Pcf11/Spt5 condensates stall RNA polymerase II to facilitate termination and piRNA-guided heterochromatin formation [RNA-Seq]

The Piwi-interacting RNA (piRNA) pathway plays a crucial role in protecting animal germ cells by repressing transposons. However, the mechanism behind this RNA-mediated epigenetic regulation that leads to heterochromatin formation is not yet fully understood. Through RNA interference screens, we discovered Pcf11 and PNUTS, two conserved termination factors of RNA polymerase II (Pol II), as essential for piRNA-guided silencing. When Pcf11 is artificially tethered to a reporter, it leads to co-transcriptional repression and Pol II stalling, both of which are dependent on an alpha-helical region of Pcf11 capable of forming condensates when triggered by the C-terminal repeat domain (CTD) of Pol II. Interestingly, an intrinsically disordered region (IDR) derived from the Plant FCA can substitute for the alpha-helical region of Pcf11 in its silencing capacity and support animal development, demonstrating a causal relationship between phase separation and Pcf11's function. Further exploration into how Pcf11 causes Pol II stalling revealed a direct interaction between Pcf11's CTD-interaction domain (CID) and a CTR region of Spt5. The dephosphorylation of Spt5-CTR by PP1/PNUTS is essential for Pcf11's recruitment during Pol II termination. It appears that the phosphorylated state of CTR inhibits Pcf11 condensate formation, while the phosphorylation of Pol II CTD at Threonine 4 enhances Pcf11's ability to undergo phase separation. In conclusion, we propose a model in which Pcf11 regulates transcriptional termination by slowing down Pol II elongation, a process that is aided by phase separation with the unphosphorylated Spt5. The ability of Pcf11 to stall Pol II, utilized by the piRNA pathway to initiate heterochromatin formation, might represent a universal strategy for nascent RNA-mediated epigenetic control. Overall design: To validate Pcf11's essential role in Panx-mediated silencing, we generated an shRNA line targeting a distinct region of the Pcf11 mRNA. We then performed gene expression profiling analysis using data obtained from RNA-seq of Pcf11 and control knockdown. Compare gene expression profiling analysis of RNA-seq data for control knockdown and Pcf11 knockdown. To explore the functional significance of the a-helical domain in Pcf11, we utilized CRISPR/Cas9 to generate a loss-of-function mutant. We create transgenic flies expression either the wild-type Pcf11, a version lacking the a-helical domain (Pcf11?-helical), a version with the a-helical domain replaced by the IDR from Arabidopsis FCA (Pcf11FCA) and a version with a-helical domain replaced with the FUS-IDR (Pcf11FUS) We then performed gene expression profiling analysis using data obtained from RNA-seq of 3 developmental stages (L1, L2 and Adult). Compare gene expression profiling analysis of RNA-seq data for wildtype rescue and its mutant (Pcf11?-helical, Pcf11FUS, Pcf11FCA).