C-terminal Domain Of RNA Polymerase Research Articles

Abstract 2823: Identification of highly selective inhibitors of cyclin-dependent kinase 12

Abstract CDK12 and CDK13 belong to the cyclin-dependent kinase (CDK) family, which includes at least 20 different human CDKs and CDK-like enzymes. One subclass of CDKs (e.g. CDK7, CDK8, CDK9, CDK12 and CDK13) preferentially regulates transcription by phosphorylation of the C-terminal domain of RNA polymerase II (RNAPII), whereas another subgroup of CDKs plays a pivotal role in controlling cell cycle progression (e.g. CDK1, CDK2, CDK4, CDK6 and CDK7). Both groups - cell cycle as well as transcriptional CDKs - have been reported to be involved in cancer development and progression. Recent reports indicated CDK12 to be involved in DNA damage repair mechanisms and linked its mutation to high-grade serous ovarian carcinoma rendering CDK12 a promising target for drug development. However, the pivotal role of individual CDKs and their complex regulation requests for highly selective inhibitors to avoid unwanted adverse effects. Non-specific first-generation CDK-inhibitors cause toxicity in vivo issues. They showed little to no therapeutic window in clinical trials and thus shifted the focus to the optimization of selective small molecule CDK inhibitors. To identify selective small molecular weight inhibitors of CDK12 and CDK13 we employed a rationale drug discovery approach based on a focused kinase library screen. Screening of slightly over 16.000 compounds identified several hit compound classes. Medicinal chemistry based optimization yielded nanomolar CDK12 inhibitors. The selectivity of these improved CDK12 inhibitors is excellent, when tested against 12 CDK family members. Such selective CDK12 inhibitors have also shown promising physicochemical properties, indicating lead- and drug-likeness. Although, potency on the target still needs to be improved, the frontrunners from our selective CDK12 inhibitor family have shown cellular inhibition of CDK12 substrate phosphorylation such as those of RNA polymerase II. Currently, potency of these CDK12 inhibitors is being improved by using structure-guided medicinal chemistry-based optimization. The resulting lead candidates will then be tested in in vivo cancer models. Citation Format: Axel Choidas, Carsten Schultz-Fademrecht, Carsten Degeenhardt, Peter Habenberger, Uwe Koch, Jan E. Eickhoff, Ann Kathrin Greifenberg, Peter Nussbaumer, Matthias Geyer, Bert M. Klebl. Identification of highly selective inhibitors of cyclin-dependent kinase 12. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2823.

Abstract 4337: Novel potent and selective orally available CDK8/19 kinase inhibitors

Abstract CDK8 and its paralog CDK19 are cyclin-dependent kinases and together with CDK7 and CDK9 belongs to the group of C-terminal domain (CTD) kinases that phosphorylate the CTD of RNA polymerase II, thus regulating transcription. CDK8 together with its partner Cyclin C, MED12 and MED13 are components of multi-protein Mediator complex which couples action of transcription factors with the molecular machinery that carries out transcription, e.g. CDK8 couple basal transcriptional machinery to sequence-specific transcription factors such as Notch, p53, β-catenin, and also repress the transcription of other genes. As Mediator independent roles, CDK8 has been shown to act as part of a separate complex behaving as a histone kinase. Moreover, CDK8 has been identified as a major kinase in the response to IFN signaling mediated STAT1-S727 phosphorylation. Several studies indicated that high overexpression and activity of CDK8 could be a driver of malignant progression in colorectal cancer being a marker of poor prognosis. Moreover, in gastric cancer CDK8 expression and the delocalization of β-catenin expression showed a significant positive correlation with carcinogenesis and tumor progression, especially lymph node metastasis. Recently, gene amplification of CDK8, CDK19, CCNC and MED13 in breast cancers has been related with poor response to adjuvant therapy. These results suggest that CDK8 inhibitors may become a unique class of anticancer drugs that could increase the efficacy of cancer therapy by blocking chemotherapy-induced production of tumor-promoting secreted factors. We have carried out a medium throughput screening campaign which led to the discovery of several low nanomolar hits belonging to 3 different chemical series. Later on, we have performed a Hit Generation phase, designing novel inhibitors within a patentable chemical space. Here, we have taken into account information from the chemical structures of the screening hits, X-ray structure of CDK8 protein co-crystalized with sorafenib, docking studies, chemical feasibility and intellectual property. We have identified a novel chemical series of CDK8/19 inhibitors. After a HtL exploration we have reached lead compounds with potency in the picomolar range and high selectivity versus a panel of 468 protein kinases. The leads display cellular activity by blocking β−catenin reporter activity and STAT1 phoshorylation in the low nanomolar range. Lead compounds have been screened in a panel of tumoral cell lines, showing a defined profile in terms of sensitivity with GI50s which range from nanomolar to low micromolar values. These inhibitors induce cell death in a dose response manner. The novel chemical series show a drug-like profile in terms of solubility, permeability, CYP450 inhibition and hERG. The identified lead compounds are orally bioavailable, with good clearance, volume of distribution and exposure levels. A selected lead compound has been used in in vivo PK-PD studies showing positive results. Citation Format: Carmen Blanco Aparicio, Oliver Renner, Elena Gomez-Casero, Maria Isabel Albarran, Antonio Cebria, Borja Barrera, Enara Aguirre, Maria del Carmen Rodriguez de Miguel, Manuel Urbano, Ana Isabel Hernandez, Cristina Gomez de la Oliva, Virginia Rivero, Rosario Concepcion Riesco, Sonia Martinez Gonzalez, Joaquin Pastor. Novel potent and selective orally available CDK8/19 kinase inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4337.

Distributive O-GlcNAcylation on the Highly Repetitive C-Terminal Domain of RNA Polymerase II.

O-GlcNAcylation is a nutrient-responsive glycosylation that plays a pivotal role in transcriptional regulation. Human RNA polymerase II (Pol II) is extensively modified by O-linked N-acetylglucosamine (O-GlcNAc) on its unique C-terminal domain (CTD), which consists of 52 heptad repeats. One approach to understanding the function of glycosylated Pol II is to determine the mechanism of dynamic O-GlcNAcylation on the CTD. Here, we discovered that the Pol II CTD can be extensively O-GlcNAcylated in vitro and in cells. Efficient glycosylation requires a minimum of 20 heptad repeats of the CTD and more than half of the N-terminal domain of O-GlcNAc transferase (OGT). Under conditions of saturated sugar donor, we monitored the attachment of more than 20 residues of O-GlcNAc to the full-length CTD. Surprisingly, glycosylation on the periodic CTD follows a distributive mechanism, resulting in highly heterogeneous glycoforms. Our data suggest that initial O-GlcNAcylation can take place either on the proximal or on the distal region of the CTD, and subsequent glycosylation occurs similarly over the entire CTD with nonuniform distributions. Moreover, removal of O-GlcNAc from glycosylated CTD is also distributive and is independent of O-GlcNAcylation level. Our results suggest that O-GlcNAc cycling enzymes can employ a similar mechanism to react with other protein substrates on multiple sites. Distributive O-GlcNAcylation on Pol II provides another regulatory mechanism of transcription in response to fluctuating cellular conditions.

CDK9 and its repressor LARP7 modulate cardiomyocyte proliferation and response to injury in the zebrafish heart

Cyclin dependent kinase (Cdk)9 acts through the positive transcription elongation factor-b (P-TEFb) complex to activate and expand transcription through RNA polymerase II. It has also been shown to regulate cardiomyocyte hypertrophy, with recent evidence linking it to cardiomyocyte proliferation. We hypothesised that modification of CDK9 activity could both impair and enhance the cardiac response to injury by modifying cardiomyocyte proliferation. Cdk9 expression and activity were inhibited in the zebrafish (Danio rerio) embryo. We show that dephosphorylation of residue Ser2 on the C-terminal domain of RNA polymerase II is associated with impaired cardiac structure and function, and cardiomyocyte proliferation and also results in impaired functional recovery following cardiac laser injury. In contrast, de-repression of Cdk9 activity, through knockdown of La-related protein (Larp7) increases phosphorylation of Ser2 in RNA polymerase II and increases cardiomyocyte proliferation. Larp7 knockdown rescued the structural and functional phenotype associated with knockdown of Cdk9. The balance of Cdk9 and Larp7 plays a key role in cardiomyocyte proliferation and response to injury. Larp7 represents a potentially novel therapeutic target to promote cardiomyocyte proliferation and recovery from injury.

Direct Analysis of Phosphorylation Sites on the Rpb1 C-Terminal Domain of RNA Polymerase II

Dynamic interactions between RNA polymerase II and various mRNA-processing and chromatin-modifying enzymes are mediated by the changing phosphorylation pattern on the C-terminal domain (CTD) of polymerase subunit Rpb1 during different stages of transcription. Phosphorylations within the repetitive heptamer sequence (YSPTSPS) of CTD have primarily been defined using antibodies, but these do not distinguish different repeats or allow comparative quantitation. Using a CTD modified for mass spectrometry (msCTD), we show that Ser5-P and Ser2-P occur throughout the length of CTD and are far more abundant than other phosphorylation sites. msCTD extracted from cells mutated in several CTD kinases or phosphatases showed the expected changes in phosphorylation. Furthermore, msCTD associated with capping enzyme was enriched for Ser5-P while that bound to the transcription termination factor Rtt103 had higher levels of Ser2-P. These results suggest a relatively sparse and simple "CTD code."

Targeting Transcriptional Dependency in Acute Myeloid Leukemia (AML) with a Covalent Inhibitor of Transcriptional Kinase CDK7

Highly aggressive acute myeloid leukemia (AML) is the most common cause of leukemia-related deaths in the United States. The development of new treatments for acute leukemias that would increase durable responses and improve poor survival rates with manageable toxicity is imperative. Certain cancers, including acute lymphoblastic leukemia (ALL), are dependent on oncogenic transcription factors and are particularly sensitive to selective CDK7 inhibitors (Kwiatkowski et al., 2014; Chipumuro et al., 2014; Christensen 2014).AML tumors harbor fewer somatic mutations compared to solid tumors and many of these mutations affect proteins critical to gene control, potentially conferring a unique vulnerability to the inhibition of transcriptional processes. CDK7 is a transcriptional kinase that phosphorylates the C-terminal domain of RNA polymerase II, enabling processive transcription. CDK7 has previously been implicated in the regulation of super-enhancer (SE) mediated circuitry.Here, we report preclinical in vitro and in vivo data in AML with a first-in-class CDK7 inhibitor, SY-351, that covalently targets a cysteine outside the kinase domain, resulting in sustained, highly selective inhibition. SY-351 shows potent inhibition of CDK7 in biochemical (Ki = 62 nM and Kinact = 11.3 hr-1) and cell-based (EC50 = 4.7 nM HL-60) assays and, when profiled against nearly 400 other kinases, exhibits limited off-target inhibition. In in vitro studies, potent and selective inhibition of CDK7 with SY-351 results in rapid induction of apoptosis in AML cell lines, while inducing little to no cell death in normal immortalized cell lines at comparable levels of target engagement.In vivo, CDK7 inhibition did not show myelosupression that has previously limited the clinical development of pan CDK inhibitors. Orthotopic AML and ALL patient-derived xenotransplants (PDX), treated with SY-351 results in complete responses (CR) where the clearance of blasts from blood and marrow was achieved without body weight changes or cell death in normal hematopoietic cells. These effects led to increased survival in mouse PDX models, and the response was sustained following removal of the drug.In summary, we describe a first-in-class CDK7 inhibitor that is potent and selective, well tolerated at efficacious doses and that leads to durable, complete responses in PDX models of AML and ALL. These data support the further exploration of this class of compounds for clinical development. DisclosuresRen:Syros Pharmaceuticals: Employment, Equity Ownership. Brown:Syros Pharmaceuticals: Employment, Equity Ownership. Hu:Syros Pharmaceuticals: Employment, Equity Ownership. Lopez:Syros Pharmaceuticals: Employment, Equity Ownership. Miljovska:Syros Pharmaceuticals: Employment, Equity Ownership. Schmidt:Syros Pharmaceuticals: Employment, Equity Ownership. Bradley:Syros Pharmaceuticals: Employment, Equity Ownership. Sprott:Sprott Biotech LLC: Other: Spouse Kam M. Sprott is a consultant; Syros Pharmaceuticals: Employment, Equity Ownership. Olson:Syros Pharmaceuticals: Employment, Equity Ownership. Fritz:Syros Pharmaceuticals: Employment, Equity Ownership. Choi:Syros Pharmaceuticals: Employment, Equity Ownership.

Residue-by-Residue View of In Vitro FUS Granules that Bind the C-Terminal Domain of RNA Polymerase II

Phase-separated states of proteins underlie ribonucleoprotein (RNP) granules and nuclear RNA-binding protein assemblies that may nucleate protein inclusions associated with neurodegenerative diseases. We report that the N-terminal low-complexity domain of the RNA-binding protein Fused in Sarcoma (FUS LC) is structurally disordered and forms a liquid-like phase-separated state resembling RNP granules. This state directly binds the C-terminal domain of RNA polymerase II. Phase-separated FUS lacks static structures as probed by fluorescence microscopy, indicating they are distinct from both protein inclusions and hydrogels. We use solution nuclear magnetic resonance spectroscopy to directly probe the dynamic architecture within FUS liquid phase-separated assemblies. Importantly, we find that FUS LC retains disordered secondary structure even in the liquid phase-separated state. Therefore, we propose that disordered protein granules, even those made of aggregation-prone prion-like domains, are dynamic and disordered molecular assemblies with transiently formed protein-protein contacts.

Abstract AS21: Ovarian carcinoma CDK12 mutations misregulate DNA repair genes via deficient formation and function of the Cdk12/CycK complex

Abstract The Cdk12/CycK complex promotes gene expression by phosphorylating the C-terminal domain of RNA polymerase II. In previous work we found that CDK12/CycK complex maintains genomic stability via regulation of expression of several key DNA damage response genes such as BRCA1, ATR, FANCD2 or FANCI. CDK12 is among only nine genes with recurrent somatic mutations in high-grade serous ovarian carcinoma. However, the influence of these mutations on the Cdk12/CycK complex and their link to cancerogenesis remain ill-defined. Here, we show that most mutations interfere with the Cdk12/CycK complex formation, rendering the kinase inactive. By examining the mutations within the Cdk12/CycK structure, we find that they likely provoke structural rearrangements detrimental to Cdk12 activation. Our mRNA expression analysis of the patient samples containing the CDK12 mutations identifies coordinated down-regulation of key DNA damage response genes. Accordingly, we observed that the mutant Cdk12 proteins fail to promote the repair of DNA double strand breaks via homologous recombination. Together, we provide the molecular basis of how mutated CDK12 ceases to function in ovarian carcinoma. We propose that CDK12 is a tumor suppressor of which the loss-of-function mutations may elicit defects in multiple DNA repair pathways, leading to genomic instability that underlies the genesis of the cancer.. Citation Format: Kingsley M. Ekumi, Hana Paculova,Vendula Pospichalova, Christian A. Bosken,Vitezslav Bryja, Matthias Geyer, Dalibor Blazek, Matjaz Barboric. Ovarian carcinoma CDK12 mutations misregulate DNA repair genes via deficient formation and function of the Cdk12/CycK complex [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr AS21.

The Emerging Role for RNA Polymerase II in Regulating Virulence Gene Expression in Malaria Parasites.

Plasmodium falciparum causes the most severe form of human malaria and is responsible for a significant public health burden in the developing world. These protozoan parasites invade circulating red blood cells (RBCs) and maintain prolonged infections through an intricate gene-expression switching mechanism that enables immune evasion through antigenic variation [1]. One of the families of genes responsible for this evasion is called var; it is made up of ~60 members, of which approximately two-thirds are found located within subtelomeric heterochromatic regions of the P. falciparum genome, with the remaining third of the family arranged in clusters located in similarly heterochromatinized areas within the internal regions of the chromosomes. The var gene family encodes P. falciparum erythrocyte membrane protein 1 (PfEMP1), a protein displayed on the surface of infected RBCs and considered the primary antigenic determinant required for cytoadherence and sequestration of infected cells, thus enabling them to avoid circulation through the spleen. Only one var gene is expressed at a time, while the other 59 remain transcriptionally silent, and which gene is active switches over the course of an infection. This process allows the parasites to maintain chronic infections through an ever-changing display of PfEMP1 antigens to the immune system. Structurally, each var gene consists of two exons flanking a single, conserved intron with a bidirectional promoter that transcribes noncoding RNAs [2–4]. Although an understanding of the mechanisms that lead to coordinated switching within the var gene family have remained elusive, it has become clear in recent years that epigenetic components, particularly histone modifications, play major roles in determining whether an individual gene will be active or silent. Many of the histone modifications involved in antigenic variation, as well as other aspects of parasite development, have been catalogued. For example, trimethylation of histone H3 at lysine 4 (H3K4me3) denotes transcriptionally active genes [5], including the single active var gene. In contrast, H3K9me3 marks the 59 silent var genes [6,7]. Interestingly, H3K9me3 and another mark, H3K36me3, are very limited in their distribution throughout the genome and are found primarily at gene families that encode variant antigens like var [5,8]. How the enzymes that deposit these marks are recruited to very limited regions of the genome is poorly understood. This short review aims to expand on recent work that sheds light on the recruitment of histone modifiers to narrow regions of chromosomes, specifically var genes, by way of the C-terminal domain of RNA polymerase II (RNA pol II CTD).

Writing and Reading the CTD Code: In Vitro Reconstruction of Post‐Translational Modifications on RNA Polymerase II

Information flow within the cell is filtered through a vast array of codes. Vital biological data is compiled at the DNA, RNA, and protein levels with additional information relayed through an ever‐expanding repertoire of post‐transcriptional and post‐translational modifications (PTM). The PTM state of proteins can impact their recognition, structure, and function as well as relay important signals to influence cellular events, as found in histone code. A newly characterized coding system in eukaryotic transcription is “CTD code.” The C‐terminal Domain of RNA Polymerase II (CTD) is a unique structure composed of repeats of the consensus heptad YSPTSPS. These residues are subject to a variety of PTMs and specific factors are recruited depending on the PTM state of CTD. Particular PTM marks are linked to transcriptional events including initiation, elongation, and termination. The PTM state of CTD is generated and altered through the action of PTM enzymes such as kinases and phosphatases. Current technologies allow the determination of isolated and simplistic PTM marks along the CTD but lack the potential for the investigation of a combinatorial code or the identification of novel PTM marks. To overcome this we propose a bottom up approach to in vitro reconstruct the CTD code. We treat CTD substrate with PTM enzymes and characterize resultant modifications using a combination of molecular biology and mass spectrometry. Our novel mass spectrometry methodologies allow for heptad level resolution and characterization of individual and combinatorial PTM marks along CTD. Using this interdisciplinary cipher we can determine the lexicon for CTD code and better understand the impact of PTM on cellular communication.

CTD Code: a Combinatorial Code for Eukaryotic Transcription

In eukaryotes, the C‐terminal domain of RNA polymerase II (CTD) orchestrates the temporal and spatial control of transcription and is involved in the epigenetic regulation of gene expression. Errors in CTD regulation can result in cell death, cancer and severe developmental defects. The CTD executes its function as transcription template through various post‐translational modifications on its heptad repeat sequences. Recently, novel modifications on new regulatory sites of CTD have been identified, setting the stage for the possibility of combinatorial mechanisms for transcription regulation. We focused on two well‐characterized modification of CTD, namely serine phosphorylation and prolyl isomerization, and discuss the interplay between the enzymes regulating these modification states. To address this question, we developed chemical compounds that can be used to mimic the cis and trans isomer states of proline residues in the CTD and therefore probe the selectivity of CTD binding proteins that are sensitive for this subtle variation. Our results established that the selectivity of prolyl isomerization state of CTD by phosphatases results in different regulatory effects and can lead to differentiated CTD phosphorylation states and eventually, transcription outcome. In particular, the phosphatases that specially recognize the minor species of cis proline will display enhanced activity upon prolyl isomerase regulation which is essential for accurate transcription termination. The application of such chemical probes can help us understand the molecular mechanism of the interplay between different post‐translational modifications with prolyl isomerization state and how that affects the conformational status of CTD in transcription.

Visualization of Positive Transcription Elongation Factor b (P-TEFb) Activation in Living Cells

Regulation of transcription elongation by positive transcription elongation factor b (P-TEFb) plays a central role in determining the state of cell activation, proliferation, and differentiation. In cells, P-TEFb exists in active and inactive forms. Its release from the inactive 7SK small nuclear ribonucleoprotein complex is a critical step for P-TEFb to activate transcription elongation. However, no good method exists to analyze this P-TEFb equilibrium in living cells. Only inaccurate and labor-intensive cell-free biochemical assays are currently available. In this study, we present the first experimental system to monitor P-TEFb activation in living cells. We created a bimolecular fluorescence complementation assay to detect interactions between P-TEFb and its substrate, the C-terminal domain of RNA polymerase II. When cells were treated with suberoylanilide hydroxamic acid, which releases P-TEFb from the 7SK small nuclear ribonucleoprotein, they turned green. Other known P-TEFb-releasing agents, including histone deacetylase inhibitors, bromodomain and extraterminal bromodomain inhibitors, and protein kinase C agonists, also scored positive in this assay. Finally, we identified 5'-azacytidine as a new P-TEFb-releasing agent. This release of P-TEFb correlated directly with activation of human HIV and HEXIM1 transcription. Thus, our visualization of P-TEFb activation by fluorescent complementation assay could be used to find new P-TEFb-releasing agents, compare different classes of agents, and assess their efficacy singly and/or in combination.

Expanding the SRI domain family: A common scaffold for binding the phosphorylated C-terminal domain of RNA polymerase II

The SRI domain is a small three-helix domain originally discovered near the C-terminus of both histone methyltransferase SETD2 and helicase RECQL5. The SRI domain binds to the C-terminal repeat domain of the largest subunit of RNA polymerase II, allowing SETD2 and RECQL5 to regulate various mechanisms associated with RNA transcription. Using original tools to detect common patterns in distantly related sequences, we have identified SRI domains in several additional proteins, most of which are involved in RNA metabolism. Combining sequence analysis with structural prediction, we show that this domain family is more diverse than previously thought and we predict critical structural and functional features.

Characterization of native protein complexes using ultraviolet photodissociation mass spectrometry.

Ultraviolet photodissociation (UVPD) mass spectrometry (MS) was used to characterize the sequences of proteins in native protein-ligand and protein-protein complexes and to provide auxiliary information about the binding sites of the ligands and protein-protein interfaces. UVPD outperformed collisional induced dissociation (CID), higher-energy collisional dissociation (HCD), and electron transfer dissociation (ETD) in terms of yielding the most comprehensive diagnostic primary sequence information about the proteins in the complexes. UVPD also generated noncovalent fragment ions containing a portion of the protein still bound to the ligand which revealed some insight into the nature of the binding sites of myoglobin/heme, eIF4E/m(7)GTP, and human peptidyl-prolyl cis-trans isomerase 1 (Pin1) in complex with the peptide derived from the C-terminal domain of RNA polymerase II (CTD). Noncovalently bound protein-protein fragment ions from oligomeric β-lactoglobulin dimers and hexameric insulin complexes were also produced upon UVPD, providing some illumination of tertiary and quaternary protein structural features.

Arabidopsis CPL4 is an essential C-terminal domain phosphatase that suppresses xenobiotic stress responses.

Eukaryotic gene expression is both promoted and inhibited by the reversible phosphorylation of the C-terminal domain of RNA polymerase II (pol II CTD). More than 20 Arabidopsis genes encode CTD phosphatase homologs, including four CTD phosphatase-like (CPL) family members. Although in vitro CTD phosphatase activity has been established for some CPLs, none have been shown to be involved in the phosphoregulation of pol II in vivo. Here we report that CPL4 is a CTD phosphatase essential for the viability of Arabidopsis thaliana. Mass spectrometry analysis identified the pol II subunits RPB1, RPB2 and RPB3 in the affinity-purified CPL4 complex. CPL4 dephosphorylates both Ser2- and Ser5-PO(4) of the CTD in vitro, with a preference for Ser2-PO(4). Arabidopsis plants overexpressing CPL4 accumulated hypophosphorylated pol II, whereas RNA interference-mediated silencing of CPL4 promoted hyperphosphorylation of pol II. A D128A mutation in the conserved DXDXT motif of the CPL4 catalytic domain resulted in a dominant negative form of CPL4, the overexpression of which inhibited transgene expression in transient assays. Inhibition was abolished by truncation of the phosphoprotein-binding Breast Cancer 1 C-terminal domain of CPL4, suggesting that both catalytic function and protein-protein interaction are essential for CPL4-mediated regulation of gene expression. We were unable to recover a homozygous cpl4 mutant, probably due to the zygotic lethality of this mutation. The reduction in CPL4 levels in CPL4(RNAi) plants increased transcript levels of a suite of herbicide/xenobiotic-responsive genes and improved herbicide tolerance, thus suggesting an additional role for CPL4 as a negative regulator of the xenobiotic detoxification pathway.

CTD Code: a Combinatorial Code for Eukaryotic Transcription

In eukaryotes, the C-terminal domain of RNA polymerase II (CTD) orchestrates the temporal and spatial control of transcription and is involved in the epigenetic regulation of gene expression. Errors in CTD regulation can result in cell death, cancer and severe developmental defects. The CTD executes its function as transcription modulator through various post-translational modifications on its heptad repeat sequences. Recently, novel modifications on new regulatory sites of CTD have been identified, setting the stage for the possibility of combinatorial mechanisms for transcription regulation. We focused on two well-characterized modification of CTD, namely serine phosphorylation and prolyl isomerization, and discuss the interplay between the enzymes regulating these modification states. Our results established that the selectivity of prolyl isomerization state of CTD on phosphatases can lead to differentiated outcome for the CTD phosphorylation state and therefore, transcription. To further investigate the prolyl selectivity, we developed chemical compounds that can be used to probe such subtle structural variation in the CTD binding proteins. These compounds closely mimic the cis or trans proline state and can be effectively recognized by CTD phosphatases. The application of such chemical probes can help us understand the molecular mechanism of the interplay between phosphorylation and prolyl isomerization state and how that affect the conformational status of CTD in transcription temporally and spatially.

Molecular Dynamics Study of the Phosphorylation Effect on the Conformational States of the C-Terminal Domain of RNA Polymerase II

The carboxyl-terminal domain (CTD) of RNA polymerase II in eukaryotes regulates mRNA processing processes by recruiting various regulation factors. A main function of the CTD relies on the heptad consensus sequence (YSPTSPS). The CTD dynamically changes its conformational state to recognize and bind different regulation factors. The dynamical conformation changes are caused by modifications, mainly phosphorylation and dephosphorylation, to the serine residues. In this study, we investigate the conformational states of the unit consensus CTD peptide with various phosphorylation patterns of the serine residues by extended ensemble simulations. The results show that the CTD without phosphorylation has a flexible disordered structure distributed between twisted and extended states, but phosphorylation tends to reduce the conformational space. It was found that phosphorylation induces a β-turn around the phosphorylated serine residue and the cis conformation of the proline residue significantly inhibits the β-turn formation. The β-turn should contribute to specific CTD binding of the different regulation factors by changing the conformation propensity combined with induced fit.

Release of Positive Transcription Elongation Factor b (P-TEFb) from 7SK Small Nuclear Ribonucleoprotein (snRNP) Activates Hexamethylene Bisacetamide-inducible Protein (HEXIM1) Transcription

By phosphorylating negative elongation factors and the C-terminal domain of RNA polymerase II (RNAPII), positive transcription elongation factor b (P-TEFb), which is composed of CycT1 or CycT2 and CDK9, activates eukaryotic transcription elongation. In growing cells, it is found in active and inactive forms. In the former, free P-TEFb is a potent transcriptional coactivator. In the latter, it is inhibited by HEXIM1 or HEXIM2 in the 7SK small nuclear ribonucleoprotein (snRNP), which contains, additionally, 7SK snRNA, methyl phosphate-capping enzyme (MePCE), and La-related protein 7 (LARP7). This P-TEFb equilibrium determines the state of growth and proliferation of the cell. In this study, the release of P-TEFb from the 7SK snRNP led to increased synthesis of HEXIM1 but not HEXIM2 in HeLa cells, and this occurred only from an unannotated, proximal promoter. ChIP with sequencing revealed P-TEFb-sensitive poised RNA polymerase II at this proximal but not the previously annotated distal HEXIM1 promoter. Its immediate upstream sequences were fused to luciferase reporters and were found to be responsive to many P-TEFb-releasing compounds. The superelongation complex subunits AF4/FMR2 family member 4 (AFF4) and elongation factor RNA polymerase II 2 (ELL2) were recruited to this proximal promoter after P-TEFb release and were required for its transcriptional effects. Thus, P-TEFb regulates its own equilibrium in cells, most likely to maintain optimal cellular homeostasis.

Inhibition of Post-Transcriptional RNA Processing by CDK Inhibitors and Its Implication in Anti-Viral Therapy

Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle and RNA polymerase II mediated transcription. Several pharmacological CDK inhibitors are currently in clinical trials as potential cancer therapeutics and some of them also exhibit antiviral effects. Olomoucine II and roscovitine, purine-based inhibitors of CDKs, were described as effective antiviral agents that inhibit replication of a broad range of wild type human viruses. Olomoucine II and roscovitine show high selectivity for CDK7 and CDK9, with important functions in the regulation of RNA polymerase II transcription. RNA polymerase II is necessary for viral transcription and following replication in cells. We analyzed the effect of inhibition of CDKs by olomoucine II on gene expression from viral promoters and compared its effect to widely-used roscovitine. We found that both roscovitine and olomoucine II blocked the phosphorylation of RNA polymerase II C-terminal domain. However the repression of genes regulated by viral promoters was strongly dependent on gene localization. Both roscovitine and olomoucine II inhibited expression only when the viral promoter was not integrated into chromosomal DNA. In contrast, treatment of cells with genome-integrated viral promoters increased their expression even though there was decreased phosphorylation of the C-terminal domain of RNA polymerase II. To define the mechanism responsible for decreased gene expression after pharmacological CDK inhibitor treatment, the level of mRNA transcription from extrachromosomal DNA was determined. Interestingly, our results showed that inhibition of RNA polymerase II C-terminal domain phosphorylation increased the number of transcribed mRNAs. However, some of these mRNAs were truncated and lacked polyadenylation, which resulted in decreased translation. These results suggest that phosphorylation of RNA polymerase II C-terminal domain is critical for linking transcription and posttrancriptional processing of mRNA expressed from extrachromosomal DNA.

Cross-Talk of Phosphorylation and Prolyl Isomerization of the C-terminal Domain of RNA Polymerase II

Post-translational modifications of the heptad repeat sequences in the C-terminal domain (CTD) of RNA polymerase II (Pol II) are well recognized for their roles in coordinating transcription with other nuclear processes that impinge upon transcription by the Pol II machinery; and this is primarily achieved through CTD interactions with the various nuclear factors. The identification of novel modifications on new regulatory sites of the CTD suggests that, instead of an independent action for all modifications on CTD, a combinatorial effect is in operation. In this review we focus on two well-characterized modifications of the CTD, namely serine phosphorylation and prolyl isomerization, and discuss the complex interplay between the enzymes modifying their respective regulatory sites. We summarize the current understanding of how the prolyl isomerization state of the CTD dictates the specificity of writers (CTD kinases), erasers (CTD phosphatases) and readers (CTD binding proteins) and how that correlates to transcription status. Subtle changes in prolyl isomerization states cannot be detected at the primary sequence level, we describe the methods that have been utilized to investigate this mode of regulation. Finally, a general model of how prolyl isomerization regulates the phosphorylation state of CTD, and therefore transcription-coupled processes, is proposed.