A PP2A-Integrator complex fine-tunes transcription by opposing CDK9

Abstract

Gene expression is tightly controlled by Cyclin-dependent kinases (CDKs) which regulate the RNA Polymerase II (RNAPII) transcription cycle at discrete checkpoints. RNAPII pausing is a CDK9-controlled rate-limiting process that occurs shortly after initiation and is required for spatio-temporal control of transcription in multicellular organisms. We discovered that CDK9-mediated RNAPII pause-release is functionally opposed by a protein phosphatase 2A (PP2A) complex. PP2A dynamically competes for key CDK9 substrates, DSIF and RNAPII, and is recruited to transcription pausing sites by INTS6, a subunit of the Integrator complex. INTS6 depletion disrupts the Integrator-PP2A association and confers resistance to CDK9 inhibition. This results in unrestrained activity of CDK9 and dysregulation of acute transcriptional responses. Pharmacological PP2A activation amplifies RNAPII pausing mediated by CDK9 inhibitors and synergizes therapeutically in a model of MLL-rearranged leukemia. These data demonstrate that finely-tuned gene expression relies on the delicate balance of kinase and phosphatase activity throughout the transcription cycle.

SPT5 stabilizes RNA polymerase II, orchestrates transcription cycles, and maintains the enhancer landscape

“…Similar to SPT5 kinase P-TEFb that phosphorylates many substrates including Pol II, the SPT5 phosphatase INTAC also targets Pol II CTD at serine residues (Huang et al, 2020;Vervoort et al, 2021;Zheng et al, 2020). Our finding that disruption of the INTAC phosphatase module still partially rescues the impaired transcription in the absence of SPT5 by promoting pause release suggests a parallel pathway mediating INTAC function in transcription independently of SPT5 (Figure 6D).…”

“…Direct comparison of gene body reads suggested a full or partial restoration of elongating Pol II for most of the genes by additionally depleting INTS8 (Figure 5H). These data indicate that, in the absence of SPT5, INTAC likely functions through targeting Pol II or other transcription regulators such as MYC (Vervoort et al, 2021) to regulate Pol II pausing and its release.…”

“…SPT5 knockdown also resulted in a reduction in RPB1 levels (Figure S1D), ruling out the possible artifacts caused by the acute protein degradation system. SPT4, the other subunit of DSIF, and Pol II binding complexes Integrator-PP2A (INTAC) (Huang et al, 2020;Vervoort et al, 2021;Zheng et al, 2020) and the negative elongation factor (NELF) were not affected (Figure 1C). The engaged Pol II tends to be phosphorylated at CTD of RPB1, with initiating/paused and elongating Pol II enriched in phosphorylation at serine 5 (Ser5) and serine 2 (Ser2), respectively (Buratowski, 2009;Devaiah and Singer, 2012;Hsin and Manley, 2012).…”

“…INTAC dephosphorylates SPT5 but regulates pausing in part through SPT5-independent mechanisms As discussed above, the phosphorylation of both S666 and CTR1 is catalyzed by P-TEFb. As for their corresponding phosphatases, in contrast with the relatively clear understanding of CTR1 dephosphorylation by PNUTS-PP1 complex (Cortazar et al, 2019;Kecman et al, 2018;Parua et al, 2018), the potential S666 phosphatases were identified recently (Huang et al, 2020;Parua et al, 2020;Vervoort et al, 2021;Zheng et al, 2020) 5C). Although the loss of SPT5 substantially decreased the Pol II levels, the addi-tional INTS8 depletion greatly enhanced the phosphorylation of the remaining Pol II (Figure 5D).…”