Regulation of both transcription and RNA turnover contribute to germline specification

The nuanced mechanisms driving primordial germ cells (PGC) specification remain incompletely understood since genome-wide transcriptional regulation in developing PGCs has previously only been defined indirectly. Here, using SLAMseq analysis, we determined genome-wide transcription rates during the differentiation of embryonic stem cells (ESCs) to form epiblast-like (EpiLC) cells and ultimately PGC-like cells (PGCLCs). This revealed thousands of genes undergoing bursts of transcriptional induction and rapid shut-off not detectable by RNAseq analysis. Our SLAMseq datasets also allowed us to infer RNA turnover rates, which revealed thousands of mRNAs stabilized and destabilized during PGCLC specification. mRNAs tend to be unstable in ESCs and then are progressively stabilized as they differentiate. For some classes of genes, mRNA turnover regulation collaborates with transcriptional regulation, but these processes opposed each other in a surprisingly high frequency of genes. To test whether regulated mRNA turnover has a physiological role in PGC development, we examined 3 genes that we found were regulated by RNA turnover: Sox2, Klf2, and Ccne1. Circumvention of their regulated RNA turnover severely impaired the ESC-to-EpiLC and EpiLC-to-PGCLC transitions. Our study demonstrates the functional importance of regulated RNA stability in germline development and provides a roadmap of transcriptional and post-transcriptional regulation during germline specification. Overall design: Mouse ES cells were induced to differentiate into EpiLCs and PGCLCs, as previously described (Hayashi et al., 2011). ESC, EpiLC, and purified PGCLC were collected for SLAMseq analysis. Libraries were prepared using the Quant-seq mRNA 3' end library preparation kit (Lexogen) according to the manufacturer's instructions. Sequencing was performed using a Illumina HiSeq 4000 in the SR100 mode.