P-TEFb, the Super Elongation Complex and Mediator Regulate a Subset of Non-paused Genes during Early Drosophila Embryo Development

Positive Transcription Elongation Factor b (P-TEFb) is a kinase consisting of Cdk9 and Cyclin T that releases RNA Polymerase II (Pol II) into active elongation. It can assemble into a larger Super Elongation Complex (SEC) consisting of additional elongation factors. Here, we use a miRNA-based approach to knock down the maternal contribution of P-TEFb and SEC components in early Drosophila embryos. P-TEFb or SEC depletion results in loss of cells from the embryo posterior and in cellularization defects. Interestingly, the expression of many patterning genes containing promoter-proximal paused Pol II is relatively normal in P-TEFb embryos. Instead, P-TEFb and SEC are required for expression of some non-paused, rapidly transcribed genes in pre-cellular embryos, including the cellularization gene Serendipity-α. We also demonstrate that another P-TEFb regulated gene, terminus, has an essential function in embryo development. Similar morphological and gene expression phenotypes were observed upon knock down of Mediator subunits, providing in vivo evidence that P-TEFb, the SEC and Mediator collaborate in transcription control. Surprisingly, P-TEFb depletion does not affect the ratio of Pol II at the promoter versus the 3’ end, despite affecting global Pol II Ser2 phosphorylation levels. Instead, Pol II occupancy is reduced at P-TEFb down-regulated genes. We conclude that a subset of non-paused, pre-cellular genes are among the most susceptible to reduced P-TEFb, SEC and Mediator levels in Drosophila embryos.

正性转录延伸因子b（P-TEFb）是一类由细胞周期蛋白依赖性激酶9（Cdk9）与细胞周期蛋白T（Cyclin T）构成的激酶，可将RNA聚合酶II（Pol II）激活并释放至活跃转录延伸状态。该因子可组装为包含更多延伸因子的大型超级延伸复合物（SEC）。本研究借助基于微RNA（miRNA）的技术手段，在早期果蝇胚胎中敲低P-TEFb及SEC组分的母源表达贡献。P-TEFb或SEC的耗竭会导致果蝇胚胎后部细胞缺失，并引发细胞化过程缺陷。值得注意的是，在P-TEFb敲低的胚胎中，众多携带启动子近端暂停Pol II的模式形成基因的表达水平相对正常。与之相反，在细胞化前胚胎中，部分非暂停型快速转录基因的表达依赖于P-TEFb与SEC，其中便包括细胞化相关基因Serendipity-α。本研究还证实，另一个受P-TEFb调控的末端基因（terminus）在胚胎发育中具有必需功能。在敲低中介体亚基的实验中，我们观测到了相似的形态学与基因表达表型，这为P-TEFb、SEC与中介体协同调控转录过程提供了体内实验依据。令人意外的是，尽管P-TEFb耗竭会影响全局Pol II的丝氨酸2磷酸化水平，但其并未改变启动子区域与3'端的Pol II占比。与之相对，在P-TEFb下调的基因位点，Pol II的占据量出现显著降低。综上，果蝇胚胎中的部分非暂停型细胞化前基因，是受P-TEFb、SEC与中介体水平下调影响最为敏感的基因子集。