by Tamoxifen injections, and profile changes in cell types and gene expression by single cell RNA-seq. In addition, make cell barcoded Cbx2 amplicons to determine cellular Cbx2 genotypes from Cbx2 transcript information.

多梳蛋白家族（Polycomb group, PcG）可维持谱系不相容基因的沉默状态，因此对胚胎发育与成年组织稳态至关重要。多梳蛋白复合物的核心功能之一是调控染色质结构。经典多梳抑制复合物1（cPRC1），尤其是其组分CBX2，可在体外实现染色质压缩与相分离。学界推测这些活性对于在细胞内构建沉默的物理微环境至关重要。尽管学界已通过细胞培养模型对这类多梳蛋白家族的活性有了诸多认知，但在活体动物中，cPRC1如何调控成年干细胞及其在组织稳态过程中的后续分化，目前仍未得到充分探索。本研究发现，CBX2会随着精原干细胞（spermatogonial stem cells）的分化而上调，并在雄性生殖系的分化型精原细胞中发挥必需作用。CBX2会形成类似此前报道的多梳小体（Polycomb-bodies）的凝聚体，该凝聚体可与分化型精原细胞中被沉默的靶基因共定位。对嵌合型Cbx2突变睾丸的单细胞分析表明，CBX2是生成分化型A1精原细胞的特异性必需因子。此外，CBX2中负责染色质压缩与相分离的结构域，是活体动物体内雄性生殖细胞长期维持所必需的。本研究结果表明，CBX2在精子发生（spermatogenesis）特定阶段对染色质结构的调控，对于持续产生精子以及最终将遗传物质传递给下一代至关重要。整体实验设计：目标1）获取成年睾丸的单细胞基因表达谱；目标2）通过他莫昔芬（Tamoxifen）注射诱导Cbx2基因敲除，并借助单细胞RNA测序（single cell RNA-seq）分析细胞类型与基因表达的变化。此外，构建带有细胞条形码的Cbx2扩增子，以从Cbx2转录本信息中确定细胞的Cbx2基因型。