PGC 1α Senses the CBC of Pre-mRNA to Dictate the Fate of Promoter-Proximally Paused RNAPII [RIP-seq footprint]. PGC 1α Senses the CBC of Pre-mRNA to Dictate the Fate of Promoter-Proximally Paused RNAPII [RIP-seq footprint]

Although PGC 1α is well-established as a transcriptional coactivator for the metabolic adaptation of mammalian cells to diverse physiological stresses, its molecular dynamics at the start and during gene transcription are incompletely understood. Previously, we defined a CBP80-binding motif (CBM) within PGC 1α that binds CBP80 at the 5′-cap of nascent transcripts deriving from PGC 1α-responsive genes. Binding verifies proper 5′-cap assembly, after which gene transcription is somehow promoted. Here, using protein immunoprecipitations, size-exclusion chromatography, PRO-seq, RNA-seq, native RIP-seq, RIP-seq footprinting and CUT&RUN, we report that PGC 1α, serving as a DNA−RNA conduit between promoter-bound ERRα and nascent transcript-bound CBP80, overcomes promoter-proximal pausing of RNAPII by competing against the premature transcription termination complex Integrator and also by recruiting P-TEFb. Using mice homozygous for five amino-acid changes in PGC 1α that destroy CBM function, we demonstrate that efficient differentiation of primary myoblasts to myofibers and effective regeneration of skeletal muscle after injury requires PGC 1α binding to CBP80. Overall design: Three biological replicates were generated to compare RNA footprints, using RIP-seq footprinting, in anti-FLAG IPs using lysates of PGC-1α KD C2C12 MBs (i.e. WT C2C12 MBs stably expressing a PGC-1α-mRNA targeting shRNA) transiently expressing FLAG alone, FLAG-PGC-1α(WT), or FLAG-PGC-1α(CBM5mut)

尽管PGC-1α作为哺乳动物细胞应对多种生理应激的代谢适应过程中的转录共激活因子已得到广泛证实，但其在基因转录起始及进行过程中的分子动态仍未被完全阐明。此前，我们在PGC-1α中鉴定出一个CBP80结合基序（CBP80-binding motif, CBM），该基序可结合源自PGC-1α应答基因的新生转录本5'帽结构处的CBP80。该结合事件可验证正确的5'帽组装过程，随后会以某种方式促进基因转录。
本研究中，我们借助蛋白质免疫沉淀、尺寸排阻色谱、PRO-seq、RNA-seq、天然RIP-seq、RIP-seq足迹实验以及CUT&RUN技术，证实PGC-1α可作为启动子结合型雌激素相关受体α（ERRα）与新生转录本结合型CBP80之间的DNA-RNA桥梁，通过与过早转录终止复合物Integrator竞争以及招募正转录延伸因子b（P-TEFb），克服RNA聚合酶II（RNAPII）的启动子近端暂停现象。
我们利用携带PGC-1α中破坏CBM功能的5个氨基酸突变的纯合子小鼠，证实原代肌母细胞向肌纤维的高效分化以及损伤后骨骼肌的有效再生，均依赖于PGC-1α与CBP80的结合。
总体实验设计：设置3次生物学重复，通过RIP-seq足迹技术对比不同组的RNA足迹：以PGC-1α敲低（KD）的C2C12肌母细胞（MBs，即稳定表达靶向PGC-1α mRNA的shRNA的野生型C2C12肌母细胞）的裂解液进行抗FLAG免疫沉淀，这些细胞分别瞬时表达单独的FLAG标签、FLAG-PGC-1α（野生型，WT）或FLAG-PGC-1α（CBM5突变体，CBM5mut）