The Integrator complex terminates promoter-proximal transcription at protein-coding genes

The transition of RNA polymerase II (Pol II) from initiation to productive elongation is a central, regulated step in metazoan gene expression. At many genes, Pol II pauses stably in early elongation, remaining engaged with the 25-60 nucleotide-long nascent RNA for many minutes while awaiting signals for release into the gene body. However, a number of genes display highly unstable promoter Pol II, suggesting that paused polymerase terminates transcription at these promoters and releases a short, non-functional RNA. Here, we investigate the mechanisms driving Pol II instability and discover that the Integrator complex targets paused Pol II and terminates transcription at selected genes. Specifically, Integrator utilizes its RNA endonuclease activity to cleave nascent RNA and destabilize elongating polymerase. Our findings uncover a previously unappreciated mechanism of metazoan gene repression, wherein promoter paused Pol II is prevented from entering productive elongation through regulated termination. Overall design: RNAseq experiments: 3 biological replicates were generated for Control (B-galactosidade treated) or IntS9-depletion (treated with double stranded RNA for IntS9) in DL1 cells. RNAi treatments were carried for 60h. 3 biological replicates were generated for Control (B-galactosidade treated) in Flag-tagged eGFP control line transgenes or IntS11-depletion (treated with double stranded RNA for IntS11) in Flag-tagged IntS11WT, E203Q mutant, and the eGFP control DL1 cell line transgenes. RNAi treatments were carried for 60h along with a final concentration of 100 µM CuSO4 to induce transgene expression. PRO-seq: 3 biological replicates were generated using DL1 cells treated with Bgal or IntS9 dsRNA for 60h. ChIP-seq: 3 biological replicates using untreated DL1 cells were generated for Input, IntS1 and IntS12 ChIP-seq datasets. ATAC-seq: 3 biological replicates using untreated S2 cells were generated.