Stochastic pausing at latent HIV-1 promoters generates transcriptional bursting

ARTICLE https://doi.org/10.1038/s41467-021-24462-5 OPEN Stochastic pausing at latent HIV-1 promoters generates transcriptional bursting 1234567890():,; Katjana Tantale1,2,7, Encar Garcia-Oliver 1,7, Marie-Cécile Robert1,2,3, Adèle L’Hostis4, Yueyuxiao Yang4, Nikolay Tsanov1,2, Rachel Topno 2,3,4, Thierry Gostan1, Alja Kozulic-Pirher1,2, Meenakshi Basu-Shrivastava1,2, Kamalika Mukherjee 1,2, Vera Slaninova 2,3, Jean-Christophe Andrau 1, Florian Mueller 5, Eugenia Basyuk 1,2,6 ✉, Ovidiu Radulescu 4 ✉ & Edouard Bertrand 1,2,3 ✉ Promoter-proximal pausing of RNA polymerase II is a key process regulating gene expression. In latent HIV-1 cells, it prevents viral transcription and is essential for latency maintenance, while in acutely infected cells the viral factor Tat releases paused polymerase to induce viral expression. Pausing is fundamental for HIV-1, but how it contributes to bursting and stochastic viral reactivation is unclear. Here, we performed single molecule imaging of HIV-1 transcription. We developed a quantitative analysis method that manages multiple time scales from seconds to days and that rapidly fits many models of promoter dynamics. We found that RNA polymerases enter a long-lived pause at latent HIV-1 promoters (>20 minutes), thereby effectively limiting viral transcription. Surprisingly and in contrast to current models, pausing appears stochastic and not obligatory, with only a small fraction of the polymerases undergoing long-lived pausing in absence of Tat. One consequence of stochastic pausing is that HIV-1 transcription occurs in bursts in latent cells, thereby facilitating latency exit and providing a rationale for the stochasticity of viral rebounds. 1 Institut de Génétique Moléculaire de Montpellier, University of Montpellier, CNRS, Montpellier, France. 2 Equipe labélisée Ligue Nationale Contre le Cancer, University of Montpellier, CNRS, Montpellier, France. 3 Institut de Génétique Humaine, University of Montpellier, CNRS, Montpellier, France. 4 LPHI, UMR CNRS 5235, University of Montpellier, Montpellier, France. 5 Unité Imagerie et Modélisation, Institut Pasteur and CNRS UMR 3691, Paris, France. 6Present address: Microbiology Fundamental and Pathogenicity CNRS UMR 5234, University of Bordeaux, Bordeaux, France. 7These authors contributed equally: Katjana Tantale, Encar Garcia-Oliver. ✉email: ; ; NATURE COMMUNICATIONS | (2021)12:4503 | https://doi.org/10.1038/s41467-021-24462-5 | www.nature.com/naturecommunications 1 ARTICLE T NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24462-5 ranscription initiation is a complex process that comprises chromatin opening, assembly of a pre-initiation complex (PIC), polymerase recruitment and its maturation into an elongation-competent form (see ref. 1 for review). In Drosophila and mammals, this last step is highly regulated and appears to be a key point in the control of gene expression (ref. 2 for review). RNA polymerase II (RNAPII) is recruited by the PIC in a hypophosphorylated form and is then loaded on a short stretch of single-stranded DNA, which is melted by TFIIH. The initiating polymerase starts elongating about a dozen of nucleotides and must undergo a number of modifications before leaving the promoter and entering productive elongation3. First, the TFIIHassociated CDK7 kinase phosphorylates the Serine 5 of the heptad repeats of the C-terminal domain (CTD) of RNAPII, thereby disrupting the interaction with Mediator and facilitating promoter escape (refs. 4,5 for reviews). The S5 phosphorylated CTD also recruits the RNA capping enzymes that access the RNA 5′-end when it emerges from the polymerase6. The polymerase then transcribes an additional 10–80 nucleotides and typically enters a paused state. Two factors appear particularly important to trigger pausing, in relation to TFIID7: DSIF (DRB sensitivity-inducing factor), which is composed of SPT4 and SPT5, and NELF (negative elongation factor), a four subunit complex that also interacts with the cap-binding complex (CBC8). A recent structure of the pausing complex indicates that the RNA-DNA hybrid adopts a tilted conformation within the polymerase that prevents further nucleotide addition9. This structure is stabilized by NELF and DSIF, which also prevent binding of TFIIS, a factor that can trigger cleavage of the RNA at the active site to restart backtracked polymerases10. Release from the paused state requires the positive transcription elongation factor b (P-TEFb), which is composed of Cyclin T1 or T2 associated with the kinase CDK911, sometimes in association with the super-elongation complex (SEC12,13). P-TEFb is activated by CDK74,5,14 and it phosphorylates a number of components of the pausing complex to enable the formation of an elongation-competent polymerase9,15,16. Phosphorylation of NELF triggers its dissociation from the polymerase, and this frees a binding site for PAF, an elongation factor that is required for transcription through chromatin. P-TEFb also phosphorylates the RNA polymerase CTD on its Serine 2, as well as the linker between the polymerase core and the CTD, creating a binding site for the elongation factor SPT69. DSIF functions both as a repressor and activator of elongation, and it is also phosphorylated by P-TEFb (17 and ref therein). The structures of the paused and active elongation complex show that DSIF adopts different conformations in the two complexes. In particular, phosphorylated DSIF frees the nascent RNA and allows the polymerase to clamp around the DNA, promoting elongation while preventing the release of the polymerase from DNA. Overall, P-TEFb mediated phosphorylation thus disrupts the pausing complex and triggers formation of an active elongation complex comprising the polymerase associated with DSIF, SPT6, and PAF. While pausing is thought to be a key regulatory point for many cellular promoters in mammals and Drosophila, it is often revealed by a peak of RNAPII near the promoter that can in fact correspond to different molecular processes such as slow elongation, polymerase arrest, or defective processivity (i.e. abortive initiation18). Recent efforts have been made to clarify these mechanisms by measuring pausing duration. These studies indicated that pausing time varies from less than a minute up to an hour in Drosophila and mammals, depending on the promoter19–23. This revealed a surprising variability in pausing kinetics, with widely different regulatory potential. Another major finding of the last 15 years is that transcription is a discontinuous process in vivo (24 see25,26 for reviews), with “active” genes going through active and inactive periods in a 2 stochastic manner, a phenomenon also called transcriptional noise or gene bursting. In particular, recent evidences suggest that for many genes, expression levels are dynamically encoded in the time domain by controlling the periods during which a gene is active, rather than by regulating the initiation rate27–29. Major (...truncated)