Productive Transcription Elongation Research Articles

A structure-based mechanism for displacement of the HEXIM adapter from 7SK small nuclear RNA

Productive transcriptional elongation of many cellular and viral mRNAs requires transcriptional factors to extract pTEFb from the 7SK snRNP by modulating the association between HEXIM and 7SK snRNA. In HIV-1, Tat binds to 7SK by displacing HEXIM. However, without the structure of the 7SK-HEXIM complex, the constraints that must be overcome for displacement remain unknown. Furthermore, while structure details of the TatNL4-3-7SK complex have been elucidated, it is unclear how subtypes with more HEXIM-like Tat sequences accomplish displacement. Here we report the structures of HEXIM, TatG, and TatFin arginine rich motifs in complex with the apical stemloop-1 of 7SK. While most interactions between 7SK with HEXIM and Tat are similar, critical differences exist that guide function. First, the conformational plasticity of 7SK enables the formation of three different base pair configurations at a critical remodeling site, which allows for the modulation required for HEXIM binding and its subsequent displacement by Tat. Furthermore, the specific sequence variations observed in various Tat subtypes all converge on remodeling 7SK at this region. Second, we show that HEXIM primes its own displacement by causing specific local destabilization upon binding — a feature that is then exploited by Tat to bind 7SK more efficiently.

HIF-1 Interacts with TRIM28 and DNA-PK to release paused RNA polymerase II and activate target gene transcription in response to hypoxia

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that acts as a regulator of oxygen (O2) homeostasis in metazoan species by binding to hypoxia response elements (HREs) and activating the transcription of hundreds of genes in response to reduced O2 availability. RNA polymerase II (Pol II) initiates transcription of many HIF target genes under non-hypoxic conditions but pauses after approximately 30–60 nucleotides and requires HIF-1 binding for release. Here we report that in hypoxic breast cancer cells, HIF-1 recruits TRIM28 and DNA-dependent protein kinase (DNA-PK) to HREs to release paused Pol II. We show that HIF-1α and TRIM28 assemble the catalytically-active DNA-PK heterotrimer, which phosphorylates TRIM28 at serine-824, enabling recruitment of CDK9, which phosphorylates serine-2 of the Pol II large subunit C-terminal domain as well as the negative elongation factor to release paused Pol II, thereby stimulating productive transcriptional elongation. Our studies reveal a molecular mechanism by which HIF-1 stimulates gene transcription and reveal that the anticancer effects of drugs targeting DNA-PK in breast cancer may be due in part to their inhibition of HIF-dependent transcription.

Abstract P237: PRT2527 is a potent and selective CDK9 inhibitor that demonstrates anti-cancer activity in preclinical models of hematological malignancies and solid tumors with MYC amplification

Abstract Cyclin-dependent kinase 9 (CDK9) is a master regulator of transcription that controls paused RNA polymerase II (RNAP2) release through phosphorylation of its carboxy-terminal domain, resulting in productive transcription elongation. CDK9 has been extensively studied as a potential target for cancer therapy in “transcriptionally addicted” tumors as transient inhibition of CDK9 primarily depletes proteins with short half-lives, such as the oncogenes MCL1 and MYC, making CDK9 a promising target in cancer. Here we show that PRT2527 is a potent and highly selective CDK9 inhibitor with moderate to high clearance that achieves optimal temporal target engagement and exhibits potent in vitro and in vivo activities. PRT2527 inhibited CDK9 enzymatic activity with an IC50 of 0.98 nM in a biochemical assay and showed high selectivity in a panel of kinases when tested at physiologically relevant 1 mM ATP concentration. In vitro, PRT2527 inhibited phosphorylation of Ser2RNAP2 in NCI-H929 cells with an IC50 of 54 nM, and an IC50 of 198 nM in a plasma assay to adjust for human plasma protein binding. Transient treatment of cells with PRT2527 inhibited pSer2RNAP2, depleted MCL1 and MYC proteins, and activated cleaved caspase-3 (CC3) in a concentration-dependent manner. In a proteomic profiling study, MCL1 was identified as one of the major down-regulated proteins following PRT2527 treatment. In a panel of hematological cancer cell lines representing B- and T-ALL, AML, and non-Hodgkin’s lymphoma (NHL), as well as subsets of sarcoma, prostate, adenoid cystic carcinoma (ACC), and non-small cell lung cancer (NSCLC) cell lines, PRT2527 treatment consistently led to a potent, concentration-dependent inhibition of proliferation. In a pharmacokinetic/pharmacodynamic (PK/PD) study, intravenous (IV) administration of PRT2527 achieved transient target engagement, depletion of MCL1 and MYC proteins, and induction of apoptosis in tumor tissue. This PK/PD correlation was successfully translated into in vivo efficacy in multiple models. Once weekly dosing of PRT2527 was well-tolerated and significantly inhibited tumor growth in various AML CDX models and induced tumor regressions in double-hit and triple-hit diffuse large B-cell lymphoma (DLBCL) CDX and PDX models carrying the MYC translocation. Combining PRT2527 with venetoclax achieved complete tumor regressions in a venetoclax resistant OCI-AML3 model. PRT2527 demonstrated potent ex vivo activity in PDX models of B-ALL and T-ALL, as well as various solid tumor PDX models with high levels of MYC amplification and overexpression, including pancreatic carcinoma, gastric and gastroesophageal carcinomas, NSCLC, and sarcoma. In vivo efficacy studies with once weekly IV administration of PRT2527 confirmed significant tumor growth inhibition in select MYC-amplified solid tumor PDX models. Taken together, this preclinical characterization supports the advancement of PRT2527 into clinical studies for transcriptionally addicted hematological malignancies and solid tumors with MYC amplification and/or dysregulation. Citation Format: Yang W. Zhang, Liang Lu, Min Wang, Dave Rominger, Stefan Ruepp, Kirsten Gallagher, William Gowen-MacDonald, Chaofeng Dai, Miles Cowart, Andrew Combs, Bruce Ruggeri, Peggy Scherle, Kris Vaddi. PRT2527 is a potent and selective CDK9 inhibitor that demonstrates anti-cancer activity in preclinical models of hematological malignancies and solid tumors with MYC amplification [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P237.

MYCN recruits the nuclear exosome complex to RNA polymerase II to prevent transcription-replication conflicts

The MYCN oncoprotein drives the development of numerous neuroendocrine and pediatric tumors. Here we show that MYCN interacts with the nuclear RNA exosome, a 3'-5' exoribonuclease complex, and recruits the exosome to its target genes. In the absence of the exosome, MYCN-directed elongation by RNA polymerase II (RNAPII) is slow and non-productive on a large group of cell-cycle-regulated genes. During the S phase of MYCN-driven tumor cells, the exosome is required to prevent the accumulation of stalled replication forks and of double-strand breaks close to the transcription start sites. Upon depletion of the exosome, activation of ATM causes recruitment of BRCA1, which stabilizes nuclear mRNA decapping complexes, leading to MYCN-dependent transcription termination. Disruption of mRNA decapping in turn activates ATR, indicating transcription-replication conflicts. We propose that exosome recruitment by MYCN maintains productive transcription elongation during S phase and prevents transcription-replication conflicts to maintain the rapid proliferation of neuroendocrine tumor cells.

Inhibition of CDK9 activity compromises global splicing in prostate cancer cells

ABSTRACT Cyclin-dependent kinase 9 (CDK9) phosphorylates RNA polymerase II to promote productive transcription elongation. Here we show that short-term CDK9 inhibition affects the splicing of thousands of mRNAs. CDK9 inhibition impairs global splicing and there is no evidence for a coordinated response between the alternative splicing and the overall transcriptome. Alternative splicing is a feature of aggressive prostate cancer (CRPC) and enables the generation of the anti-androgen resistant version of the ligand-independent androgen receptor, AR-v7. We show that CDK9 inhibition results in the loss of AR and AR-v7 expression due to the defects in splicing, which sensitizes CRPC cells to androgen deprivation. Finally, we demonstrate that CDK9 expression increases as PC cells develop CRPC-phenotype both in vitro and also in patient samples. To conclude, here we show that CDK9 inhibition compromises splicing in PC cells, which can be capitalized on by targeting the PC-specific addiction androgen receptor.

Promoter-proximal elongation regulates transcription in archaea

Recruitment of RNA polymerase and initiation factors to the promoter is the only known target for transcription activation and repression in archaea. Whether any of the subsequent steps towards productive transcription elongation are involved in regulation is not known. We characterised how the basal transcription machinery is distributed along genes in the archaeon Saccharolobus solfataricus. We discovered a distinct early elongation phase where RNA polymerases sequentially recruit the elongation factors Spt4/5 and Elf1 to form the transcription elongation complex (TEC) before the TEC escapes into productive transcription. TEC escape is rate-limiting for transcription output during exponential growth. Oxidative stress causes changes in TEC escape that correlate with changes in the transcriptome. Our results thus establish that TEC escape contributes to the basal promoter strength and facilitates transcription regulation. Impaired TEC escape coincides with the accumulation of initiation factors at the promoter and recruitment of termination factor aCPSF1 to the early TEC. This suggests two possible mechanisms for how TEC escape limits transcription, physically blocking upstream RNA polymerases during transcription initiation and premature termination of early TECs.

Topoisomerase IIα represses transcription by enforcing promoter-proximal pausing.

SummaryAccumulation of topological stress in the form of DNA supercoiling is inherent to the advance of RNA polymerase II (Pol II) and needs to be resolved by DNA topoisomerases to sustain productive transcriptional elongation. Topoisomerases are therefore considered positive facilitators of transcription. Here, we show that, in contrast to this general assumption, human topoisomerase IIα (TOP2A) activity at promoters represses transcription of immediate early genes such as c-FOS, maintaining them under basal repressed conditions. Thus, TOP2A inhibition creates a particular topological context that results in rapid release from promoter-proximal pausing and transcriptional upregulation, which mimics the typical bursting behavior of these genes in response to physiological stimulus. We therefore describe the control of promoter-proximal pausing by TOP2A as a layer for the regulation of gene expression, which can act as a molecular switch to rapidly activate transcription, possibly by regulating the accumulation of DNA supercoiling at promoter regions.

SPT6 promotes epidermal differentiation and blockade of an intestinal-like phenotype through control of transcriptional elongation

In adult tissue, stem and progenitor cells must tightly regulate the balance between proliferation and differentiation to sustain homeostasis. How this exquisite balance is achieved is an area of active investigation. Here, we show that epidermal genes, including ~30% of induced differentiation genes already contain stalled Pol II at the promoters in epidermal stem and progenitor cells which is then released into productive transcription elongation upon differentiation. Central to this process are SPT6 and PAF1 which are necessary for the elongation of these differentiation genes. Upon SPT6 or PAF1 depletion there is a loss of human skin differentiation and stratification. Unexpectedly, loss of SPT6 also causes the spontaneous transdifferentiation of epidermal cells into an intestinal-like phenotype due to the stalled transcription of the master regulator of epidermal fate P63. Our findings suggest that control of transcription elongation through SPT6 plays a prominent role in adult somatic tissue differentiation and the inhibition of alternative cell fate choices.

JMJD6 cleaves MePCE to release positive transcription elongation factor b (P-TEFb) in higher eukaryotes.

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.

Integrator of stress cues.

The Integrator complex fine-tunes mRNA levels in response to stress by attenuating productive transcription elongation

A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects (TE<sup>deff</sup>) in Cancers

We have previously reported that a subset of cancers is defined by global transcriptional deregulations with widespread deficiencies in mRNA transcription elongation (TE)-we call such cancers as TEdeff. Notably, TEdeff cancers are characterized by spurious transcription and faulty mRNA processing in a large set of genes, such as interferon/JAK/STAT and TNF/NF-κB pathways, leading to their suppression. The TEdeff subtype of tumors in renal cell carcinoma and metastatic melanoma patients significantly correlate with poor response and outcome in immunotherapy. Given the importance of investigating TEdeff cancers-as it portends a significant roadblock against immunotherapy-the goal of this protocol is to establish an in vitro TEdeff mouse model to study these widespread, non-genetic transcriptional abnormalities in cancers and gain new insights, novel uses for existing drugs, or find new strategies against such cancers. We detail the use of chronic flavopiridol mediated CDK9 inhibition to abrogate phosphorylation of serine 2 residue on the C-terminal repeat domain (CTD) of RNA polymerase II (RNA Pol II), suppressing the release of RNA Pol II into productive transcription elongation. Given that TEdeff cancers are not classified under any specific somatic mutation, a pharmacological model is advantageous, and best mimics the widespread transcriptional and epigenetic defects observed in them. The use of an optimized sublethal dose of flavopiridol is the only efficacious strategy in creating a generalizable model of non-genetic widespread disruption in transcription elongation and mRNA processing defects, closely mimicking the clinically observed TEdeff characteristics. Therefore, this model of TEdeff can be leveraged to dissect, cell-autonomous factors enabling them in resisting immune-mediated cell attack.

SREBP1-dependent de novo fatty acid synthesis gene expression is elevated in malignant melanoma and represents a cellular survival trait

de novo fatty acid biosynthesis (DNFA) is a hallmark adaptation of many cancers that supports survival, proliferation, and metastasis. Here we elucidate previously unexplored aspects of transcription regulation and clinical relevance of DNFA in cancers. We show that elevated expression of DNFA genes is characteristic of many tumor types and correlates with poor prognosis, especially in melanomas. Elevated DNFA gene expression depends on the SREBP1 transcription factor in multiple melanoma cell lines. SREBP1 predominantly binds to the transcription start sites of DNFA genes, regulating their expression by recruiting RNA polymerase II to promoters for productive transcription elongation. We find that SREBP1-regulated DNFA represents a survival trait in melanoma cells, regardless of proliferative state and oncogenic mutation status. Indeed, malignant melanoma cells exhibit elevated DNFA gene expression after the BRAF/MEK signaling pathway is blocked (e.g. by BRAF inhibitors), and DNFA expression remains higher in melanoma cells resistant to vemurafenib treatment than in untreated cells. Accordingly, DNFA pathway inhibition, whether by direct targeting of SREBP1 with antisense oligonucleotides, or through combinatorial effects of multiple DNFA enzyme inhibitors, exerts potent cytotoxic effects on both BRAFi-sensitive and -resistant melanoma cells. Altogether, these results implicate SREBP1 and DNFA enzymes as enticing therapeutic targets in melanomas.

Abstract 1521: Role of Trim28 in prostate cancer

Abstract TRIM28 is an essential gene during embryonic development, which is involved in a wide range of biologic processes. From the pathologic viewpoint, TRIM28 overexpression was proved to broadly exist in pancreatic, liver, breast, glioma and lung cancer, and often expression of TRIM28 is correlated with clinical stage. Functional assays have demonstrated that TRIM28 promotes cancer cell growth and metastasis, further supporting TRIM28 as a promising target for therapeutic intervention. For past decade, TRIM28 is considered to be a transcriptional co-repressor due to TRIM28-dependent recruitment of histone modifiers such as CHD1/NuRD complex and SETDB1 for histone methylation and heterochromatin formation to achieve gene silencing. Surprisingly, transcriptome study revealed that TRIM28 can activate the expression of its target genes as much as it represses, hypothesizing the main role of TRIM28 may not lie in transcriptional repression. In addition, recent findings demonstrated that TRIM28 interacts with 7SK snRNP complex, which facilitates P-TEFb kinase module recruitment and productive transcription elongation at inducible genes. This evidence implicated that TRIM28 may form distinct complexes to induce or repress gene expression in context-dependent manner. On top of that, it remains unclear about the role of TRIM28 in prostate cancer. Our preliminary study revealed that TRIM28 is upregulated in castration-resistant prostate cancer (CRPC). TRIM28 knockdown markedly decreased prostate cancer cell proliferation, clonogenicity as well as CRPC-xenograft tumor growth. Moreover, proteomic analysis in LNCaP prostate cancer cells revealed that TRIM28 associates with a number of transcription co-activators. Gene expression profiling showed that androgen signaling is positively regulated by TRIM28. We proposed that TRIM28 forms the transcription activation complexes that modulate gene expression of various oncogenic pathway and promote PCa progression. Citation Format: Ka-Wing Fong, Jonathan Zhao, Bin Zheng, Jindan Yu. Role of Trim28 in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1521.

SUMO suppresses and MYC amplifies transcription globally by regulating CDK9 sumoylation.

Regulation of transcription is fundamental to the control of cellular gene expression and function. Although recent studies have revealed a role for the oncoprotein MYC in amplifying global transcription, little is known as to how the global transcription is suppressed. Here we report that SUMO and MYC mediate opposite effects upon global transcription by controlling the level of CDK9 sumoylation. On one hand, SUMO suppresses global transcription via sumoylation of CDK9, the catalytic subunit of P-TEFb kinase essential for productive transcriptional elongation. On the other hand, MYC amplifies global transcription by antagonizing CDK9 sumoylation. Sumoylation of CDK9 blocks its interaction with Cyclin T1 and thus the formation of active P-TEFb complex. Transcription profiling analyses reveal that SUMO represses global transcription, particularly of moderately to highly expressed genes and by generating a sumoylation-resistant CDK9 mutant, we confirm that sumoylation of CDK9 inhibits global transcription. Together, our data reveal that SUMO and MYC oppositely control global gene expression by regulating the dynamic sumoylation and desumoylation of CDK9.

Specific Recognition of Arginine Methylated Histone Tails by JMJD5 and JMJD7

We have reported that JMJD5 and JMJD7 (JMJD5/7) are responsible for the clipping of arginine methylated histone tails to generate “tailless nucleosomes”, which could release the pausing RNA polymerase II (Pol II) into productive transcription elongation. JMJD5/7 function as endopeptidases that cleave histone tails specifically adjacent to methylated arginine residues and continue to degrade N-terminal residues of histones via their aminopeptidase activity. Here, we report structural and biochemical studies on JMJD5/7 to understand the basis of substrate recognition and catalysis mechanism by this JmjC subfamily. Recognition between these enzymes and histone substrates is specific, which is reflected by the binding data between enzymes and substrates. High structural similarity between JMJD5 and JMJD7 is reflected by the shared common substrates and high binding affinity. However, JMJD5 does not bind to arginine methylated histone tails with additional lysine acetylation while JMJD7 does not bind to arginine methylated histone tails with additional lysine methylation. Furthermore, the complex structures of JMJD5 and arginine derivatives revealed a Tudor domain-like binding pocket to accommodate the methylated sidechain of arginine, but not lysine. There also exists a glutamine close to the catalytic center, which may suggest a unique imidic acid mediated catalytic mechanism for proteolysis by JMJD5/7.

Crosstalk between the RNA Methylation and Histone-Binding Activities of MePCE Regulates P-TEFb Activation on Chromatin

RNAP II switching from the paused to the productive transcription elongation state is a pivotal regulatory step that requires specific phosphorylations catalyzed by the P-TEFb kinase. Nucleosolic P-TEFb activity is inhibited by its interaction with the ribonuclear protein complex built around the 7SK small nuclear RNA (7SK snRNP). MePCE is the RNA methyltransferase that methylates and stabilizes 7SK in the nucleosol. Here, we report that MePCE also binds chromatin through the histone H4 tail to serve as a P-TEFb activator at specific genes important for cellular identity. Notably, this histone binding abolishes MePCE's RNA methyltransferase activity toward 7SK, which explains why MePCE-bound P-TEFb on chromatin may not be associated with the full 7SK snRNP and is competent for RNAP II activation. Overall, our results suggest that crosstalk between the histone-binding and RNA methylation activities of MePCE regulates P-TEFb activation on chromatin in a 7SK- and Brd4-independent manner.

Nuclear EGFR and Integrator/Super Elongation Complex concurrently binds to Immediate Early Genes for gene transactivation.

The gene transactivation function of nuclear EGFR (nEGFR) has been studied by investigating the genomic co-occupancies of nEGFR and RNA Polymerase II (RNAPII). However, due to RNAPII pausing, the co-recruitment of RNAPII and nEGFR does not necessarily represent productive transactivation. In this study, we integrated gatekeepers of productive transcriptional elongation such as Integrator and Super Elongation Complex (SEC) to interrogate the function of nEGFR. By analyzing publicly available ChIP-seq and RNA-seq data, we aims to 1) explore the function of nEGFR, 2) unravel nEGFR target genes, and 3) discuss potential mechanisms of nEGFR chromatin recruitment. EGF treatment in HeLa cells instigated chromatin recruitment of nEGFR, ERK, RNAPII, Integrator, and SEC in a cluster of 61 EGF-responsive genes. The function of nEGFR was identified as gene-activating rather than gene-repressing. Within the cluster of EGF-responsive genes, nEGFR targeted eleven Immediate Early Genes (IEGs) — JUN, EGR1, JUNB, IER2, KLF2, FOS, FOSL1, RHOB, CCNL1, DUSP2, and DUSP5, which up-regulated >2-fold after EGF stimulation. The promoter of these target genes commonly harbors AT-rich minimal consensus sequences for nEGFR binding. In addition, TCGA data analysis demonstrated positive correlations between EGFR and JUN/FOSL1/RHOB expressions, as well as clinical correlations in specific cancer types. To our knowledge, this is the first study to compare the genome-wide distribution of nEGFR versus Integrator and SEC, providing novel insight into supporting the gene-activating function of nEGFR. We revealed a panel of eleven nEGFR target genes, which concurrently recruited nEGFR, RNAPII, Integrator, and SEC for productive transcriptional elongation.

Chemical control of transcription

Chemical Biology Friedreich's ataxia, a devastating neurodegenerative disease with no effective therapy, is caused by an expansion of intronic repeats and hence a reduced expression of the FXN gene. Erwin et al. synthesized a molecule that specifically targets the expanded repressive repeats. This molecule thereby licenses productive transcription elongation and restores FXN expression to normal levels. In the future, similar interventions may be effective in a diverse array of diseases caused by unstable expansions in microsatellite repeats. Science , this issue p. [1617][1] [1]: /lookup/doi/10.1126/science.aan6414

Synthetic transcription elongation factors license transcription across repressive chromatin.

The release of paused RNA polymerase II into productive elongation is highly regulated, especially at genes that affect human development and disease. To exert control over this rate-limiting step, we designed sequence-specific synthetic transcription elongation factors (Syn-TEFs). These molecules are composed of programmable DNA-binding ligands flexibly tethered to a small molecule that engages the transcription elongation machinery. By limiting activity to targeted loci, Syn-TEFs convert constituent modules from broad-spectrum inhibitors of transcription into gene-specific stimulators. Here we present Syn-TEF1, a molecule that actively enables transcription across repressive GAA repeats that silence frataxin expression in Friedreich's ataxia, a terminal neurodegenerative disease with no effective therapy. The modular design of Syn-TEF1 defines a general framework for developing a class of molecules that license transcription elongation at targeted genomic loci.

272 - Global disruption of productive transcriptional elongation via targeted BET protein degradation

272 - Global disruption of productive transcriptional elongation via targeted BET protein degradation