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. 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.

原始生殖细胞（primordial germ cells, PGC）特化的精细调控机制仍未完全阐明，因为此前对发育中PGC的全基因组转录调控仅能通过间接方式界定。本研究通过SLAMseq测序分析，定量了胚胎干细胞（embryonic stem cells, ESCs）分化为上胚层样细胞（epiblast-like cells, EpiLCs）并最终形成原始生殖细胞样细胞（PGC-like cells, PGCLCs）过程中的全基因组转录速率。该分析揭示了数千个基因出现转录激活爆发与快速沉默的动态变化，这一现象是RNA测序（RNAseq）分析无法捕捉到的。本研究的SLAMseq数据集还支持我们推导RNA周转速率，由此揭示了在PGCLC特化过程中数千条mRNA的稳定性发生上调与下调。mRNA在ESCs中通常稳定性较差，随着分化进程其稳定性逐渐升高。对于部分基因类别而言，mRNA周转调控与转录调控协同发挥作用，但在占比高得出人意料的基因中，二者的调控效应却彼此拮抗。为验证受调控的mRNA周转在PGC发育中是否具有生理功能，我们选取了3个经发现受RNA周转调控的基因：Sox2、Klf2及Ccne1进行实验验证。干扰这些基因的受调控RNA周转过程会严重阻碍ESC向EpiLC以及EpiLC向PGCLC的分化过渡过程。本研究证实了受调控的RNA稳定性在生殖系发育中的功能重要性，并为生殖细胞特化过程中的转录及转录后调控提供了完整的调控蓝图。本研究按照此前报道的方法（Hayashi等，2011），将小鼠ES细胞诱导分化为EpiLCs与PGCLCs。收集ESC、EpiLC及纯化后的PGCLC样本用于SLAMseq测序分析。文库构建采用Quant-seq mRNA 3'端文库制备试剂盒（Lexogen），并严格遵循制造商提供的操作指南。测序工作在Illumina HiSeq 4000平台上以SR100单端测序模式完成。