Integrator Recruits Protein Phosphatase 2A to Prevent Pause Release and Enable Transcription Termination

Efficient release of promoter-proximally paused Pol II into productive elongation is essential for gene expression. Recently, we reported that the Integrator complex can bind paused Pol II and drive premature transcription termination, potently attenuating the activity of target genes. Premature termination requires RNA cleavage by the endonuclease subunit of Integrator, but the roles of other Integrator subunits in gene regulation have yet to be elucidated. Here, we report that Integrator subunit 8 (IntS8) is critical for transcription repression through its association with Protein Phosphatase 2A (PP2A). We find that Integrator-bound PP2A dephosphorylates the Pol II C-terminal domain and Spt5, and prevents the transition to productive elongation. Blocking PP2A association with Integrator thus stimulates pause release and gene activation. These results reveal a second catalytic function associated with Integrator-mediated transcription termination, and suggest a new model for the control of productive elongation involving active competition between transcriptional kinases and phosphatases. PRO-seq: Examination of 8 samples (2 biological replicate sets of Drosophila DL1 cells: control, IntS8-depleted, IntS8-depleted + wildtype IntS8 rescue, IntS8-depleted + IntS8 WFEF/A mutant rescue); RNA-seq: Examination of 6 samples in human cell lines (3 biological replicate sets of 293T cells: control knockdown; IntS8 knockdown) and 6 samples in Drosophila DL1 cells treated with copper (3 biological replicate sets of control, IntS8-depleted, IntS8-depleted + wildtype IntS8 rescue, IntS8-depleted + IntS8 WFEF/A mutant rescue) plus 2 samples not treated with copper (3 biological replicate sets of control and IntS8-depleted). The copper treatment was used to induce transgene expression in the DL1 rescue RNA-seq.