RNA Polymerase II Research Articles

Transcriptomic profiling implicates PAF1 in both active and repressive immune regulatory networks

BackgroundSitting at the interface of gene expression and host-pathogen interaction, polymerase associated factor 1 complex (PAF1C) is a rising player in the innate immune response. The complex localizes to the nucleus and associates with chromatin to modulate RNA polymerase II (RNAPII) elongation of gene transcripts. Performing this function at both proximal and distal regulatory elements, PAF1C interacts with many host factors across such sites, along with several microbial proteins during infection. Therefore, translating the ubiquity of PAF1C into specific impacts on immune gene expression remains especially relevant.ResultsAdvancing past work, we treat PAF1 knockout cells with a slate of immune stimuli to identify key trends in PAF1-dependent gene expression with broad analytical depth. From our transcriptomic data, we confirm PAF1 is an activator of traditional immune response pathways as well as other cellular pathways correlated with pathogen defense. With this model, we employ computational approaches to refine how PAF1 may contribute to both gene activation and suppression. Specifically focusing on transcriptional motifs and regulons, we predict gene regulatory elements strongly associated with PAF1, including those implicated in an immune response. Overall, our results suggest PAF1 is involved in innate immunity at several distinct axes of regulation.ConclusionsBy identifying PAF1-dependent gene expression across several pathogenic contexts, we confirm PAF1C to be a key mediator of innate immunity. Combining these transcriptomic profiles with potential regulatory networks corroborates the previously identified functions of PAF1C. With this, we foster new avenues for its study as a regulator of innate immunity, and our results will serve as a basis for targeted study of PAF1C in future validation studies.

Structural basis of RNA polymerase II transcription on the chromatosome containing linker histone H1

In chromatin, linker histone H1 binds to nucleosomes, forming chromatosomes, and changes the transcription status. However, the mechanism by which RNA polymerase II (RNAPII) transcribes the DNA in the chromatosome has remained enigmatic. Here we report the cryo-electron microscopy (cryo-EM) structures of transcribing RNAPII-chromatosome complexes (forms I and II), in which RNAPII is paused at the entry linker DNA region of the chromatosome due to H1 binding. In the form I complex, the H1 bound to the nucleosome restricts the linker DNA orientation, and the exit linker DNA is captured by the RNAPII DNA binding cleft. In the form II complex, the RNAPII progresses a few bases ahead by releasing the exit linker DNA from the RNAPII cleft, and directly clashes with the H1 bound to the nucleosome. The transcription elongation factor Spt4/5 masks the RNAPII DNA binding region, and drastically reduces the H1-mediated RNAPII pausing.

HEXIM1 Overexpression Alters GATA1 Activity and Promotes Fetal Globin Expression

Erythropoiesis is a highly organized process whereby multipotent HSCs become committed to the erythroid linage and mature into enucleate erythrocytes. This process is associated with significant changes in gene expression as non-erythroid genes are switched off while expression of erythroid genes is maintained. HEXIM1 is a key regulator of RNA Polymerase II (RNPII) activity that is essential for erythropoiesis. Following transcription initiation, RNPII transcribes 30-50 base pairs before pausing. Phosphorylation of the c-terminal domain of RNPII by the dimer pTEFb (positive transcription elongation factor beta) is necessary for RNPII to resume active transcription elongation. HEXIM1 associates with pTEFb in the 7SK complex and renders it inactive. Phosphorylation of HEXIM1 releases pTEFb from the 7SK complex, allowing it to act on RNPII. As 7SK can be targeted to specific sequences, HEXIM1/7SK mediated pTEFb delivery is a crucial mechanism to facilitate on-site pTEFb activation and RNPII pause release. Recent work from our lab demonstrated that HEXIM1 is essential in the regulation of erythroid proliferation, viability and gene expression, and also promotes the expression of GATA1 target genes (Murphy Blood 2021). New analyses of HEXIM1 overexpression (OE) in HUDEP2 cells revealed a significant increase of gamma globin production, with a higher proportion of cells expressing fetal hemoglobin. These changes were accompanied by downregulation of beta globin and multiple key repressors of fetal hemoglobin, including BCL11A, MYB, and SOX6. These phenotypes were dependent on pTEFb activity, as the pTEFb (CDK9) inhibitor NVP-2 rescued the proliferative advantage granted by HEXIM1 OE and greatly decreased both gamma globin levels and the portion of fetal hemoglobin expressing cells. Further suggesting the effects of HEXIM1 OE are pTEFb-dependent, overexpression of HEXIM1 with a tyrosine to alanine mutation (Y271A) that prevents phosphorylation of HEXIM1 and subsequent release of pTEFb cannot enhance proliferation or gamma globin production. To gain insights into the mechanisms underlying the increased fetal globin expression, we performed CUT&RUN assays for Ser5 phosphorylated and total RNPII in HEXIM1 OE and control cells. Surprisingly, OE of HEXIM1 had only modest impacts on RNPII pausing, but instead lead to changes in the genes occupied by RNPII. Notably, the gene that lost the most RNPII was HBB and the gene that gained the most RNPII was HBG. Of note, GATA1 motifs were highly enriched in regions that gained RNPII occupancy. CUT&RUN identified peaks of GATA1 in the HEXIM1 OE cells at regions of increased RNPII occupancy that were not present in the EV or Y271A controls. These novel GATA1 sites occurred in regions of previously closed chromatin, suggesting that HEXIM1 OE enhanced the pioneer ability of GATA1. Similar to RNPII, GATA1 occupancy shifted from the beta globin gene in cells transduced with EV or HEXIM Y271A to the gamma globin genes in cells transduced with WT HEXIM1. Novel RNPII and GATA1 peaks were also identified in other genes associated with a fetal erythroid phenotype, most notably ARID3A and LIN28B. Together, these results support a model where elevated HEXIM1 levels promote the pioneer activity of GATA1, and facilitate the expression of a fetal transcriptional program in adult cells in a pTEFb-dependent manner.

RF11 | PSAT369 CDK7 and CDK9 Inhibition Induces RNA Polymerase II Ubiquitination and Proteasome Degradation in Thyroid Cancer Cells

Abstract "Superhenhanced" transcription of specific oncogenes by aberrant looping of upstream enhancer elements to marked transcriptional regulatory regions is a mechanism of oncogene overexpression. Therefore, compounds that target key positive regulators of RNA polymerase II (RNAPII) activity, such as CDK7 and CDK9, can relatively selectively reduce expression of such superenhanced oncogenes and are in clinical trials. We have previously reported activity of the CDK7 inhibitor THZ1 in medullary thyroid cancer (MTC) along with identification of a superenhancer in RET. CDK7 inhibitors also are reported to be active in anaplastic thyroid cancer cell lines. In the present study, we extend this work to both CDK7 and CDK9 inhibitors, and to a more extensive set of thyroid cancer cell lines. We selected 8 thyroid cancer cell lines (TPC1, FTC133, BCPAP, SW1376; K1, THJ-16T, C643, and 8505C) from a variety of histological subtypes with different driver mutations for treatment with a CDK7 (THZ1) or CDK9 (AZD4573) inhibitors. Cell viability assays were performed to determine IC50 and Western blots were performed for target validation. IC50 values ranged from 5-100 nM for both compounds for all cell lines demonstrating broad activity and robust activity. Unexpectedly, both compounds inhibited both CDK7 RNAPII phosphorylation (Ser5) and CDK9 RNAPII phosphorylation (Ser2) due to a loss of total RNAPII protein levels in all cell lines. Further time course studies two cell lines, 8505C (BRAFV600E) and TPC1 (RET/PTC) showed the loss off RNAPII occurred rapidly (1-3 hours) with stable RNAPII mRNA levels by qRTPCR. We therefore hypothesized that RNAPII stalling induced by inhibition of CDK7 may cause loss of RNAPII protein stability through ubiquitination and proteasome-mediated degradation. Immunoprecipitation-Western blots demonstrated variable baseline levels of RNAPII ubiquitination in 8505C (low) and TPC1 (high) cells. THZ1 treatment induced ubiquitination of RNAPII in 8505C cells signficantly, while a modest increase beyond the higher baseline occurred in TPC1. Bortezomib (proteasome inhibitor) treatment rescued THZ1-reduced RNAPII protein levels in both cell lines. In summary, human thyroid cancer cell lines are broadly sensitive to CDK7 and CDK9 inhibitors. CDK 7 and 9 inhibitors reduce RNAPII levels and THZ1 induces RNAPII ubiquitination and proteasome degradation demonstrating a new mode of action for the reduced RNAPII activity induced by these inhibitors. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m., Saturday, June 11, 2022 1:42 p.m. - 1:47 p.m.

SETD1A regulates transcriptional pause release of heme biosynthesis genes in leukemia.

Histone methyltransferase SETD1A is critical for acute myeloid leukemia (AML) cell survival, but the molecular mechanism driving SETD1A gene regulation remains elusive. To delineate the role of SETD1A, we utilize a protein degrader technology to induce rapid SETD1A degradation in AML cell lines. SETD1A degradation results in immediate downregulation of transcripts associated with DNA repair and heme biosynthesis pathways. CRISPR-based functional analyses and metabolomics reveal an essential role of SETD1A to maintain mitochondrial respiration in AML cells. These SETD1A targets are enriched in head-to-head (H2H) genes. SETD1A degradation disrupts a non-enzymatic SETD1A domain-dependent cyclin K function, increases the Ser5P RNA polymerase II (RNAPII) at the transcriptional start site (TSS), and induces the promoter-proximal pausing of RNAPII in a strand-specific manner. This study reveals a non-enzymatic role for SETD1A in transcriptional pause release and provides insight into the mechanism of RNAPII pausing and its function in cancer.

Integrator endonuclease drives promoter-proximal termination at all RNA polymerase II-transcribed loci

RNA polymerase II (RNAPII) pausing in early elongation is critical for gene regulation. Paused RNAPII can be released into productive elongation by the kinase P-TEFb or targeted for premature termination by the Integrator complex. Integrator comprises endonuclease and phosphatase activities, driving termination by cleavage of nascent RNA and removal of stimulatory phosphorylation. We generated a degron system for rapid Integrator endonuclease (INTS11) depletion to probe the direct consequences of Integrator-mediated RNA cleavage. Degradation of INTS11 elicits nearly universal increases in active early elongation complexes. However, these RNAPII complexes fail to achieve optimal elongation rates and exhibit persistent Integrator phosphatase activity. Thus, only short transcripts are significantly upregulated following INTS11 loss, including transcription factors, signaling regulators, and non-coding RNAs. We propose a uniform molecular function for INTS11 across all RNAPII-transcribed loci, with differential effects on particular genes, pathways, or RNA biotypes reflective of transcript lengths rather than specificity of Integrator activity.

DNA Damage-Induced RNAPII Degradation and Its Consequences in Gene Expression.

RPB1, the major and catalytic subunit of human RNA Polymerase II (RNAPII), is specifically degraded by the ubiquitin-proteasome system upon induction of DNA damage by different agents, such as ultraviolet (UV) light. The "last resort" model of RNAPII degradation states that a persistently stalled RNAPII is degraded at the site of the DNA lesion in order to facilitate access to Nucleotide Excision Repair (NER) factors, thereby promoting repair in template strands of active genes. Recent identification and mutation of the lysine residue involved in RPB1 ubiquitylation and degradation unveiled the relevance of RNAPII levels in the control of gene expression. Inhibition of RNAPII degradation after UV light exposure enhanced RNAPII loading onto chromatin, demonstrating that the mere concentration of RNAPII shapes the gene expression response. In this review, we discuss the role of RNAPII ubiquitylation in NER-dependent repair, recent advances in RPB1 degradation mechanisms and its consequences in gene expression under stress, both in normal and repair deficient cells.

The Role of S. cerevisiae Sub1/PC4 in Transcription Elongation Depends on the C-Terminal Region and Is Independent of the ssDNA Binding Domain.

Saccharomyces cerevisiae Sub1 (ScSub1) has been defined as a transcriptional stimulatory protein due to its homology to the ssDNA binding domain (ssDBD) of human PC4 (hPC4). Recently, PC4/Sub1 orthologues have been elucidated in eukaryotes, prokaryotes, and bacteriophages with functions related to DNA metabolism. Additionally, ScSub1 contains a unique carboxyl–terminal region (CT) of unknown function up to date. Specifically, it has been shown that Sub1 is required for transcription activation, as well as other processes, throughout the transcription cycle. Despite the progress that has been made in understanding the mechanism underlying Sub1′s functions, some questions remain unanswered. As a case in point: whether Sub1’s roles in initiation and elongation are differentially predicated on distinct regions of the protein or how Sub1′s functions are regulated. Here, we uncover some residues that are key for DNA–ScSub1 interaction in vivo, localized in the ssDBD, and required for Sub1 recruitment to promoters. Furthermore, using an array of genetic and molecular techniques, we demonstrate that the CT region is required for transcription elongation by RNA polymerase II (RNAPII). Altogether, our data indicate that Sub1 plays a dual role during transcription—in initiation through the ssDBD and in elongation through the CT region.

Mechanism and treatment of α-amanitin poisoning.

Amanita poisoning has a high mortality rate. The α-amanitin toxin in Amanita is the main lethal toxin. There is no specific detoxification drug for α-amanitin, and the clinical treatment mainly focuses on symptomatic and supportive therapy. The pathogenesis of α-amanitin mainly includes: α-amanitin can inhibit the activity of RNA polymeraseII in the nucleus, including the inhibition of the largest subunit of RNA polymeraseII, RNApb1, bridge helix, and trigger loop. In addition, α-amanitin acts in vivo through the enterohepatic circulation and transport system. α-Amanitin can cause the cell death. The existing mechanisms of cell damage mainly focus on apoptosis, oxidative stress, and autophagy. In addition to the pathogenic mechanism, α-amanitin also has a role in cancer treatment, which is the focus of current research. The mechanism of action of α-amanitin on the body is still being explored.

Rtr1 is required for Rpb1-Rpb2 assembly of RNAPII and prevents their cytoplasmic clump formation.

RNA polymerase II (RNAPII) is an essential machinery for catalyzing mRNA synthesis and controlling cell fate in eukaryotes. Although the structure and function of RNAPII have been relatively defined, the molecular mechanism of its assembly process is not clear. The identification and functional analysis of assembly factors will provide new understanding to transcription regulation. In this study, we identify that RTR1, a known transcription regulator, is a new multicopy genetic suppressor of mutants of assembly factors Gpn3, Gpn2, and Rba50. We demonstrate that Rtr1 is directly required to assemble the two largest subunits of RNAPII by coordinating with Gpn3 and Npa3. Deletion of RTR1 leads to cytoplasmic clumping of RNAPII subunit and multiple copies of RTR1 can inhibit the formation of cytoplasmic clump of RNAPII subunit in gpn3-9 mutant, indicating a new layer function of Rtr1 in checking proper assembly of RNAPII. In addition, we find that disrupted activity of Rtr1 phosphatase does not trigger the formation of cytoplasmic clump of RNAPII subunit in a catalytically inactive mutant of RTR1. Based on these results, we conclude that Rtr1 cooperates with Gpn3 and Npa3 to assemble RNAPII core.

Targeting RNA elongation in endothelial-to-mesenchymal transition

Abstract Background and purpose EndMT describes a process in which endothelial cells lose their endothelial features such as the maintenance of the vascular barrier function and trans-differentiate into mesenchymal like cells. ECs which have underwent EndMT are enriched in vulnerable plaques in humans. Several stress stimuli such as hypoxia or inflammation demand immediate induction of adaptive gene expression to improve cell survival. In contrast to the usual induction of gene expression which requires several hours, release of paused RNA Polymerase II (RNAPII) from promoter allows rapid gene induction via RNA elongation. This rapid response mechanism is mediated by the super elongation complex (SEC), which is built around the scaffold proteins AFF1 and AFF4. Here, we hypothesized that RNA elongation regulates EndMT and therefore investigated on the contribution of the SEC scaffold proteins during the process of EndMT. Methods and results After induction of EndMT in human primary endothelial cells (EC) the elongation factors AFF1 and AFF4 were induced (AFF1: 1.9-fold and AFF4: 2.7-fold, P<0.05). Overexpression of AFF4 resulted in increased expression of the mesenchymal marker genes SM22 (1.8-fold, P<0.05) and VCAN (2.0-fold, P<0.05). Accordingly, treatment of EC with two small molecules inhibiting the SEC reduced the expression of several mesenchymal markers SM22 (−87.6%, P<0.05) and Calponin (−93.8%, P<0.05) during EndMT. After induction of EndMT, a large number of newly synthesized RNA transcripts were upregulated, indicating regulation of gene expression on the level of RNA elongation during EndMT. Sequencing of newly transcribed RNA (4sU-sequencing) after treatment with the SEC inhibitors revealed a lower transcript density downstream of promoter sites, suggesting a decreased RNA elongation rate in ECs. SEC inhibitors decreased the expression of the majority of EndMT-induced transcripts (63.6%). Among these rapid induced genes we found the mRNA of RE1 (+10,8-fold). We validated the regulation of RE1 by qPCR (2.8-fold, P<0.05) and single-cell sequencing. Chromatin immunoprecipitation (ChIP) sequencing revealed a reduced occupation of RNAPII in proximity of promotor sites of RE1 upon induction of EndMT. These data suggest a SEC-mediated regulation on the level of RNA elongation of RE1 during EndMT. Conclusion SEC scaffold proteins AFF1 and AFF4 are upregulated during EndMT. Targeting the SEC during EndMT reduces the expression of common mesenchymal markers, consistent with an overall higher occupancy of paused RNAPII at promoter-proximal sites. We identify RE1 as a rapidly induced gene during EndMT that is mediated by the SEC scaffold proteins AFF1 and AFF4. Our findings suggest that RNA elongation is an inducer of EndMT. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)

Coordinating transcription and replication to mitigate their conflicts in early Drosophila embryos.

Collisions between transcribing RNA polymerases and DNA replication forks are disruptive. The threat of collisions is particularly acute during the rapid early embryonic cell cycles of Drosophila when S phase occupies the entirety of interphase. We hypothesize that collision-avoidance mechanisms safeguard this early transcription. Real-time imaging of endogenously tagged RNA polymerase II (RNAPII) and a reporter for nascent transcripts in unperturbed embryos shows clustering of RNAPII at around 2min after mitotic exit, followed by progressive dispersal as associated nascent transcripts accumulate later in interphase. Abrupt inhibition of various steps in DNA replication, including origin licensing, origin firing, and polymerization, suppresses post-mitotic RNAPII clustering and transcription in nuclear cycles. We propose that replication dependency defers the onset of transcription so that RNAPII transcribes behind advancing replication forks. The resulting orderly progression can explain how early embryos circumvent transcription-replication conflicts to express essential developmental genes.

DNA double-strand break-derived RNA drives TIRR/53BP1 complex dissociation

Tudor-interacting repair regulator (TIRR) is an RNA-binding protein and a negative regulator of the DNA-repair factor p53-binding protein 1 (53BP1). In non-damage conditions, TIRR is bound to 53BP1. After DNA damage, TIRR and 53BP1 dissociate, and 53BP1 binds the chromatin at the double-strand break (DSB) to promote non-homologous end joining (NHEJ)-mediated repair. However, the exact mechanistic details of this dissociation after damage are unknown. Increasing evidence has implicated RNA as a crucial factor in the DNA damage response (DDR). Here, we show that RNA can separate TIRR/53BP1. Specifically, RNA with a hairpin secondary structure, transcribed at the DSB by RNA polymerase II (RNAPII), promotes TIRR/53BP1 complex separation. This hairpin RNA binds to the same residues on TIRR as 53BP1. Our results uncover a role of DNA-damage-derived RNA in modulating a protein-protein interaction and contribute to our understanding of DSB repair.

Distinct role of subunits of the Arabidopsis RNA polymerase II elongation factor PAF1C in transcriptional reprogramming.

Transcript elongation by RNA polymerase II (RNAPII) is dynamic and highly regulated, thereby contributing to the implementation of gene expression programs during plant development or in response to environmental cues. The heterohexameric polymerase-associated factor 1 complex (PAF1C) stabilizes the RNAPII elongation complex promoting efficient transcript synthesis. In addition, PAF1C links transcriptional elongation with various post-translational histone modifications at transcribed loci. We have exposed Arabidopsis mutants deficient in the PAF1C subunits ELF7 or CDC73 to elevated NaCl concentrations to provoke a transcriptional response. The growth of elf7 plants was reduced relative to that of wildtype under these challenging conditions, whereas cdc73 plants exhibited rather enhanced tolerance. Profiling of the transcriptional changes upon NaCl exposure revealed that cdc73 responded similar to wildtype. Relative to wildtype and cdc73, the transcriptional response of elf7 plants was severely reduced in accord with their greater susceptibility to NaCl. The data also imply that CDC73 is more relevant for the transcription of longer genes. Despite the fact that both ELF7 and CDC73 are part of PAF1C the strikingly different transcriptional response of the mutants upon NaCl exposure suggests that the subunits have (partially) specific functions.

The Paf1 complex is required for RNA polymerase II removal in response to DNA damage

Rpb1, the largest subunit of RNA polymerase II (RNAPII), is rapidly polyubiquitinated and degraded in response to DNA damage; this process is considered to be a "mechanism of last resort'' employed by cells. The underlying mechanism of this process remains elusive. Here, we uncovered a previously uncharacterized multistep pathway in which the polymerase-associated factor 1 (Paf1) complex (PAF1C, composed of the subunits Ctr9, Paf1, Leo1, Cdc73, and Rtf1) is involved in regulating the RNAPII pool by stimulating Elongin-Cullin E3 ligase complex-mediated Rpb1 polyubiquitination and subsequent degradation by the proteasome following DNA damage. Mechanistically, Spt5 is dephosphorylated following DNA damage, thereby weakening the interaction between the Rtf1 subunit and Spt5, which might be a key step in initiating Rpb1 degradation. Next, Rad26 is loaded onto stalled RNAPII to replace the Spt4/Spt5 complex in an RNAPII-dependent manner and, in turn, recruits more PAF1C to DNA lesions via the binding of Rad26 to the Leo1 subunit. Importantly, the PAF1C, assembled in a Ctr9-mediated manner, coordinates with Rad26 to localize the Elongin-Cullin complex on stalled RNAPII, thereby inducing RNAPII removal, in which the heterodimer Paf1/Leo1 and the subunit Cdc73 play important roles. Together, our results clearly revealed a new role of the intact PAF1C in regulating the RNAPII pool in response to DNA damage.

Slow RNAPII Transcription Elongation Rate, Low Levels of RNAPII Pausing, and Elevated Histone H1 Content at Promoters Associate with Higher m6A Deposition on Nascent mRNAs.

N6-methyladenosine modification (m6A) fine-tunes RNA fate in a variety of ways, thus regulating multiple fundamental biological processes. m6A writers bind to chromatin and interact with RNA polymerase II (RNAPII) during transcription. To evaluate how the dynamics of the transcription process impact m6A deposition, we studied RNAPII elongation rates in mouse embryonic stem cells with altered chromatin configurations, due to reductions in linker histone H1 content. We found that genes transcribed at slow speed are preferentially methylated and display unique signatures at their promoter region, namely high levels of histone H1, together with marks of bivalent chromatin and low RNAPII pausing. They are also highly susceptible to m6A loss upon histone H1 reduction. These results indicate that RNAPII velocity links chromatin structure and the deposition of m6A, highlighting the intricate relationship between different regulatory layers on nascent mRNA molecules.

Chromatin-associated microprocessor assembly is regulated by the U1 snRNP auxiliary protein PRP40.

In plants, microRNA (miRNA) biogenesis involves cotranscriptional processing of RNA polymerase II (RNAPII)-generated primary transcripts by a multi-protein complex termed the microprocessor. Here, we report that Arabidopsis (Arabidopsis thaliana) PRE-MRNA PROCESSING PROTEIN 40 (PRP40), the U1 snRNP auxiliary protein, positively regulates the recruitment of SERRATE, a core component of the plant microprocessor, to miRNA genes. The association of DICER-LIKE1 (DCL1), the microprocessor endoribonuclease, with chromatin was altered in prp40ab mutant plants. Impaired cotranscriptional microprocessor assembly was accompanied by RNAPII accumulation at miRNA genes and retention of miRNA precursors at their transcription sites in the prp40ab mutant plants. We show that cotranscriptional microprocessor assembly, regulated by AtPRP40, positively affects RNAPII transcription of miRNA genes and is important to reach the correct levels of produced miRNAs.

CtIP-dependent nascent RNA expression flanking DNA breaks guides the choice of DNA repair pathway

The RNA world is changing our views about sensing and resolution of DNA damage. Here, we develop single-molecule DNA/RNA analysis approaches to visualize how nascent RNA facilitates the repair of DNA double-strand breaks (DSBs). RNA polymerase II (RNAPII) is crucial for DSB resolution in human cells. DSB-flanking, RNAPII-generated nascent RNA forms RNA:DNA hybrids, guiding the upstream DNA repair steps towards favouring the error-free Homologous Recombination (HR) pathway over Non-Homologous End Joining. Specific RNAPII inhibitor, THZ1, impairs recruitment of essential HR proteins to DSBs, implicating nascent RNA in DNA end resection, initiation and execution of HR repair. We further propose that resection factor CtIP interacts with and helps re-activate RNAPII when paused by the RNA:DNA hybrids, collectively promoting faithful repair of chromosome breaks to maintain genomic integrity.

THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours

BackgroundGastrointestinal stromal tumours (GISTs) are the most common mesenchymal tumours of the gastrointestinal tract and are characterized by activating mutations of c-KIT or PDGFRa receptor tyrosine kinases (RTKs). Despite the clinical success of tyrosine kinase inhibitors (TKIs), more than half of GIST patients develop resistance due to a second mutation. Cyclin-dependent kinase 7 (CDK7) is the catalytic subunit of CDK-activating kinase (CAK), and it plays an important role in the regulation of cell cycle transitions and gene transcription. THZ1, a CDK7 inhibitor, exhibits a dose-dependent inhibitory effect in various cancers.MethodsData from the public GEO database and tissue microarray were used to analyse the gene expression levels of CDKs in GISTs. The impact of CDK7 knockdown and the CDK7 inhibitor THZ1 on GIST progression was investigated in vitro using CCK-8, colony formation, and flow cytometry assays and in vivo using a xenograft mouse model. RNA sequencing was performed to investigate the mechanism of GIST cell viability impairment mediated by THZ1 treatment.ResultsOur study demonstrated that CDK7 is relatively overexpressed in high-risk GISTs and predicts a poor outcome. A low concentration of THZ1 exhibited a pronounced antineoplastic effect in GIST cells in vivo and in vitro. Moreover, THZ1 exerted synergistic anticancer effects with imatinib. THZ1 treatment resulted in transcriptional modulation by inhibiting the phosphorylation of Ser2, Ser5, and Ser7 within RNA polymerase II (RNAPII). c-KIT, an oncogene driver of GIST, was transcriptionally repressed by THZ1 treatment or CDK7 knockdown. Transcriptome sequencing analysis showed that OSR1 acts as a downstream target of CDK7 and regulates c-KIT expression. Taken together, our results highlight elevated CDK7 expression as a predictor of poor outcome in GIST and present the combination of CDK7 and RTK inhibitors as a potent therapeutic strategy to improve the efficacy of GIST treatment.3dK7zp1xpFMvfXo5mnDRH-Video abstract

P10.12.A CDK12/CDK13 inhibition disrupts a transcriptional program critical for glioblastoma survival

Abstract Background Glioblastoma multiforme (GBM) is the most prevalent and aggressive malignant tumor of the central nervous system. With a median survival of only one year, GBM patients have a particularly poor prognosis, highlighting a clear need for novel therapeutic strategies to target this disease. Transcriptional cyclin-dependent kinases (CDK), which phosphorylate key residues of RNA polymerase II (RNAPII) C-terminal domain (CTD), play a major role in sustaining aberrant transcriptional programs that are key to development and maintenance of cancer cells. Material and Methods We used pharmacological inhibition and genetic ablation to study effects of CDK12/CDK13 depletion on the proliferatory and migratory capacity of GBM cells and mouse xenografts. SLAM-seq, CUT&RUN and cell cycle assays were used to study the mechanistic effects of CDK12/CDK13 depletion in GBM cells. Results CDK12/CDK13 depletion markedly reduced the proliferatory and migratory capacity of GBM cells, as well as in vivo growth. CDK12/CDK13 inhibition potentiated existing chemotherapeutic treatments. Mechanistically, inhibition of CDK12/CDK13 leads to a genome-wide abrogation of RNAPII CTD phosphorylation, which in turn disrupts transcription and cell cycle progression in GBM cells. Conclusion These results provide proof-of-concept for the potential of CDK12 and CDK13 as therapeutic targets for GBM.