Qiang Zhou

Corrigendum: The m6A methyltransferase METTL16 inhibits the proliferation of pancreatic adenocarcinoma cancer cells via the p21 signaling pathway

DNA damage shuts down genome-wide transcription to prevent transcriptional mutagenesis and to initiate repair signalling, but the mechanism to stall elongating RNA polymerase II (Pol II) is not fully understood. Central to the DNA damage response, poly(ADP-ribose) polymerase 1 (PARP1) initiates DNA repair by translocating to the lesions where it catalyses protein poly(ADP-ribosylation). Here we report that PARP1 inhibits Pol II elongation by inactivating the transcription elongation factor P-TEFb, a CDK9–cyclin T1 (CycT1) heterodimer. After sensing damage, the activated PARP1 binds to transcriptionally engaged P-TEFb and modifies CycT1 at multiple positions, including histidine residues that are rarely used as an acceptor site. This prevents CycT1 from undergoing liquid–liquid phase separation that is required for CDK9 to hyperphosphorylate Pol II and to stimulate elongation. Functionally, poly(ADP-ribosylation) of CycT1 promotes DNA repair and cell survival. Thus, the P-TEFb–PARP1 signalling plays a protective role in transcription quality control and genomic stability maintenance after DNA damage.

HEXIM1 is often down-regulated in human triple-negative breast cancer (TNBC), and these cancer cells are highly sensitive to Hsp90 inhibitors. However, the mechanistic link between the two has not been determined. Our study provides a mechanistic explanation for the increased sensitivity of TNBC to Hsp90 inhibition.

The Siah1 and Siah2 ubiquitin ligases are implicated in diverse biological processes ranging from cellular stress responses, signaling to transcriptional regulation. A key substrate of Siah1 is ELL2, which undergoes proteolysis upon polyubiquitination. ELL2 stimulates transcriptional elongation and is a subunit of the Super Elongation Complex (SEC) essential for HIV-1 transactivation. Previously, multiple transcriptional and post-translational mechanisms are reported to control Siah's expression and activity. Here we show that the activity of Siah1/2 can also be suppressed by host cell factor 1 (HCF1), and the hitherto poorly characterized HCF2, which themselves are not degraded but can bind and block the substrate-binding domain (SBD) of Siah1/2 to prevent their autoubiquitination and trans-ubiquitination of downstream targets including ELL2. This effect stabilizes ELL2 and enhances the ELL2-SEC formation for robust HIV-1 transactivation. Thus, our study not only identifies HCF1/2 as novel activators of HIV-1 transcription through inhibiting Siah1 to stabilize ELL2, but also reveals the SBD of Siah1/2 as a previously unrecognized new target for HCF1/2 to exert this inhibition.

More than 30% of genes in higher eukaryotes are regulated by promoter-proximal pausing of RNA polymerase II (Pol II). Phosphorylation of Pol II CTD by positive transcription elongation factor b (P-TEFb) is a necessary precursor event that enables productive transcription elongation. The exact mechanism on how the sequestered P-TEFb is released from the 7SK snRNP complex and recruited to Pol II CTD remains unknown. In this report, we utilize mouse and human models to reveal methylphosphate capping enzyme (MePCE), a core component of the 7SK snRNP complex, as the cognate substrate for Jumonji domain-containing 6 (JMJD6)’s novel proteolytic function. Our evidences consist of a crystal structure of JMJD6 bound to methyl-arginine, enzymatic assays of JMJD6 cleaving MePCE in vivo and in vitro, binding assays, and downstream effects of Jmjd6 knockout and overexpression on Pol II CTD phosphorylation. We propose that JMJD6 assists bromodomain containing 4 (BRD4) to recruit P-TEFb to Pol II CTD by disrupting the 7SK snRNP complex.

More than 30% of genes in higher eukaryotes are regulated by promoter-proximal pausing of RNA polymerase II (Pol II). Phosphorylation of Pol II-CTD by positive transcription elongation factor (P-TEFb) is a necessary precursor event that enables productive transcription elongation. The exact mechanism on how the sequestered P-TEFb is released from the 7SK snRNP complex and recruited to Pol II-CTD remains unknown. In this report, we reveal methylphosphate capping enzyme (MePCE), a core component of the 7SK snRNP complex, as the cognate substrate for Jumonji domain-containing 6 (JMJD6)’s novel proteolytic function. Our evidences consist of a crystal structure of JMJD6 bound to methyl-arginine, enzymatic assays of JMJD6 cleaving MePCE in vivo and in vitro , binding assays, and downstream effects of Jmjd6 knockout and overexpression on Pol II-CTD phosphorylation. We propose that JMJD6 assists bromodomain containing 4 (BRD4) to recruit P-TEFb to Pol II-CTD by disrupting the 7SK snRNP complex.

The general transcription factor P-TEFb, a master regulator of RNA polymerase (Pol) II elongation, phosphorylates the C-terminal domain (CTD) of Pol II and negative elongation factors to release Pol II from promoter-proximal pausing. We show here that P-TEFb surprisingly inhibits the myoblast differentiation into myotubes, and that P-TEFb and its two positive complexes are eliminated in this process. In contrast, DYRK1A, another CTD kinase known to control transcription of a subset of genes important for development and tissue homeostasis, is found to activate transcription of key myogenic genes. We show that active DYRK1A exists in a complex with the WD40-repeat protein DCAF7 that stabilizes and tethers DYRK1A to Pol II, so that DYRK1A–DCAF7 can co-migrate with and phosphorylate Pol II along the myogenic gene loci. Thus, DCAF7 modulates the kinase signaling output of DYRK1A on Pol II to stimulate myogenic transcription after active P-TEFb function is shut off.

We recently reported that the cyclin T1 histidine-rich domain creates a phase-separated environment to promote hyperphosphorylation of RNA polymerase II C-terminal domain and robust transcriptional elongation by P-TEFb. Here, we discuss this and several other recent discoveries to demonstrate that phase separation is important for controlling various aspects of transcription.

The establishment of HIV-1 latency gives rise to persistent chronic infection that requires life-long treatment. To reverse latency for viral eradiation, the HIV-1 Tat protein and its associated ELL2-containing Super Elongation Complexes (ELL2-SECs) are essential to activate HIV-1 transcription. Despite efforts to identify effective latency-reversing agents (LRA), avenues for exposing latent HIV-1 remain inadequate, prompting the need to identify novel LRA targets. Here, by conducting a CRISPR interference-based screen to reiteratively enrich loss-of-function genotypes that increase HIV-1 transcription in latently infected CD4⁺ T cells, we have discovered a key role of the proteasome in maintaining viral latency. Downregulating or inhibiting the proteasome promotes Tat-transactivation in cell line models. Furthermore, the FDA-approved proteasome inhibitors bortezomib and carfilzomib strongly synergize with existing LRAs to reactivate HIV-1 in CD4⁺ T cells from antiretroviral therapy-suppressed individuals without inducing cell activation or proliferation. Mechanistically, downregulating/inhibiting the proteasome elevates the levels of ELL2 and ELL2-SECs to enable Tat-transactivation, indicating the proteasome-ELL2 axis as a key regulator of HIV-1 latency and promising target for therapeutic intervention. To cure chronic HIV-1 infection requires reversal of HIV-1 latency from latently infected CD4⁺ T cells. A key step in HIV latency reversal is the recruitment of Super Elongation Complexes (SECs) that contain ELL2 by an HIV-encoded protein, Tat, to activate proviral transcription. To identify novel drug targets, we conducted a CRISPRi-based screen to enrich the sgRNAs that increase HIV transcription in latently infected CD4⁺ T cells. Three of the six most prominent hits in our screen are proteasome subunits. We further proved that antagonizing the proteasome promotes Tat-induced HIV-1 transcription in cell line-based latency models. Furthermore, we found that two FDA-approved proteasome inhibitors strongly synergize with existing LRAs ex vivo without inducing cell activation or proliferation. We further found that antagonizing the proteasome elevates the levels of ELL2 and ELL2-containing SECs in the cells, thus enabling Tat-transactivation. These results indicate that the proteasome-ELL2 axis is a key regulator of HIV-1 latency could potentially be targeted for therapeutic interventions.