Jak/Stat3 regulated global gene expression dynamics during late-stage reprogramming process

We performed RNAseq analysis to dissect the dynamic transcriptome change during mouse iPSC induction, with or without blocking the Jak/Stat3 activity. We described Jak/Stat3 activity-specific global gene expression patterns, biological events, and regulation of key pluripotent genes, epigenetic modulators, and non-coding RNAs during the reprogramming process. We further demonstrated that Jak/Stat3 activity is essential for proper imprint of Dlk1-Dio3 region that is necessary for complete pluripotency establishment. Functional analysis revealed that one Jak/Stat3 downstream targets identified in our study - Esrrb significantly rescues reprogramming despite the inhibition of Jak/Stat3 activity. Our data illustrated novel mechanism in Jak/Stat3 promoted pluripotency establishment, which is valuable for further improvement of naïve-state iPSC generation across different species. Overall design: Examination of the RNA samples collected from reprogrammed cells at 18 days after retroviral OKSM transduction of OG-MEFs and cultured in reprogramming medium with LIF, with either Jaki or DMSO (control) treatment starting at day 3 (named hereafter Jaki-Stage 1 (S1) or DMSO-S1, respectively), or from iPSC colonies picked at day 21 and expanded one more passage (p2) (named hereafter Jaki-S2 or DMSO-S2, respectively)

本研究通过RNA测序（RNAseq）分析，解析了小鼠诱导多能干细胞（induced pluripotent stem cell, iPSC）诱导过程中的动态转录组变化，设置了阻断Jak/Stat3信号通路活性与未阻断的两组对照。本研究阐明了重编程过程中，受Jak/Stat3活性特异性调控的全局基因表达模式、生物学事件，以及关键多能基因、表观遗传调控因子与非编码RNA的调控机制。本研究进一步证实，Jak/Stat3活性对于Dlk1-Dio3区域的正常印记修饰至关重要，而该修饰是完全建立多能性的必要条件。功能分析显示，本研究鉴定出的一个Jak/Stat3下游靶基因Esrrb，可在Jak/Stat3活性被抑制的情况下显著挽救重编程过程。本研究的数据揭示了Jak/Stat3促进多能性建立的全新机制，该机制对于优化跨物种的幼稚态iPSC生成方法具有重要参考价值。实验整体设计：本研究采集的RNA样本来自两类细胞：其一为OG-MEFs经逆转录病毒介导的OKSM转导后，在添加白血病抑制因子（LIF）的重编程培养基中培养至第18天的重编程细胞，且从第3天起分别用Jaki或二甲基亚砜（DMSO，对照组）处理，对应样本命名为Jaki-阶段1（S1）与DMSO-S1；其二为第21天挑取的iPSC克隆，再传代培养一次（p2），对应样本命名为Jaki-S2与DMSO-S2。