Dependent Transcriptional Activity Research Articles

An IDR-dependent mechanism for nuclear receptor control of Mediator interaction with RNA polymerase II

The essential Mediator (MED) coactivator complex plays a well-understood role in regulation of basal transcription in all eukaryotes, but the mechanism underlying its role in activator-dependent transcription remains unknown. We investigated modulation of metazoan MED interaction with RNA polymerase II (RNA Pol II) by antagonistic effects of the MED26 subunit and the CDK8 kinase module (CKM). Biochemical analysis of CKM-MED showed that the CKM blocks binding of the RNA Pol II carboxy-terminal domain (CTD), preventing RNA Pol II interaction. This restriction is eliminated by nuclear receptor (NR) binding to CKM-MED, which enables CTD binding in a MED26-dependent manner. Cryoelectron microscopy (cryo-EM) and crosslinking-mass spectrometry (XL-MS) revealed that the structural basis for modulation of CTD interaction with MED relates to a large intrinsically disordered region (IDR) in CKM subunit MED13 that blocks MED26 and CTD interaction with MED but is repositioned upon NR binding. Hence, NRs can control transcription initiation by priming CKM-MED for MED26-dependent RNA Pol II interaction.

Regulation of RHOV signaling by interaction with SH3 domain-containing adaptor proteins and phosphorylation by PKA

RHOV and RHOU are considered atypical Rho-family small GTPases because of the existence of N- and C-terminal extension regions, abnormal GDP/GTP cycling, and post-translational modification. Particularly, RHOV and RHOU both have a proline-rich (PR) motif in the N-terminal region. It has been reported that the PR motif of RHOU interacts with GRB2, a SH3 domain-containing adaptor protein, and regulates its activity through EGF receptor signaling. However, it is unknown whether RHOV, like RHOU, interacts with SH3 domain-containing adaptor proteins. In this study, we investigated the interactions between RHOV and SH3 domain-containing adaptor proteins, including GRB2 and NCK2. The RHOV-induced serum response factor (SRF)-dependent gene transcriptional activity was attenuated in cells co-expressing either GRB2 or NCK2 compared to cells expressing RHOV alone. From the results of experiments using various gene mutants of RHOV and GRB2, it appears that the PR motif of the N-terminal region of RHOV is the crucial binding site for the SH3 domain-containing proteins. Furthermore, we found that Ser25 in the N-terminal region of RHOV is phosphorylated by PKA and that its phosphorylation is suppressed by interaction with NCK2 but not GRB2. We have found a novel regulatory mechanism for the phosphorylation of RHOV and its interaction with SH3 domain-containing adaptor proteins.

Hypoxia-driven deSUMOylation of EXOSC10 promotes adaptive changes in the transcriptome profile

Reduced oxygen availability (hypoxia) triggers adaptive cellular responses via hypoxia-inducible factor (HIF)-dependent transcriptional activation. Adaptation to hypoxia also involves transcription-independent processes like post-translational modifications; however, these mechanisms are poorly characterized. Investigating the involvement of protein SUMOylation in response to hypoxia, we discovered that hypoxia strongly decreases the SUMOylation of Exosome subunit 10 (EXOSC10), the catalytic subunit of the RNA exosome, in an HIF-independent manner. EXOSC10 is a multifunctional exoribonuclease enriched in the nucleolus that mediates the processing and degradation of various RNA species. We demonstrate that the ubiquitin-specific protease 36 (USP36) SUMOylates EXOSC10 and we reveal SUMO1/sentrin-specific peptidase 3 (SENP3) as the enzyme-mediating deSUMOylation of EXOSC10. Under hypoxia, EXOSC10 dissociates from USP36 and translocates from the nucleolus to the nucleoplasm concomitant with its deSUMOylation. Loss of EXOSC10 SUMOylation does not detectably affect rRNA maturation but affects the mRNA transcriptome by modulating the expression levels of hypoxia-related genes. Our data suggest that dynamic modulation of EXOSC10 SUMOylation and localization under hypoxia regulates the RNA degradation machinery to facilitate cellular adaptation to low oxygen conditions.

Abstract LB279: Targeting microglial metabolic rewiring synergizes with immune checkpoint blockade therapy for glioblastoma

Abstract Glioblastoma (GBM) constitutes the most lethal primary brain tumor for which immunotherapy has provided limited benefit. The unique brain immune landscape is reflected in a complex tumor immune microenvironment (TIME) in GBM. Here, single cell sequencing of the GBM TIME revealed that microglia were under severe oxidative stress, which induced nuclear receptor subfamily 4 group A member 2 (NR4A2)-dependent transcriptional activity in microglia. Heterozygous Nr4a2 (Nr4a2 +/−) or microglia-specific Nr4a2 (Nr4a2 fl/fl Cx3cr1 cre) genetic targeting reshaped microglia plasticity in vivo by reducing alternatively activated microglia and enhancing antigen presentation capacity for CD8+ T cells in GBM. In microglia, NR4A2 activated squalene monooxygenase (SQLE) to dysregulate cholesterol homeostasis. Pharmacological NR4A2 inhibition attenuated the pro-tumorigenic TIME, and targeting the NR4A2 or SQLE enhanced therapeutic efficacy of immune checkpoint blockade in vivo. Collectively, oxidative stress promotes tumor growth through NR4A2-SQLE activity in microglia, informing novel immune therapy paradigms in brain cancer. Citation Format: Zengpanpan Ye, Xiaolin Ai, Linjie Zhao, Jeremy N. Rich, Shengtao Zhou. Targeting microglial metabolic rewiring synergizes with immune checkpoint blockade therapy for glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB279.

Toxic effect and inability of L-homoserine to be a nitrogen source for growth of Escherichia coli resolved by a combination of in vivo evolution engineering and omics analyses.

L-homoserine is a pivotal intermediate in the carbon and nitrogen metabolism of E. coli. However, this non-canonical amino acid cannot be used as a nitrogen source for growth. Furthermore, growth of this bacterium in a synthetic media is potently inhibited by L-homoserine. To understand this dual effect, an adapted laboratory evolution (ALE) was applied, which allowed the isolation of a strain able to grow with L-homoserine as the nitrogen source and was, at the same time, desensitized to growth inhibition by this amino acid. Sequencing of this evolved strain identified only four genomic modifications, including a 49 bp truncation starting from the stop codon of thrL. This mutation resulted in a modified thrL locus carrying a thrL* allele encoding a polypeptide 9 amino acids longer than the thrL encoded leader peptide. Remarkably, the replacement of thrL with thrL* in the original strain MG1655 alleviated L-homoserine inhibition to the same extent as strain 4E, but did not allow growth with this amino acid as a nitrogen source. The loss of L-homoserine toxic effect could be explained by the rapid conversion of L-homoserine into threonine via the thrL*-dependent transcriptional activation of the threonine operon thrABC. On the other hand, the growth of E. coli on a mineral medium with L-homoserine required an activation of the threonine degradation pathway II and glycine cleavage system, resulting in the release of ammonium ions that were likely recaptured by NAD(P)-dependent glutamate dehydrogenase. To infer about the direct molecular targets of L-homoserine toxicity, a transcriptomic analysis of wild-type MG1655 in the presence of 10 mM L-homoserine was performed, which notably identified a potent repression of locomotion-motility-chemotaxis process and of branched-chain amino acids synthesis. Since the magnitude of these effects was lower in a ΔthrL mutant, concomitant with a twofold lower sensitivity of this mutant to L-homoserine, it could be argued that growth inhibition by L-homoserine is due to the repression of these biological processes. In addition, L-homoserine induced a strong upregulation of genes in the sulfate reductive assimilation pathway, including those encoding its transport. How this non-canonical amino acid triggers these transcriptomic changes is discussed.

Abstract 957: Cadherin-11 contributes to the heterogenous and dynamic Wnt-β-catenin pathway activation in Ewing sarcoma

Abstract Introduction: Metastatic Ewing sarcoma (ES) has a dismal prognosis, and clarifying the molecular and cellular mechanism of metastasis is the most pressing need to improving prognosis. Recently, the heterogenous expression of the driving fusion gene EWS-FLI1, as well as canonical Wnt/β-catenin signaling activity has been increasingly recognized as a potential mediator of metastatic progression. However, targeting the pathway has been difficult due to its complexity and cross-activity, and not much is known of the mechanism of how ES cells maintain Wnt signaling heterogeneity. Cadherin-11 (CDH11) is a member of the cadherin super-family, which modulates calcium ion-dependent cell-cell adhesion and signal transduction, and is highly expressed in ES. CDH11 has been found to regulate the expression level of β-catenin, and play a key role in enhancing the stem cell, migration and invasion in other carcinomas, however the relationship between CDH11 and β-catenin in ES is not clear. Methods: Wnt/β-catenin dependent transcriptional activity of ES cells in vitro and in vivo were assessed by flow cytometry, immune fluorescence, as well as single cell RNA-seq. β-catenin and CDH11 knock-down cell lines were generated, and affected on invasion/migration, and metastatic progression was assessed in vitro and in vivo. Furthermore, the interaction of CDH11 with β-catenin, and the contribution of the protein-protein interaction to the heterogenous activation of Wnt/β-catenin was assessed by immunofluorescence, flowcytometry, Western blot, and co-immunoprecipitation. Results: At baseline, only a small fraction of ES cells have activated Wnt/β-catenin dependent transcriptional activity both in vitro and in vivo, which can respond rapidly with Wnt ligand stimulation. β-catenin knockdown reduced clonogenicity in vitro, and metastatic tumor burden in the orthotopic implantation amputation mouse model of ES. We further identified that most of the tested ES cells had abundant β-catenin bound to CDH11 in the cell membrane, which would rapidly be released through the cytoplasm and into the nucleus within 2 hours of adding exogenous Wnt ligand. CDH11 knockdown reduced β-catenin expression, rapid activation of signaling by Wnt ligands, and metastasis progression. Conclusion: Our data suggests that the cell surface CDH11- β-catenin complex contributes to the heterogenous and dynamic activation of the Wnt/β-catenin pathway in ES that drives ES metastasis. Targeting the CDH11- β-catenin complex can present a novel strategy to interfere with Wnt driven metastasis in ES. Citation Format: Ryota Shirai, Tyler Biebighauser, Sarah Nelson-Taylor, Avery Bodlak, Timothy Porfilio, Deandra Walker, Naoki Oike, Masanori Hayashi. Cadherin-11 contributes to the heterogenous and dynamic Wnt-β-catenin pathway activation in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 957.

Genome Editing Targets for Improving Nutrient Use Efficiency and Nutrient Stress Adaptation.

In recent years, the development of RNA-guided genome editing (CRISPR-Cas9 technology) has revolutionized plant genome editing. Under nutrient deficiency conditions, different transcription factors and regulatory gene networks work together to maintain nutrient homeostasis. Improvement in the use efficiency of nitrogen (N), phosphorus (P) and potassium (K) is essential to ensure sustainable yield with enhanced quality and tolerance to stresses. This review outlines potential targets suitable for genome editing for understanding and improving nutrient use (NtUE) efficiency and nutrient stress tolerance. The different genome editing strategies for employing crucial negative and positive regulators are also described. Negative regulators of nutrient signalling are the potential targets for genome editing, that may improve nutrient uptake and stress signalling under resource-poor conditions. The promoter engineering by CRISPR/dead (d) Cas9 (dCas9) cytosine and adenine base editing and prime editing is a successful strategy to generate precise changes. CRISPR/dCas9 system also offers the added advantage of exploiting transcriptional activators/repressors for overexpression of genes of interest in a targeted manner. CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) are variants of CRISPR in which a dCas9 dependent transcription activation or interference is achieved. dCas9-SunTag system can be employed to engineer targeted gene activation and DNA methylation in plants. The development of nutrient use efficient plants through CRISPR-Cas technology will enhance the pace of genetic improvement for nutrient stress tolerance of crops and improve the sustainability of agriculture.

Lysine Deacetylases and Demethylases: Early Epigenetic Regulators of Intermittent Hypoxia induced Sympathetic Activation and Blood Pressure

Intermittent hypoxia (IH) is a hallmark manifestation of obstructive sleep apnea (OSA). Long term IH (LT‐IH) triggers epigenetic reprogramming of the redox state involving DNA hypermethylation leading to persistent sympathetic activation and hypertension. Present study examined whether IH also activates epigenetic mechanism(s) other than DNA methylation. Lysine modifications of histones is another major epigenetic mechanism associated with gene regulation. Interplay between lysine deacetylases (HDACs) and demethylases (KDMs) differentially regulate gene expression. Lysine acetylation of histone opens up the chromatin thereby facilitating binding of transcription factors. On the other hand, KDMs regulate transcription by demethylating histone lysines which activate gene expression. Here we report that exposure of rat pheochromocytoma (PC)‐12 cells to IH in vitro exhibit reduced HDAC5 and increased KDM6B enzyme activity leading to increased HIF‐1α stability and transcriptional activity. Treatment of rats with Trichostatin A (TSA), an inhibitor of HDACs increased HIF‐1 dependent NADPH oxidase (NOX)‐4 transcription, in adrenal medullae (AM), resulting in elevated plasma catecholamines and blood pressure. Treating IH exposed rats with GSKJ4 an inhibitor of KDMs blocked HIF‐1 dependent transcriptional activation of NOX4, as well as absence of elevated plasma catecholamines and hypertension. These findings indicate a hitherto uncharacterized role of HDACs and KDMs as early epigenetic regulators in IH‐augmented sympathetic nerve activation and hypertension in rodent models of IH.

MED1 Downregulation Contributes to TGFβ-Induced Metastasis by Inhibiting SMAD2 Ubiquitination Degradation in Cutaneous Melanoma

Metastasis is the main reason for the high mortality of patients and indeed a difficult task in the treatment of cutaneous melanoma. Therefore, it is of great clinical value to explore the molecular mechanism of cutaneous metastatic melanoma and develop novel therapies. MED1, acting as a factor required for activator-dependent transcription, is reported to be involved in carcinogenesis and progression. In this study, we found that MED1 was highly expressed in patients with cutaneous melanoma. MED1 downregulation could induce cellular epithelial-to-mesenchymal transition and promote migration, invasion, and metastasis of cutaneous melanoma invivo and invitro. Further analysis showed that in Med1 knockdown cells, the TGFβ/SMAD2 signaling pathway mediated an increase in epithelial-to-mesenchymal transition phenotype and migration. The opposite results were observed after treatment with TGFβ inhibitors. To further explore the mechanism, we found that MED1 interacted with SMAD2, and MED1 downregulation could protect SMAD2 from degradation by inhibiting SMAD2 ubiquitination. Together, these results suggest that MED1 inhibited TGFβ signaling pathway to reduce cell epithelial-to-mesenchymal transition phenotype and migration through SMAD2 ubiquitination in the metastasis of cutaneous melanoma. Our findings elucidated the role of MED1 in the metastasis of cutaneous melanoma and provided a target for the therapeutic strategies of cutaneous melanoma.

Autophagy-related gene 5 (Atg5) is required for maintaining subcellular calcium homeostasis during acute cardiac stress

Abstract Background Autophagy exerts protective effects during cardiac stress inflicted by β-adrenergic stimulation and pressure overload. Contrarily, deletion of the cardiac-specific Atg5 gene contributes to left ventricular hypertrophy and contractile dysfunction, resulting in dilated cardiomyopathy. Heart failure is characterized by perturbations of excitation-contraction coupling (ECC) underlying alterations in intracellular calcium signaling. Purpose In this study, we aimed to understand whether loss of basal autophagy due to Atg5 deletion specifically in cardiomyocytes contributes directly to disturbances in subcellular calcium cycling. Methods Cardiac-specific Atg5−/− and Atg5+/+ mice were used to isolate ventricular cardiomyocytes, which were loaded with 8 μM Ca2+ indicator Fluo4/AM. Isolated cells were electrically stimulated and subjected to elevated workload by increasing pacing frequencies from 1 to 4 Hz. Cytosolic and nuclear calcium transients (CaTs) were recorded in line-scan mode using a laser scanning confocal microscope. Pharmacological inhibition of autophagic flux was performed by an intraperitoneal injection of 40 mg/kg leupeptin to Atg5+/+ mice. Results At baseline (1 Hz) stimulation the amplitude and kinetics of calcium transients were comparable between Atg5−/− and Atg5+/+ cells. However, after progressive increase of the pacing rate from 1 Hz to 4 Hz, Atg5−/− cardiomyocytes displayed reduced cytoplasmic and nuclear CaT amplitude, but increased nuclear time-averaged CaTs compared to controls. Such increase in nuclear calcium load stimulates Ca2+-dependent transcriptional activity, and likely involves CaMKII-mediated pro-hypertrophic gene program thus contributing to cardiac remodelling. In addition, we observed increased occurrence of arrhythmic events in Atg5−/− hearts at high pacing frequency, indicative of intracellular calcium overload and disturbed ECC. Acute pharmacological autophagy inhibition failed to elicit direct changes in time-averaged CaTs in the cytoplasm and nucleoplasm, suggesting that loss of Atg5-dependent autophagy seems to induce adverse cardiac remodelling that involves increased CaMKII activity due to the imbalances in nuclear Ca2+ levels. Conclusion Specific loss of ATG5 protein impairs calcium cycling during acute stress exposure by reducing CaT amplitudes, promoting arrhythmia, and increasing nuclear calcium load, which may induce hypertrophic gene expression. Further work is needed to identify key components mediating autophagy-related cardioprotection to develop strategies against cardiac remodeling and its progression to heart failure. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Austrian Science Fund (FWF)

Structural Insights into the Loss-of-Function R288H Mutant of Human PPARγ.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor and the molecular target of thiazolidinedione-class antidiabetic drugs. It has been reported that the loss of function R288H mutation in the human PPARγ ligand-binding domain (LBD) may be associated with the onset of colon cancer. A previous in vitro study showed that this mutation dampens 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2, a natural PPARγ agonist)-dependent transcriptional activation; however, it is poorly understood why the function of the R288H mutant is impaired and what role this arginine (Arg) residue plays. In this study, we found that the apo-form of R288H PPARγ mutant displays several altered conformational arrangements of the amino acid side chains in LBD: 1) the loss of a salt bridge between Arg288 and Glu295 leads to increased helix 3 movement; 2) closer proximity of Gln286 and His449 via a hydrogen bond, and closer proximity of Cys285 and Phe363 via hydrophobic interaction, stabilize the helix 3-helix 11 interaction; and 3) there is steric hindrance between Cys285/Gln286/Ser289/His449 and the flexible ligands 15d-PGJ2, 6-oxotetracosahexaenoic acid (6-oxoTHA), and 17-oxodocosahexaenoic acid (17-oxoDHA). These results suggest why Arg288 plays an important role in ligand binding and why the R288H mutation is disadvantageous for flexible ligand binding.

Sodium fluoride activates the extrinsic apoptosis via regulating NOX4/ROS-mediated p53/DR5 signaling pathway in lung cells both in vitro and in vivo

An extensive body of research has demonstrated that pulmonary toxicity induced by fluoride is related to cell apoptosis. Although induction of death receptor-initiated extrinsic apoptosis by sodium fluoride (NaF) has been reported, its mechanism of action is still not clearly defined. Herein, we found that NaF treatment induced activation of caspase-8 in BEAS-2B cells, resulting in apoptosis, which was markedly reduced by blocking caspase-8 using small interfering RNA (siRNA). In this study, we report that death receptor 5 (DR5), a major component of the extrinsic apoptotic pathway, is markedly induced upon NaF stimulation. Enhanced DR5 induction was necessary for the apoptotic effects of NaF, inasmuch as transfected BEAS-2B cells with DR5 siRNA attenuated NaF-induced caspase-8 activation in lung cells. Mechanism investigation indicated that the induction of DR5, following NaF exposure, was mediated by tumor protein 53 (p53)-dependent transcriptional activation. Notably, we demonstrated that NaF could induce a significant increase in intracellular reactive oxygen species (ROS) level derived from nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4). Specifically, NOX4 knockdown inhibited NaF-induced the activation of p53/DR5 axis by reducing NOX4-derived ROS production. Further in vivo investigation demonstrated that NOX4 deficiency markedly attenuates NaF-induced lung injury, apoptosis, and ROS levels in the lung. Moreover, the expressions of p53 and DR5 were significantly reduced after NaF treatment in NOX4 knockout mice compared with the wild type mice. Taken together, our findings provide a novel insight into for the pulmonary apoptosis in response to NaF exposure.

An organelle-tethering mechanism couples flagellation to cell division in bacteria.

In some free-living and pathogenic bacteria, problems in the synthesis and assembly of early flagellar components can cause cell-division defects. However, the mechanism that couples cell division with the flagellar biogenesis has remained elusive. Herein, we discover the regulator MadA that controls transcription of flagellar and cell-division genes in Caulobacter crescentus. We demonstrate that MadA, a small soluble protein, binds the type III export component FlhA to promote activation of FliX, which in turn is required to license the conserved σ54-dependent transcriptional activator FlbD. While in the absence of MadA, FliX and FlbD activation is crippled, bypass mutations in FlhA restore flagellar biogenesis and cell division. Furthermore, we demonstrate that MadA safeguards the divisome stoichiometry to license cell division. We propose that MadA has a sentinel-type function that senses an early flagellar biogenesis event and, through cell-division control, ensures that a flagellated offspring emerges.

Chromosome Y pericentric heterochromatin is a primary target of HSF1 in male cells

The heat shock factor 1 (HSF1)-dependent transcriptional activation of human pericentric heterochromatin in heat-shocked cells is the most striking example of transcriptional activation of heterochromatin. Until now, pericentric heterochromatin of chromosome 9 has been identified as the primary target of HSF1, in both normal and tumor heat-shocked cells. Transcriptional awakening of this large genomic region results in the nuclear accumulation of satellite III (SATIII) noncoding RNAs (ncRNAs) and the formation in cis of specific structures known as nuclear stress bodies (nSBs). Here, we show that, in four different male cell lines, including primary human fibroblasts and amniocytes, pericentric heterochromatin of chromosome Y can also serve as a unique primary site of HSF1-dependent heterochromatin transcriptional activation, production of SATIII ncRNA, and nucleation of nuclear stress bodies (nSBs) upon heat shock. Our observation suggests that the chromosomal origin of SATIII transcripts in cells submitted to heat shock is not a determinant factor as such, but that transcription of SATIII repetitive units or the SATIII ncRNA molecules is the critical element of HSF1-dependent transcription activation of constitutive heterochromatin.

Identification of a cross-talk between EGFR and Wnt/beta-catenin signaling pathways in HepG2 liver cancer cells

EGFRis a transmembrane receptor tyrosine kinase involved in regulating cell proliferation, differentiation and survival. EGFR is actively pursued as a therapeutic target because its aberrant expression or activity has been reported in several cancers. Several studies have reported the nuclear localization of the EGFR in various cell types, however, its exact nuclear functions are not clear yet. In this study, we have generated GFP fusion constructs of EGFR and its mutants to analyze their subcellular localizationin normal and cancer cells and impact of its sub-cellular location on its various activities using immunoblotting, confocal microscopy, reporter assays, loss-of-function EGFR mutants, and EGFR specific small molecule inhibitors. We show that EGFR is involved in modulating TCF dependent β-catenin transcriptional activity in HepG2 cells in a similar fashion as IGF1R tyrosine kinase. Moreover, we show that cytoplasmic and nuclear functions are two independent activities of EGFR.

Dynamics of RNA polymerase II and elongation factor Spt4/5 recruitment during activator-dependent transcription

In eukaryotes, RNA polymerase II (RNApII) transcribes messenger RNA from template DNA. Decades of experiments have identified the proteins needed for transcription activation, initiation complex assembly, and productive elongation. However, the dynamics of recruitment of these proteins to transcription complexes, and of the transitions between these steps, are poorly understood. We used multiwavelength single-molecule fluorescence microscopy to directly image and quantitate these dynamics in a budding yeast nuclear extract that reconstitutes activator-dependent transcription in vitro. A strong activator (Gal4-VP16) greatly stimulated reversible binding of individual RNApII molecules to template DNA. Binding of labeled elongation factor Spt4/5 to DNA typically followed RNApII binding, was NTP dependent, and was correlated with association of mRNA binding protein Hek2, demonstrating specificity of Spt4/5 binding to elongation complexes. Quantitative kinetic modeling shows that only a fraction of RNApII binding events are productive and implies a rate-limiting step, probably associated with recruitment of general transcription factors, needed to assemble a transcription-competent preinitiation complex at the promoter. Spt4/5 association with transcription complexes was slowly reversible, with DNA-bound RNApII molecules sometimes binding and releasing Spt4/5 multiple times. The average Spt4/5 residence time was of similar magnitude to the time required to transcribe an average length yeast gene. These dynamics suggest that a single Spt4/5 molecule remains associated during a typical transcription event, yet can dissociate from RNApII to allow disassembly of abnormally long-lived (i.e., stalled) elongation complexes.

Resistance to anti-EGFR targeted therapy mediated by oncogenetic mutations in colorectal cancer: Revision of the dogma?

Traditionally, systemic treatment for high stage colorectal carcinoma (CRC) is mainly fluorouracil-based chemotherapy [1]. The anti-epidermal growth factor receptor (EGFR) monoclonal antibodies, by acting on specific molecular pathways in tumor growth or modulating immune response towards tumor cells, provide a more targeted response, a better side effect profile and greater impact on patient survival compared with conventional molecules. This monoclonal antibody that binds the extracellular domain of epidermal growth factor receptor, is known to be effective only in a subset of KRAS wild-type colorectal cancers. Patients with mutations in either KRAS or NRAS gene are not eligible for anti-EGFR monoclonal antibody therapy [3]. This is due to downstream activation of the Ras/Raf/MAPK pathway by mutated RAS protein, leading to cell proliferation which cannot be sufficiently inhibited by anti-EGFR receptor monoclonal antibodies [4]. With the increasing choices of targeted agents, more and more biomarkers are tested. Currently, the standard recommended biomarker panel for colorectal carcinoma would include KRAS, NRAS, BRAF gene hotspot mutation detection and microsatellite instability test [5]. With the advances in genomic profiling and sequencing and the understanding of the resistance mechanisms, the contraindication of anti-EGFR therapy in mutant KRAS patients may be revised. Based on the fact that the KRAS mutation in CRC suppresses the phosphorylation of the AMP-activated protein kinase (AMPK) known to be toxic for the tumor cells, Hua et al obtained a satisfactory response to for the anti-EGFR antibodies in mutant KRAS CRC xenograft models after reactivation of the AMPK [6]. Knickelbein et al demonstrated that the anti-EGFR antibodies induce the death of CRC cells via a p73- dependent transcriptional activation of the pro-apoptotic Bcl-2 family protein (PUMA). This action is abolished in case of KRAS mutation. These authors admitted that the restoration of this pathway by inhibiting aurora kinases preferentially kills mutant KRAS CRC cells and overcomes KRAS-mediated resistance to anti-EGFR antibodies [7]. In fifty-one CRC patient-derived xenografts study, Lee et al showed that KRAS mutants expressed remarkably elevated autocrine levels of high-affinity EGFR ligands compared with wild-type KRAS. The use of an anti-EGFR IgG1 antibody that displays potent inhibitory effects on highaffinity EGFR ligand enhanced CRC KRAS mutant cells cytotoxicity [8]. Dealing with the resistance to targeted therapies in CRC patient looks feasible. It could allow to broaden the indications of anti-EGFR therapy and provide a better survival for a larger group of CRC patients. In this era of precision and personalized medicine, a complete case specific tumor profiling and the comprehensive study of the tumorogenesis mechanisms should allow to overcome the intrinsic and acquired resistance to these targeted high effective therapies.

Dehydroxyhispolon Methyl Ether, A Hispolon Derivative, Inhibits WNT/β-Catenin Signaling to Elicit Human Colorectal Carcinoma Cell Apoptosis.

Colorectal cancer (CRC) is the fourth leading cause of cancer mortality worldwide. Aberrant activation of WNT/β-catenin signaling present in the vast majority of CRC cases is indispensable for CRC initiation and progression, and thus is a promising target for CRC therapeutics. Hispolon is a fungal-derived polyphenol with a pronounced anticancer effect. Several hispolon derivatives, including dehydroxyhispolon methyl ether (DHME), have been chemically synthesized for developing lead molecules with stronger anticancer activity. Herein, a DHME-elicited anti-CRC effect with the underlying mechanism is reported for the first time. Specifically, DHME was found to be more cytotoxic than hispolon against a panel of human CRC cell lines, while exerting limited toxicity to normal human colon cell line CCD 841 CoN. Additionally, the cytotoxic effect of DHME appeared to rely on inducing apoptosis. This notion was evidenced by DHME-elicited upregulation of poly (ADP-ribose) polymerase (PARP) cleavage and a cell population positively stained by annexin V, alongside the downregulation of antiapoptotic B-cell lymphoma 2 (BCL-2), whereas the blockade of apoptosis by the pan-caspase inhibitor z-VAD-fmk attenuated DHME-induced cytotoxicity. Further mechanistic inquiry revealed the inhibitory action of DHME on β-catenin-mediated, T-cell factor (TCF)-dependent transcription activity, suggesting that DHME thwarted the aberrantly active WNT/β-catenin signaling in CRC cells. Notably, ectopic expression of a dominant–active β-catenin mutant (∆N90-β-catenin) abolished DHME-induced apoptosis while also restoring BCL-2 expression. Collectively, we identified DHME as a selective proapoptotic agent against CRC cells, exerting more potent cytotoxicity than hispolon, and provoking CRC cell apoptosis via suppression of the WNT/β-catenin signaling axis.

Activation by NarL at the Escherichia coli ogt promoter.

The Escherichia coli NarX/NarL two-component response-regulator system regulates gene expression in response to nitrate ions and the NarL protein is a global transcription factor, which activates transcript initiation at many target promoters. One such target, the E. coli ogt promoter, which controls the expression of an O6-alkylguanine-DNA-alkyltransferase, is dependent on NarL binding to two DNA targets centred at positions −44.5 and −77.5 upstream from the transcript start. Here, we describe ogt promoter derivatives that can be activated solely by NarL binding either at position −44.5 or position −77.5. We show that NarL can also activate the ogt promoter when located at position −67.5. We present data to argue that NarL-dependent activation of transcript initiation at the ogt promoter results from a direct interaction between NarL and a determinant in the C-terminal domain of the RNA polymerase α subunit. Footprinting experiments show that, at the −44.5 promoter, NarL and the C-terminal domain of the RNA polymerase α subunit bind to opposite faces of promoter DNA, suggesting an unusual mechanism of transcription activation. Our work suggests new organisations for activator-dependent transcription at promoters and future applications for biotechnology.

CTRP9 Mediates Protective Effects in Cardiomyocytes via AMPK- and Adiponectin Receptor-Mediated Induction of Anti-Oxidant Response.

The C1q/tumor necrosis factor-alpha-related protein 9 (CTRP9) has been reported to exert cardioprotective effects, but its role in the right ventricle (RV) remains unclear. To investigate the role of CTRP9 in RV hypertrophy and failure, we performed pulmonary artery banding in weanling rats to induce compensatory RV hypertrophy seven weeks after surgery and RV failure 22 weeks after surgery. CTRP9 expression, signal transduction and mechanisms involved in protective CTRP9 effects were analyzed in rat and human RV tissue and cardiac cells. We demonstrate that CTRP9 was induced during compensatory RV hypertrophy but almost lost at the stage of RV failure. RV but not left ventricular (LV) cardiomyocytes or RV endothelial cells demonstrated increased intracellular reactive oxygen species (ROS) and apoptosis activation at this stage. Exogenous CTRP9 induced AMP-activated protein kinase (AMPK)-dependent transcriptional activation of the anti-oxidant thioredoxin-1 (Trx1) and superoxide dismutase-2 (SOD2) and reduced phenylephrine-induced ROS. Combined knockdown of adiponectin receptor-1 (AdipoR1) and AdipoR2 or knockdown of calreticulin attenuated CTRP9-mediated anti-oxidant effects. Immunoprecipitation showed an interaction of AdipoR1 with AdipoR2 and the co-receptor T-cadherin, but no direct interaction with calreticulin. Thus, CTRP9 mediates cardioprotective effects through inhibition of ROS production induced by pro-hypertrophic agents via AMPK-mediated activation of anti-oxidant enzymes.