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