PRC1 Mediates the Transcriptional Repressive Function of POGZ in Neurodevelopmental Gene Regulation

Polycomb Repressive Complex 1 (PRC1) is a family of fundamental epigenetic regulators crucial for gene silencing and mammalian development. Elucidating the PRC1 composition and function in context- and stage-specific functions during differentiation and development is crucial in understanding the impact of PRC1-mediated transcriptional activity in these processes. This study focuses on the identification and characterization of POGZ, a high-risk Autism risk gene, as a novel component of the PRC1 complex in neural cells. Our biochemical analyses demonstrate that POGZ specifically interacts with the PRC1.6 complex to form the novel PRC1-POGZ complex. Functional assays indicate that POGZ elicits strong transcriptional repressor activity that is dependent on RING1B expression. ChIP-Seq studies determined that PRC1-POGZ localizes at neurodevelopmental genes in the embryonic mouse cortex. Although POGZ knockout (KO) does not compromise mESC pluripotency, POGZ ablation in neural precursor cells resulted in severe dysregulation of transcriptomic gene expression, marked by failed activation of key neural marker genes. Mechanistically, PRC1-POGZ target genes involved in multiple aspects of neuronal differentiation were differentially expressed in POGZ KO NPCs, which may explain the observed failed differentiation. The insights gained herein provide critical insights toward understanding how PRC1-POGZ function influences neural cell fate determination. Overall design: To investigate the effects POGZ KO may have on the transcriptome of WT embryonic stem cells (ESCs) and differentiated neural precursor cells (NPCs). We generated a POGZ KO ESC line via CRISPR/Cas9 technology. POGZ WT and KO ESCs were differentiated in NPCs using a previously established protocol (Hanafiah, et al. 2020). We performed RNA-Seq on POGZ WT and KO ESCs (Day 0) and NPCs (Day 7). Duplicates of each genotype and time point were included in this experiment.

多梳抑制复合体1（Polycomb Repressive Complex 1，PRC1）是一类核心表观遗传调控因子，在基因沉默与哺乳动物发育过程中发挥关键作用。阐明PRC1在细胞分化与发育进程中所具备的情境特异性与阶段特异性的组成与功能，对于理解PRC1介导的转录活性在这些过程中的调控作用至关重要。本研究聚焦于鉴定并表征POGZ——一种自闭症（Autism）高风险易感基因——作为神经细胞中PRC1复合体的新型组分。我们的生化分析实验证实，POGZ可与PRC1.6复合体特异性结合，进而形成新型的PRC1-POGZ复合体。功能实验结果显示，POGZ具备强效的转录抑制活性，且该活性依赖于RING1B的表达。染色质免疫共沉淀测序（ChIP-Seq）实验结果表明，PRC1-POGZ复合体定位于小鼠胚胎大脑皮层中的神经发育相关基因位点。尽管POGZ敲除（KO）不会影响小鼠胚胎干细胞（mouse embryonic stem cell, mESC）的多能性，但在神经前体细胞（neural precursor cell, NPC）中敲除POGZ会导致转录组基因表达出现严重失调，其特征为关键神经标志物基因的激活失败。从机制层面来看，参与神经元分化多个环节的PRC1-POGZ靶基因在POGZ KO神经前体细胞中出现了差异表达，这或可解释观测到的分化失败现象。本研究获得的认知为理解PRC1-POGZ的功能如何调控神经细胞命运决定提供了重要依据。

整体实验设计：为探究POGZ敲除对野生型胚胎干细胞（embryonic stem cell, ESC）以及分化得到的神经前体细胞转录组的影响。我们通过CRISPR/Cas9技术构建了POGZ敲除的胚胎干细胞系。将POGZ野生型与敲除型胚胎干细胞按照已发表的方案（Hanafiah 等，2020）诱导分化为神经前体细胞。我们对POGZ野生型与敲除型胚胎干细胞（第0天）以及神经前体细胞（第7天）进行了RNA测序（RNA-Seq）。本实验中每个基因型与时间点均设置了生物学重复。