C-terminal Domain Of RNA Polymerase Research Articles

The small RNA biogenesis in rice is regulated by MAP kinase-mediated OsCDKD phosphorylation.

CDKs are the master regulator of cell division and their activity is controlled by the regulatory subunit cyclins and phosphorylation by the CAKs. However, the role of MAP kinases in regulating plant cell cycle or CDKs have not been explored. Here, we report that the MAP kinases OsMPK3, OsMPK4, and OsMPK6 physically interact and phosphorylate OsCDKD and its regulatory subunit OsCYCH in rice. MAP kinases phosphorylate CDKD at Ser-168 and Thr-235 residues in OsCDKD. The MAP kinase-mediated phosphorylation of OsCDKD is required for its activation to control the small RNA biogenesis. The phosphodead version of OsCDKD fails to activate the C-terminal domain of RNA Polymerase II, thereby negatively impacting small RNA transcription. Further, the overexpression lines of wild-type (WT) OsCDKD and phosphomimic OsCDKD show increased root growth, plant height, tiller number, panicle number, and seed number in comparison to WT, phosphodead OsCDKD-OE, and kinase-dead OsCDKD-OE plants. In a nutshell, our study establishes a novel regulation of OsCDKD by MAPK-mediated phosphorylation in rice. The phosphorylation of OsCDKD by MAPKs imparts a positive effect on rice growth and development by regulating miRNAs transcription.

CDK7 kinase activity promotes RNA polymerase II promoter escape by facilitating initiation factor release

Cyclin-dependent kinase 7 (CDK7), part of the general transcription factor TFIIH, promotes gene transcription by phosphorylating the C-terminal domain of RNA polymerase II (RNA Pol II). Here, we combine rapid CDK7 kinase inhibition with multi-omics analysis to unravel the direct functions of CDK7 in human cells. CDK7 inhibition causes RNA Pol II retention at promoters, leading to decreased RNA Pol II initiation and immediate global downregulation of transcript synthesis. Elongation, termination, and recruitment of co-transcriptional factors are not directly affected. Although RNA Pol II, initiation factors, and Mediator accumulate at promoters, RNA Pol II complexes can also proceed into gene bodies without promoter-proximal pausing while retaining initiation factors and Mediator. Further downstream, RNA Pol II phosphorylation increases and initiation factors and Mediator are released, allowing recruitment of elongation factors and an increase in RNA Pol II elongation velocity. Collectively, CDK7 kinase activity promotes the release of initiation factors and Mediator from RNA Pol II, facilitating RNA Pol II escape from the promoter.

Abstract LB168: A selective, potent, and orally bioavailable CDK7 inhibitor demonstrates superior anti-cancer activity in colorectal cancer models

Abstract Dysregulated cell cycle and gene expression are pivotal factors in cancer development and the regulators of these processes are attractive targets for cancer treatment. The success of CDK4/6 inhibitors such as palbociclib, ribociclib, and abemaciclib underscores the promising role of targeting cell cycle regulators in anti-cancer therapy. On the other hand, a robust correlation has been established between the overexpression of oncogenes and super-enhancer (SE). The genes associated with SE show increased vulnerability to transcription factor disruption compared to genes with typical enhancers. It has been demonstrated in previous studies that inhibiting transcription factors can effectively decrease the expression of SE-associated oncogenes, including c-MYC, thereby highlighting potential anticancer effects for oncogene-addicted cancers. Currently, agents targeting transcription factors are undergoing clinical trials, offering promising therapeutic options for patients with various types of cancers. CDK7 plays a crucial role in orchestrating cell cycle progression and transcription by phosphorylating CDKs, the C-terminal domain of RNA polymerase II, and hormone receptors like estrogen and androgen receptors. The inhibition of CDK7 has been shown to effectively inhibit tumor growth through various mechanisms, including inducing cell cycle arrest and inhibiting transcription, thereby leading to the death of cancer cells. Notably, ongoing clinical studies have demonstrated that small-molecule CDK7 inhibitors effectively control cancer progression as a stand-alone and in combination with other anti-cancer agents. C-16661, a selective, potent, orally bioavailable, and reversible ATP-competitive inhibitor of CDK7, displayed a single-digit nanomolar IC50 value in both biochemical and cellular assays. It exhibited excellent selectivity in a kinase panel assay, and remarkable potency in a human cancer cell line panel assay. C-16661 treatment resulted in strong inhibition of both its direct and indirect downstream targets including pS5-POLII, pT161-CDK1, c-MYC, and MCL1 in HCT116, a human colorectal carcinoma cell line. While C-16661 exhibited good PK profiles in animals, it demonstrated excellent tumor growth inhibition without any tolerability issues in a colorectal cancer CDX mouse efficacy model. Citation Format: Heuijoon Park, Kyoungwan Seo, Jihyun Yu, Jieun Kim, Min Sung Joo, Sungeun Kim, Joonhyung Lee, Jeonghyeon Lee, Eun-Jung Kim, Joonwoo Nam, Wooseok Han. A selective, potent, and orally bioavailable CDK7 inhibitor demonstrates superior anti-cancer activity in colorectal cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB168.

CDK9 Inhibitor Induces Apoptosis, Autophagy, and Suppression of Tumor Growth in Adult T-Cell Leukemia/Lymphoma.

Adult T-cell leukemia/lymphoma (ATL) is a hematopoietic malignancy with a poor prognosis that develops in approximately 5% of human T-cell leukemia virus type 1 (HTLV-1) carriers. Cyclin-dependent kinase 9 (CDK9), together with Cyclin T, forms a transcription elongation factor, positive transcription elongation factor b (P-TEFb). P-TEFb promotes transcriptional elongation by phosphorylating the second serine (Ser2) of the seven amino acid repeat sequence in the C-terminal domain of RNA polymerase II (RNAP II). CDK9 inhibitors suppress cell proliferation by inducing apoptosis in chronic lymphocytic leukemia and breast cancer but there are no reports on autophagy of CDK9 inhibitors. Here, we investigated the effect of LY2857785, a novel CDK9 selective inhibitor, on cell death in ATL-related cell lines in vitro, freshly isolated cells from ATL patients ex vivo, and on ATL tumor xenografts in NOD/SCID mice in vivo. LY2857785 significantly reduced cell viability and induced apoptosis, as shown by annexin V-positive cells, cleaved poly(ADP-ribose) polymerase (PARP), and cleaved caspase-3, and suppressed the levels of anti-apoptotic protein myeloid cell leukemia-1 (MCL-1). LY2857785 decreased RNAP II Ser2 phosphorylation and downstream c-Myc protein levels. Interestingly, LY2857785 also increased microtubule-associated proteins 1A/1B light chain 3B (LC3)-II binding to autophagosome membranes. Furthermore, LY2857785 decreased the viability of freshly isolated ATL cells and induced apoptosis. Finally, LY2857785 significantly decreased the growth of ATL tumor xenografts. These results suggest that LY2857785 induces cell death of ATL cells by MCL-1-dependent apoptosis and autophagy and has anti-tumor activity.

Structure and phase separation of the C-terminal domain of RNA polymerase II.

The repetitive heptads in the C-terminal domain (CTD) of RPB1, the largest subunit of RNA Polymerase II (Pol II), play a critical role in the regulation of Pol II-based transcription. Recent findings on the structure of the CTD in the pre-initiation complex determined by cryo-EM and thenovel phase separation properties of key transcription components offers an expanded mechanistic interpretation of the spatiotemporal distribution of Pol II during transcription. Current experimental evidence further suggests an exquisite balance between CTD's local structure and an array of multivalent interactions that drive phase separation of Pol II and thus shape its transcriptional activity.

CDK12 and Integrator-PP2A complex modulates LEO1 phosphorylation for processive transcription elongation.

Cyclin-dependent kinase 12 (CDK12) interacts with cyclin K to form a functional nuclear kinase that promotes processive transcription elongation through phosphorylation of the C-terminal domain of RNA polymerase II (Pol II). To gain a comprehensive understanding of CDK12's cellular function, we used chemical genetic and phosphoproteomic screening to identify a landscape of nuclear human CDK12 substrates, including regulators of transcription, chromatin organization, and RNA splicing. We further validated LEO1, a subunit of the polymerase-associated factor 1 complex (PAF1C), as a bona fide cellular substrate of CDK12. Acute depletion of LEO1, or substituting LEO1 phosphorylation sites with alanine, attenuated PAF1C association with elongating Pol II and impaired processive transcription elongation. Moreover, we discovered that LEO1 interacts with and is dephosphorylated by the Integrator-PP2A complex (INTAC) and that INTAC depletion promotes the association of PAF1C with Pol II. Together, this study reveals an uncharacterized role for CDK12 and INTAC in regulating LEO1 phosphorylation, providing important insights into gene transcription and its regulation.

Abstract 1635: OKI-1546 is a selective, orally bioavailable inhibitor of CDK12 that causes tumor regression in multiple preclinical cancer models

Abstract Background: CDK12 regulates the expression of multiple genes in the DNA damage response (DDR) pathway by phosphorylating the C-terminal domain of RNA polymerase II (CTD pSer2), and it is a potential therapeutic target to exacerbate DNA damage and cell death in DDR-addicted, HER2-amplified, MYC-overexpressing, or EWS-FLI1-positive cancers. PARP inhibitor resistance is often driven by dysregulation of DDR signaling, and CDK12 inhibition may induce a state of DDR deficiency and increase the effectiveness of PARP inhibition. Materials and methods: Biochemical activity was assayed by ADP-glo (Promega), cellular proliferation was quantified by CellTiter-Glo (Promega), and CTD pSer2 was quantified by MSD immunoassay (Meso Scale Discovery). Kinase panels and in vivo studies are described in figure legends. Results: OKI-1546 has a biochemical IC50 of 15 nM against CDK12 and is highly selective for CDK12 and CDK13 when compared to other closely related CDKs; it is 649-, 81-, 11-, and 64-fold selective over CDKs 1, 2, 7, and 9, respectively. This selectivity was further confirmed via testing OKI-1546 against an affinity-based kinase panel. OKI-1546 potently inhibited proliferation of multiple cancer cell lines, with IC50 values that correlated with 50% inhibition of CTD pSer2. In CD-1 mice, OKI-1546 showed 77% oral bioavailability (%F) with a half-life of 2.7 hrs, and SD rats had 81 %F. Furthermore, a single 10 mg/kg dose reached plasma concentrations that surpassed the plasma protein binding-adjusted IC50 of CTD pSer2, which was confirmed in tumor-bearing mice showing 60% CTD pSer2 inhibition in tumors 4 hours after a single oral dose. That same dose translated into 119% tumor growth inhibition following 21 days of daily administration in the BRCA1 wild-type MDA-MB-231 tumor model and 121% tumor growth inhibition following 21 days of daily administration in the BRCA1-mutant MDA-MB-436 tumor model in mice. Four different PARP inhibitors synergistically combined with OKI-1546 to inhibit cell growth in multiple in vitro cancer models. Conclusion: OKI-1546 is a novel, covalent inhibitor of CDK12 and CDK13 that is potent, specific, orally available, and shows in vivo target engagement and tumor growth inhibition in preclinical cancer models. OKI-1546 showed activity in vivo as a single agent and cellular data suggest that inhibition of CDK12 has the potential to enhance the effects of other DDR-targeting therapeutic agents. Citation Format: Alexander A. Strait, David A. Mareska, Jyothi Mahadevan, Christie A. Schulte, Yevgeniy Izrayelit, Mark L. Boys, Richard Woessner. OKI-1546 is a selective, orally bioavailable inhibitor of CDK12 that causes tumor regression in multiple preclinical cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1635.

Effects of phosphorylation to the conformational space of C-terminal domain of RNA polymerase II.

Intrinsically disordered proteins (IDPs) are known as proteins without a defined secondary structure and linked with many neurological diseases. Exploring the conformational space of IDPs is one of the major challenges faced by the experimental and computational studies due to its disordered nature. Obtaining an accurate conformational space is very important to identify the structural properties of IDPs linked with these biological phenomena. RNA polymerase II (Pol II) C-terminal domain (CTD) is an intrinsically disordered low complexity domain that has a heptapeptide repeating sequence with number of repeats varies according to the organism. CTD was documented to undergo liquid-liquid phase separation, which is affected by its length and phosphorylation level. In this study, we investigated the conformational space of CTD and the effects of phosphorylation to its conformation using enhanced sampling molecular dynamics (MD) simulations. We used CTD models with two repeating units with non-phosphorylated and phosphorylated states. In addition, we study a longer synthetic CTD sequence, which has available experimental observables as a reference point to compare with the simulations. Our results suggested that phosphorylation has complex effects on the conformations of the CTD that depend on the length of the CTD, spacing between the multiple phosphorylation sites, ion coordination and interactions with the nearby residues. With this study we provide a detailed explanation of conformational changes of CTD models upon their phosphorylation.

The Phantom Mark: Enigmatic roles of phospho-Threonine 4 modification of the C-terminal domain of RNA polymerase II.

The largest subunit of RNA polymerase II (Pol II) has an unusual carboxyl-terminal domain (CTD). This domain is composed of a tandemly repeating heptapeptide, Y1 S2 P3 T4 S5 P6 S7 , that has multiple roles in regulating Pol II function and processing newly synthesized RNA. Transient phosphorylation of Ser2 and Ser5 of the YS2 PTS5 PS repeat have well-defined roles in recruiting different protein complexes and coordinating sequential steps in gene transcription. As such, these phospho-marks encipher a molecular recognition code, colloquially termed the CTD code. In contrast, the contribution of phospho-Threonine 4 (pThr4/pT4) to the CTD code remains opaque and contentious. Fuelling the debate on the relevance of this mark to gene expression are the findings that replacing Thr4 with a valine or alanine has varied impact on cellular function in different species and independent proteomic analyses disagree on the relative abundance of pThr4 marks. Yet, substitution with negatively charged residues is lethal and even benign mutations selectively disrupt synthesis and 3' processing of distinct sets of coding and non-coding transcripts. Suggestive of non-canonical roles, pThr4 marked Pol II regulates distinct gene classes in a species- and signal-responsive manner. Hinting at undiscovered roles of this elusive mark, multiple signal-responsive kinases phosphorylate Thr4 at target genes. Here, we focus on this under-explored residue and postulate that the pThr4 mark is superimposed on the canonical CTD code to selectively regulate expression of targeted genes without perturbing genome-wide transcriptional processes. This article is categorized under: RNA Processing > 3' End Processing RNA Processing > Processing of Small RNAs RNA Processing > Splicing Regulation/Alternative Splicing.

CDK8 and CDK19 act redundantly to control the CFTR pathway in the intestinal epithelium.

CDK8 and CDK19 form a conserved cyclin-dependent kinase subfamily that interacts with the essential transcription complex, Mediator, and also phosphorylates the C-terminal domain of RNA polymerase II. Cells lacking either CDK8 or CDK19 are viable and have limited transcriptional alterations, but whether the two kinases redundantly control cell proliferation and differentiation is unknown. Here, we find in mice that CDK8 is dispensable for regulation of gene expression, normal intestinal homeostasis, and efficient tumourigenesis, and is largely redundant with CDK19 in the control of gene expression. Their combined deletion in intestinal organoids reduces long-term proliferative capacity but is not lethal and allows differentiation. However, double-mutant organoids show mucus accumulation and increased secretion by goblet cells, as well as downregulation of expression of the cystic fibrosis transmembrane conductance regulator (CFTR) and functionality of the CFTR pathway. Pharmacological inhibition of CDK8/19 kinase activity in organoids and in mice recapitulates several of these phenotypes. Thus, the Mediator kinases are not essential for cell proliferation and differentiation in an adult tissue, but they cooperate to regulate specific transcriptional programmes.

Cyclin-dependent kinase inhibitor fadraciclib (CYC065) depletes anti-apoptotic protein and synergizes with venetoclax in primary chronic lymphocytic leukemia cells

Fadraciclib (CYC065) is a second-generation aminopurine CDK2/9 inhibitor with increased potency and selectivity toward CDK2 and CDK9 compared to seliciclib (R-roscovitine). In chronic lymphocytic leukemia (CLL), a disease that depends on the over-expression of anti-apoptotic proteins for its survival, inhibition of CDK9 by fadraciclib reduced phosphorylation of the C-terminal domain of RNA polymerase II and blocked transcription in vitro; these actions depleted the intrinsically short-lived anti-apoptotic protein Mcl-1 and induced apoptosis. While the simulated bone marrow and lymph node microenvironments induced Mcl-1 expression and protected CLL cells from apoptosis, these conditions did not prolong the turnover rate of Mcl-1, and fadraciclib efficiently abrogated the protective effect. Further, fadraciclib was synergistic with the Bcl-2 antagonist venetoclax, inducing more profound CLL cell death, especially in samples with 17p deletion. While fadraciclib, venetoclax, and the combination each had distinct kinetics of cell death induction, their activities were reversible, as no additional cell death was induced upon removal of the drugs. The best combination effects were achieved when both drugs were maintained together. Altogether, this study provides a rationale for the clinical development of fadraciclib in CLL, either alone or in combination with a Bcl-2 antagonist.

Abstract P5-08-05: Preclinical activity of KB-0742, an oral, highly selective, CDK9 inhibitor, in cell lines and in MYC-high expressing, patient-derived models of multiple breast cancer subtypes

Abstract KB-0742 is an orally available, potent, and selective inhibitor of cyclin-dependent kinase 9 (CDK9). Sensitivity profiling across a panel of 800 adherent and suspension immortalized pan-cancer cell lines using the Broad Institute PRISM platform had previously demonstrated MYC amplification as being a driver of KB-0742 sensitivity. We sought to further determine the activity of KB-0742 in breast cancer cell lines, patient-derived cell and organoid cultures, and patient-derived xenograft (PDX) models. KB-0742 decreased the viability of cell lines derived from primary and metastatic breast cancers, including triple-negative breast cancers (TNBCs). In a panel of 17 breast cancer cell lines, including 2 human epidermal growth factor receptor 2-positive (HER2+), 7 estrogen-receptor positive (ER+), 1 HER2+/ER+ and 7 TNBCs, the effect of KB-0742 on viability was either cytostatic or cytotoxic over the duration of the culture period. KB-0742 was also tested in patient-derived cell lines derived from primary and metastatic tumors including TNBC, and in 2 TNBC MYC-high expressing patient-derived organoid cultures. KB-0742 demonstrated cytostatic and cytotoxic effects on cell growth and superior inhibitory effects on cell growth in the TNBC organoid cultures as compared with paclitaxel and gemcitabine. Since sensitivity to KB-0742 was observed in breast cancer cell lines and patient-derived models, including TNBCs, we evaluated efficacy and target engagement in several MYC-high expressing TNBC PDX animal models. Treatment with KB-0742 using an intermittent dosing schedule (3-days on/4-days off) was well tolerated and resulted in anti-tumor activity comparable to that observed with standard of care chemotherapeutic agents. Tumors collected at 2 and 8 hours post-final dose displayed a decrease in phosphorylated serine residue 2 (pSER2) in the C-terminal domain of RNA polymerase II (RNA pol-II) and decreased MYC protein levels consistent with CDK9 inhibition. These data demonstrate the efficacy of KB-0742 in preclinical models of breast cancer and supports clinical testing in TNBC patients. KB-0742 is currently being evaluated in a phase I dose-escalation trial in patients with relapsed or refractory solid tumors or non-Hodgkin lymphoma (NCT04718675). Citation Format: Douglas C. Saffran, Melinda A.L. Day, Nathalie Rioux, Tom Chen, Christina Lee, Suha Naffar-Abu Amara, David B. Freeman, Tressa Hood, Charles Y. Lin, Pavan Kumar, Jorge DiMartino. Preclinical activity of KB-0742, an oral, highly selective, CDK9 inhibitor, in cell lines and in MYC-high expressing, patient-derived models of multiple breast cancer subtypes [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-05.

Reconstitution of 3' end processing of mammalian pre-mRNA reveals a central role of RBBP6.

The 3' ends of almost all eukaryotic mRNAs are generated in an essential two-step processing reaction: endonucleolytic cleavage of an extended precursor followed by the addition of a poly(A) tail. By reconstituting the reaction from overproduced and purified proteins, we provide a minimal list of 14 polypeptides that are essential and two that are stimulatory for RNA processing. In a reaction depending on the polyadenylation signal AAUAAA, the reconstituted system cleaves pre-mRNA at a single preferred site corresponding to the one used in vivo. Among the proteins, cleavage factor I stimulates cleavage but is not essential, consistent with its prominent role in alternative polyadenylation. RBBP6 is required, with structural data showing it to contact and presumably activate the endonuclease CPSF73 through its DWNN domain. The C-terminal domain of RNA polymerase II is dispensable. ATP, but not its hydrolysis, supports RNA cleavage by binding to the hClp1 subunit of cleavage factor II with submicromolar affinity.

CDK12 Is Necessary to Promote Epidermal Differentiation Through Transcription Elongation.

Proper differentiation of the epidermis is essential to prevent water loss and to protect the body from the outside environment. Perturbations in this process can lead to a variety of skin diseases that impacts 1 in 5 people. While transcription factors that control epidermal differentiation have been well characterized, other aspects of transcription control such as elongation are poorly understood. Here we show that of the two cyclin-dependent kinases (CDK12 and CDK13), that are known to regulate transcription elongation, only CDK12 is necessary for epidermal differentiation. Depletion of CDK12 led to loss of differentiation gene expression and absence of skin barrier formation in regenerated human epidermis. CDK12 binds to genes that code for differentiation promoting transcription factors (GRHL3, KLF4, and OVOL1) and is necessary for their elongation. CDK12 is necessary for elongation by promoting Ser2 phosphorylation on the C-terminal domain of RNA polymerase II and the stabilization of binding of the elongation factor SPT6 to target genes. Our results suggest that control of transcription elongation by CDK12 plays a prominent role in adult cell fate decisions.

Phosphorylation of RNA Polymerase II by CDKC;2 Maintains the Arabidopsis Circadian Clock Period.

The circadian clock is an internal timekeeping system that governs about 24 h biological rhythms of a broad range of developmental and metabolic activities. The clocks in eukaryotes are thought to rely on lineage-specific transcriptional–translational feedback loops. However, the mechanisms underlying the basic transcriptional regulation events for clock function have not yet been fully explored. Here, through a combination of chemical biology and genetic approaches, we demonstrate that phosphorylation of RNA polymerase II by CYCLIN DEPENDENT KINASE C; 2 (CDKC;2) is required for maintaining the circadian period in Arabidopsis. Chemical screening identified BML-259, the inhibitor of mammalian CDK2/CDK5, as a compound lengthening the circadian period of Arabidopsis. Short-term BML-259 treatment resulted in decreased expression of most clock-associated genes. Development of a chemical probe followed by affinity proteomics revealed that BML-259 binds to CDKC;2. Loss-of-function mutations of cdkc;2 caused a long period phenotype. In vitro experiments demonstrated that the CDKC;2 immunocomplex phosphorylates the C-terminal domain of RNA polymerase II, and BML-259 inhibits this phosphorylation. Collectively, this study suggests that transcriptional activity maintained by CDKC;2 is required for proper period length, which is an essential feature of the circadian clock in Arabidopsis.

Abemaciclib is a potent inhibitor of DYRK1A and HIP kinases involved in transcriptional regulation

Homeodomain-interacting protein kinases (HIPKs) belong to the CMGC kinase family and are closely related to dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs). HIPKs are regulators of various signaling pathways and involved in the pathology of cancer, chronic fibrosis, diabetes, and multiple neurodegenerative diseases. Here, we report the crystal structure of HIPK3 in its apo form at 2.5 Å resolution. Recombinant HIPKs and DYRK1A are auto-activated and phosphorylate the negative elongation factor SPT5, the transcription factor c-Myc, and the C-terminal domain of RNA polymerase II, suggesting a direct function in transcriptional regulation. Based on a database search, we identified abemaciclib, an FDA-approved Cdk4/Cdk6 inhibitor used for the treatment of metastatic breast cancer, as potent inhibitor of HIPK2, HIPK3, and DYRK1A. We determined the crystal structures of HIPK3 and DYRK1A bound to abemaciclib, showing a similar binding mode to the hinge region of the kinase as observed for Cdk6. Remarkably, DYRK1A is inhibited by abemaciclib to the same extent as Cdk4/Cdk6 in vitro, raising the question of whether targeting of DYRK1A contributes to the transcriptional inhibition and therapeutic activity of abemaciclib.

C-terminal domain phosphatase-like 1 (CPL1) is involved in floral transition in Arabidopsis

BackgroundRNA polymerase II plays critical roles in transcription in eukaryotic organisms. C-terminal Domain Phosphatase-like 1 (CPL1) regulates the phosphorylation state of the C-terminal domain of RNA polymerase II subunit B1, which is critical in determining RNA polymerase II activity. CPL1 plays an important role in miRNA biogenesis, plant growth and stress responses. Although cpl1 mutant showes delayed-flowering phenotype, the molecular mechanism behind CPL1’s role in floral transition is still unknown.ResultsTo study the role of CPL1 during the floral transition, we first tested phenotypes of cpl1-3 mutant, which harbors a point-mutation. The cpl1-3 mutant contains a G-to-A transition in the second exon, which results in an amino acid substitution from Glu to Lys (E116K). Further analyses found that the mutated amino acid (Glu) was conserved in these species. As a result, we found that the cpl1-3 mutant experienced delayed flowering under both long- and short-day conditions, and CPL1 is involved in the vernalization pathway. Transcriptome analysis identified 109 genes differentially expressed in the cpl1 mutant, with 2 being involved in floral transition. Differential expression of the two flowering-related DEGs was further validated by qRT-PCR.ConclusionsFlowering genetic pathways analysis coupled with transciptomic analysis provides potential genes related to floral transition in the cpl1-3 mutant, and a framework for future studies of the molecular mechanisms behind CPL1’s role in floral transition.

TDRD3 promotes DHX9 chromatin recruitment and R-loop resolution.

R-loops, which consist of a DNA/RNA hybrid and a displaced single-stranded DNA (ssDNA), are increasingly recognized as critical regulators of chromatin biology. R-loops are particularly enriched at gene promoters, where they play important roles in regulating gene expression. However, the molecular mechanisms that control promoter-associated R-loops remain unclear. The epigenetic ‘reader’ Tudor domain-containing protein 3 (TDRD3), which recognizes methylarginine marks on histones and on the C-terminal domain of RNA polymerase II, was previously shown to recruit DNA topoisomerase 3B (TOP3B) to relax negatively supercoiled DNA and prevent R-loop formation. Here, we further characterize the function of TDRD3 in R-loop metabolism and introduce the DExH-box helicase 9 (DHX9) as a novel interaction partner of the TDRD3/TOP3B complex. TDRD3 directly interacts with DHX9 via its Tudor domain. This interaction is important for recruiting DHX9 to target gene promoters, where it resolves R-loops in a helicase activity-dependent manner to facilitate gene expression. Additionally, TDRD3 also stimulates the helicase activity of DHX9. This stimulation relies on the OB-fold of TDRD3, which likely binds the ssDNA in the R-loop structure. Thus, DHX9 functions together with TOP3B to suppress promoter-associated R-loops. Collectively, these findings reveal new functions of TDRD3 and provide important mechanistic insights into the regulation of R-loop metabolism.

Pharmaceutical Interference of the EWS-FLI1-driven Transcriptome By Cotargeting H3K27ac and RNA Polymerase Activity in Ewing Sarcoma.

The EWSR1-FLI1 t(11;22)(q24;q12) translocation is the hallmark genomic alteration of Ewing sarcoma, a malignancy of the bone and surrounding tissue, predominantly affecting children and adolescents. Although significant progress has been made for the treatment of localized disease, patients with metastasis or who relapse after chemotherapy have less than a 30% five-year survival rate. EWS-FLI1 is currently not clinically druggable, driving the need for more effective targeted therapies. Treatment with the H3K27 demethylase inhibitor, GSK-J4, leads to an increase in H3K27me and a decrease in H3K27ac, a significant event in Ewing sarcoma because H3K27ac associates strongly with EWS-FLI1 binding at enhancers and promoters and subsequent activity of EWS-FLI1 target genes. We were able to identify targets of EWS-FLI1 tumorigenesis directly inhibited by GSK-J4. GSK-J4 disruption of EWS-FLI1-driven transcription was toxic to Ewing sarcoma cells and slowed tumor growth in patient-derived xenografts (PDX) of Ewing sarcoma. Responses were markedly exacerbated by cotreatment with a disruptor of RNA polymerase II activity, the CDK7 inhibitor THZ1. This combination together suppressed EWS-FLI1 target genes and viability of ex vivo PDX Ewing sarcoma cells in a synergistic manner. In PDX models of Ewing Sarcoma, the combination shrank tumors. We present a new therapeutic strategy to treat Ewing sarcoma by decreasing H3K27ac at EWS-FLI1-driven transcripts, exacerbated by blocking phosphorylation of the C-terminal domain of RNA polymerase II to further hinder the EWS-FLI1-driven transcriptome.

Phosphorylation of OGFOD1 by Cell Cycle-Dependent Kinase 7/9 Enhances the Transcriptional Activity of RNA Polymerase II in Breast Cancer Cells.

Simple SummaryAmong the causes of accelerating cancer properties, dysregulated transcription is considerably prominent in many cancers. However, it is difficult to target transcriptional machineries due to their fundamental importance. Compared to breast cancer cell lines, we found that OGFOD1 aggravates cancers by enhancing RNA polymerase II transcriptional activity and it is improved by cell cycle-dependent kinases. Overall, we uncovered the novel mechanism for how OGFOD1 maliciously functions in breast cancers, suggesting it as a rational cancer treatment target protein.2-oxoglutarate and iron-dependent oxygenase domain-containing protein 1 (OGFOD1) expression is upregulated in a variety of cancers and has been related to poor prognosis. However, despite this significance to cancer progression, the precise oncogenic mechanism of OGFOD1 is not understood. We demonstrated that OGFOD1 plays a role in enhancing the transcriptional activity of RNA polymerase II in breast cancer cells. OGFOD1 directly binds to the C-terminal domain of RNA polymerase II to alter phosphorylation status. The elimination of OGFOD1 resulted in decreased tumor development. Additionally, cell cycle-dependent kinase 7 and cell cycle-dependent kinase 9, critical enzymes for activating RNA polymerase II, phosphorylated serine 256 of OGFOD1, whereas a non-phosphorylated mutant OGFOD1 failed to enhance transcriptional activation and tumor growth. Consequently, OGFOD1 helps promote tumor growth by enhancing RNA polymerase II, whereas simultaneous phosphorylation of OGFOD1 by CDK enzymes is essential in stimulating RNA polymerase II-mediated transcription both in vitro and in vivo, and expression of target genes.