Monitoring rapid degradation of NANOG reveals UTP15 maintains pluripotency by regulating nascent transcripts

Master transcription factors (MTFs) are key regulators in cell fate determination. However, an approach to profile MTF's direct transcriptional targets together with their associated RNA-binding proteins (RBPs), is still lacking. Here, we applied 5-ethynyluridine RNA metabolic labelling and click chemistry to map the dynamics of the transcriptional targets and the RBPs interacting with the newly transcribed RNAs upon acute NANOG degradation in mouse embryonic stem cells (mESCs). We identified UTP15, a classic rRNA-biogenesis regulator, acts as a key activator of pluripotency-associated gene transcription independently of rRNA biogenesis. Importantly, NANOG-regulated transcription enhances UTP15 binding to transcription start sites (TSSs), associated with increased Pol II binding and more active transcription. Moreover, UTP15 promotes the assembly of Pol II biomolecular condensates, thereby potentially driving pluripotency gene transcription. Collectively, our study uncovers a NANOG-nascent transcript-UTP15 regulatory axis to activate pluripotency gene transcription, providing a distinct approach for studying MTF's function during cell fate determination. Overall design: To investigate the cooperative function NANOG/UTP15 in the regulation of pluripotency, we established NANOG-AID and UTP15-AID cell lines in which each target protein was allowed to be acute degradation.We then performed gene transcription profiling analysis using data obtained from nascent RNA-seq at different time points after target protein degradation.Comparative gene transcription profiling analysis of nascent RNA-seq data from NANOG-AID/UTP15-AID cells at different time points after target protein degradation

主转录因子（Master transcription factors, MTFs）是细胞命运决定过程中的关键调控因子。然而，当前仍缺乏可同时解析主转录因子直接转录靶标及其关联RNA结合蛋白（RNA-binding proteins, RBPs）的研究策略。本研究采用5-乙炔基尿苷RNA代谢标记结合点击化学技术，在小鼠胚胎干细胞（mouse embryonic stem cells, mESCs）中开展NANOG急性降解实验，绘制了转录靶标的动态变化图谱以及与新生RNA互作的RNA结合蛋白谱。本研究鉴定发现，经典核糖体RNA生物发生调控因子UTP15可独立于核糖体RNA生物发生过程，作为多能性相关基因转录的关键激活因子。值得注意的是，NANOG调控的转录可增强UTP15与转录起始位点（transcription start sites, TSSs）的结合，该过程伴随RNA聚合酶II（Pol II）结合水平提升及转录活性增强。此外，UTP15可促进RNA聚合酶II生物分子凝聚体的组装，进而潜在驱动多能性基因的转录。综上，本研究揭示了一条“NANOG-新生转录本-UTP15”调控轴以激活多能性基因转录，为解析主转录因子在细胞命运决定过程中的功能提供了全新的研究思路。整体实验设计：为探究NANOG与UTP15在多能性调控中的协同功能，我们分别构建了NANOG-生长素诱导降解（auxin-inducible degron, AID）细胞系与UTP15-AID细胞系，可实现靶标蛋白的急性降解。随后，我们在靶标蛋白降解后的不同时间点，通过新生RNA测序获取的数据开展基因转录组分析，并对靶标蛋白降解后不同时间点的NANOG-AID/UTP15-AID细胞的新生RNA测序数据开展对比基因转录组分析。