Early response to loss of Argonaute proteins in embryonic stem cells activates the Tgf-ß/Smad Transcriptional Network [ChIP-Seq]

Argonaute (Ago) proteins, which act in post-transcriptional gene regulation directed by small RNAs, are vital for normal stem cell biology. Here we report the genomic characterization of stable Ago-deficient mouse embryonic stem cells (mESC) and determine the direct and system level response to loss of Ago-mediated regulation. We find mESCs lacking all four Ago proteins are viable, do not repress microRNA (miRNA)-targeted cellular RNAs, and show distinctive gene network signatures. Profiling of RNA expression and epigenetic activity in an Ago mutant genetic series indicates that early responses to Ago loss are driven by transcriptional regulatory networks, in particular the Tgf-ß/Smad transcriptional network. This finding is confirmed using a time course analysis of Ago depletion and Ago rescue experiments. Detailed analysis places Tgf-ß/Smad activation upstream of cell cycle regulator activation, such as Cdkn1a, and repression of the c-Myc transcriptional network. The Tgf-ß/Smad pathway is directly controlled by multiple low-affinity miRNA interactions with Tgf-ß/Activin receptor mRNAs and receptor-mediated activation is required for Tgf-ß/Smad target induction with Ago loss. Our characterization reveals the interplay of post-transcriptional regulatory pathways with transcriptional networks in maintaining cell state and likely coordinating cell state transitions. ChIP seq from stable genetic Ago mutant mouse embryonic stem cells.

Argonaute（Ago）蛋白通过小RNA介导的转录后基因调控发挥功能，对正常干细胞生物学过程至关重要。本研究对稳定Ago缺陷型小鼠胚胎干细胞（mouse embryonic stem cells，mESC）开展基因组特征分析，并解析Ago介导的调控功能丧失后的直接响应与系统级响应。研究发现，缺失全部四种Ago蛋白的mESC仍可存活，无法抑制受微小RNA（microRNA，miRNA）靶向的细胞RNA，并呈现独特的基因网络特征。对Ago突变体遗传系列的RNA表达与表观遗传活性进行组学谱分析，结果表明Ago缺失后的早期响应由转录调控网络驱动，尤以转化生长因子β（TGF-β）/Smad转录网络为核心。通过Ago敲除与Ago回复实验的时间进程分析，验证了这一发现。详细分析显示，TGF-β/Smad的激活位于细胞周期调控因子（如Cdkn1a）激活以及c-Myc转录网络抑制的上游。TGF-β/Smad通路直接受多个低亲和力miRNA与TGF-β/激活素受体mRNA的互作调控，且在Ago缺失时，受体介导的激活是TGF-β/Smad靶基因诱导所必需的。本研究的特征分析揭示了转录后调控通路与转录网络在维持细胞状态、协调细胞状态转换过程中的相互作用。数据集包含稳定遗传Ago突变型小鼠胚胎干细胞的染色质免疫沉淀测序（ChIP-seq）数据。