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

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. Two biological replicates were generated to compare at single-nucleotide resolution, using PRO-seq, the gene localization of transcribing RNAPII in control (CTL) C2C12 myoblasts (MBs) (i.e. WT C2C12 MBs stably expressing a control GFP-mRNA-targeting shRNA) transiently expressing FLAG alone, and in 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)