A new insight into MYC action: control of RNA polymerase II methylation and transcription termination

MYC oncoprotein deregulation is a common catastrophic event in human cancer and limiting its activity restrains tumor development and maintenance, as clearly shown via Omomyc, an MYC-interfering 90 amino acid mini-protein. MYC is a multifunctional transcription factor that regulates many aspects of transcription by RNA polymerase II (RNAPII), such as transcription activation, pause release, and elongation. MYC directly associates with Protein Arginine Methyltransferase 5 (PRMT5), a protein that methylates a variety of targets, including RNAPII at the arginine residue R1810 (R1810me2s), crucial for proper transcription termination and splicing of transcripts. Therefore, we asked whether MYC controls termination as well, by affecting R1810me2S. We show that MYC overexpression strongly increases R1810me2s, while Omomyc, an MYC shRNA, or a PRMT5 inhibitor and siRNA counteract this phenomenon. Omomyc also impairs Serine 2 phosphorylation in the RNAPII carboxyterminal domain, a modification that sustains transcription elongation. ChIP-seq experiments show that Omomyc replaces MYC and reshapes RNAPII distribution, increasing occupancy at promoter and termination sites. It is unclear how this may affect gene expression. Transcriptomic analysis shows that transcripts pivotal to key signaling pathways are both up- or down-regulated by Omomyc, whereas genes directly controlled by MYC and belonging to a specific signature are strongly down-regulated. Overall, our data point to an MYC/PRMT5/RNAPII axis that controls termination via RNAPII symmetrical dimethylation and contributes to rewiring the expression of genes altered by MYC overexpression in cancer cells. It remains to be clarified which role this may have in tumor development

Novel statistical approaches for non-normal censored immunological data: analysis of cytokine and gene expression data

Background: For several immune-mediated diseases, immunological analysis will become more complex in the future with datasets in which cytokine and gene expression data play a major role. These data have certain characteristics that require sophisticated statistical analysis such as strategies for non-normal distribution and censoring. Additionally, complex and multiple immunological relationships need to be adjusted for potential confounding and interaction effects. Objective: We aimed to introduce and apply different methods for statistical analysis of non-normal censored cytokine and gene expression data. Furthermore, we assessed the performance and accuracy of a novel regression approach in order to allow adjusting for covariates and potential confounding. Methods: For non-normally distributed censored data traditional means such as the Kaplan-Meier method or the generalized Wilcoxon test are described. In order to adjust for covariates the novel approach named Tobit regression on ranks was introduced. Its performance and accuracy for analysis of non-normal censored cytokine/gene expression data was evaluated by a simulation study and a statistical experiment applying permutation and bootstrapping. Results: If adjustment for covariates is not necessary traditional statistical methods are adequate for non-normal censored data. Comparable with these and appropriate if additional adjustment is required, Tobit regression on ranks is a valid method. Its power, type-I error rate and accuracy were comparable to the classical Tobit regression. Conclusion: Non-normally distributed censored immunological data require appropriate statistical methods. Tobit regression on ranks meets these requirements and can be used for adjustment for covariates and potential confounding in large and complex immunological datasets

The Protein Arginine Methyltransferases 1 and 5 affect Myc properties in glioblastoma stem cells

Protein Arginine (R) methylation is the most common post-translational methylation in mammalian cells. Protein Arginine Methyltransferases (PRMT) 1 and 5 dimethylate their substrates on R residues, asymmetrically and symmetrically, respectively. They are ubiquitously expressed and play fundamental roles in tumour malignancies, including glioblastoma multiforme (GBM) which presents largely deregulated Myc activity. Previously, we demonstrated that PRMT5 associates with Myc in GBM cells, modulating, at least in part, its transcriptional properties. Here we show that Myc/PRMT5 protein complex includes PRMT1, in both HEK293T and glioblastoma stem cells (GSCs). We demonstrate that Myc is both asymmetrically and symmetrically dimethylated by PRMT1 and PRMT5, respectively, and that these modifications differentially regulate its stability. Moreover, we show that the ratio between symmetrically and asymmetrically dimethylated Myc changes in GSCs grown in stem versus differentiating conditions. Finally, both PRMT1 and PRMT5 activity modulate Myc binding at its specific target promoters. To our knowledge, this is the first work reporting R asymmetrical and symmetrical dimethylation as novel Myc post-translational modifications, with different functional properties. This opens a completely unexplored field of investigation in Myc biology and suggests symmetrically dimethylated Myc species as novel diagnostic and prognostic markers and druggable therapeutic targets for GBM

Myc and Omomyc functionally associate with the Protein Arginine Methyltransferase 5 (PRMT5) in glioblastoma cells

The c-Myc protein is dysregulated in many human cancers and its function has not been fully elucitated yet. The c-Myc inhibitor Omomyc displays potent anticancer properties in animal models. It perturbs the c-Myc protein network, impairs c-Myc binding to the E-boxes, retaining transrepressive properties and inducing histone deacetylation. Here we have employed Omomyc to further analyse c-Myc activity at the epigenetic level. We show that both Myc and Omomyc stimulate histone H4 symmetric dimethylation of arginine (R) 3 (H4R3me2s), in human glioblastoma and HEK293T cells. Consistently, both associated with protein Arginine Methyltransferase 5 (PRMT5)-the catalyst of the reaction-and its co-factor Methylosome Protein 50 (MEP50). Confocal experiments showed that Omomyc co-localized with c-Myc, PRMT5 and H4R3me2s-enriched chromatin domains. Finally, interfering with PRMT5 activity impaired target gene activation by Myc whereas it restrained Omomyc-dependent repression. The identification of a histone-modifying complex associated with Omomyc represents the first demonstration of an active role of this miniprotein in modifying chromatin structure and adds new information regarding its action on c-Myc targets. More importantly, the observation that c-Myc may recruit PRMT5-MEP50, inducing H4R3 symmetric di-methylation, suggests previously unpredictable roles for c-Myc in gene expression regulation and new potential targets for therapy

Wheeze in Preschool Age Is Associated with Pulmonary Bacterial Infection and Resolves after Antibiotic Therapy

BACKGROUND: Neonates with airways colonized by Haemophilus influenzae, Streptococcus pneumoniae or Moraxella catarrhalis are at increased risk for recurrent wheeze which may resemble asthma early in life. It is not clear whether chronic colonization by these pathogens is causative for severe persistent wheeze in some preschool children and whether these children might benefit from antibiotic treatment. We assessed the relevance of bacterial colonization and chronic airway infection in preschool children with severe persistent wheezing and evaluated the outcome of long-time antibiotic treatment on the clinical course in such children. METHODOLOGY/PRINCIPAL FINDINGS: Preschool children (n = 42) with severe persistent wheeze but no symptoms of acute pulmonary infection were investigated by bronchoscopy and bronchoalveolar lavage (BAL). Differential cell counts and microbiological and virological analyses were performed on BAL samples. Patients diagnosed with bacterial infection were treated with antibiotics for 2-16 weeks (n = 29). A modified ISAAC questionnaire was used for follow-up assessment of children at least 6 months after bronchoscopy. Of the 42 children with severe wheezing, 34 (81%) showed a neutrophilic inflammation and 20 (59%) of this subgroup had elevated bacterial counts (≥ 10⁴ colony forming units per milliliter) suggesting infection. Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis were the most frequently isolated species. After treatment with appropriate antibiotics 92% of patients showed a marked improvement of symptoms upon follow-up examination. CONCLUSIONS/SIGNIFICANCE: Chronic bacterial infections are relevant in a subgroup of preschool children with persistent wheezing and such children benefit significantly from antibiotic therapy

Comparison of physical fitness between healthy and mild‐to‐moderate asthmatic children with exercise symptoms: A cross‐sectional study

.Objective Asthma is a chronic disease that may affect physical fitness, although its primary effects on exercise capacity, muscle strength, functionality and lifestyle, in children and adolescents, are still poorly understood. This study aimed to evaluate the differences in cardiorespiratory fitness, muscle strength, lifestyle, lung function, and functionality between asthmatics with exercise symptoms and healthy children. In addition, we have analyzed the association between clinical history and the presence of asthma. Study Design Cross-sectional study including 71 patients with a diagnosis of asthma and 71 healthy children and adolescents (7–17 years of age). Anthropometric data, clinical history, disease control, lifestyle (KIDMED and physical activity questionnaires), lung function (spirometry), exercise-induced bronchoconstriction test, aerobic fitness (cardiopulmonary exercise test), muscle strength and functionality (timed up and go; timed up and down stairs) were evaluated. Results Seventy-one patients with asthma (mean age 11.5 ± 2.7) and 71 healthy subjects (mean age 10.7 ± 2.5) were included. All asthmatic children had mild to moderate and stable asthma. EIB occurred in 56.3% of asthmatic children. Lung function was significantly (p < .05) lower in the asthmatic group when compared to healthy peers, as well as the cardiorespiratory fitness, muscle strength, lifestyle and functionality. Moreover, asthmatic children were more likely to have atopic dermatitis, allergic reactions, food allergies, and a family history of asthma when compared to healthy children. Conclusions Children with mild-to-moderate asthma presenting exercise symptoms show a reduction in cardiorespiratory fitness, muscle strength, lung function, functionality, and lifestyle when compared to healthy peers. The study provides data for pediatricians to support exercise practice aiming to improve prognosis and quality of life in asthmatic children.S

T Regulatory Cells in Cord Blood—FOXP3 Demethylation as Reliable Quantitative Marker

Regulatory T-cells (Tregs), characterized as CD4+CD25(hi) T-cells expressing FOXP3, play a crucial role in controlling healthy immune development during early immune maturation. Recently, FOXP3 demethylation was suggested to be a novel marker for natural Tregs in adults. In cord blood, the role and function of Tregs and its demethylation is poorly understood. We assessed FOXP3 demethylation in cord blood in relation to previously used Treg markers such as CD4+CD25(hi), FOXP3 mRNA, protein expression, and suppressive Treg function

In Situ Spatiotemporal Mapping of Flow Fields around Seeded Stem Cells at the Subcellular Length Scale

A major hurdle to understanding and exploiting interactions between the stem cell and its environment is the lack of a tool for precise delivery of mechanical cues concomitant to observing sub-cellular adaptation of structure. These studies demonstrate the use of microscale particle image velocimetry (μ-PIV) for in situ spatiotemporal mapping of flow fields around mesenchymal stem cells, i.e. murine embryonic multipotent cell line C3H10T1/2, at the subcellular length scale, providing a tool for real time observation and analysis of stem cell adaptation to the prevailing mechanical milieu. In the absence of cells, computational fluid dynamics (CFD) predicts flow regimes within 12% of μ-PIV measures, achieving the technical specifications of the chamber and the flow rates necessary to deliver target shear stresses at a particular height from the base of the flow chamber. However, our μ-PIV studies show that the presence of cells per se as well as the density at which cells are seeded significantly influences local flow fields. Furthermore, for any given cell or cell seeding density, flow regimes vary significantly along the vertical profile of the cell. Hence, the mechanical milieu of the stem cell exposed to shape changing shear stresses, induced by fluid drag, varies with respect to proximity of surrounding cells as well as with respect to apical height. The current study addresses a previously unmet need to predict and observe both flow regimes as well as mechanoadaptation of cells in flow chambers designed to deliver precisely controlled mechanical signals to live cells. An understanding of interactions and adaptation in response to forces at the interface between the surface of the cell and its immediate local environment may be key for de novo engineering of functional tissues from stem cell templates as well as for unraveling the mechanisms underlying multiscale development, growth and adaptation of organisms

Association of the polygenic scores for personality traits and response to selective serotonin reuptake inhibitors in patients with major depressive disorder

Studies reported a strong genetic correlation between the Big Five personality traits and major depressive disorder (MDD). Moreover, personality traits are thought to be associated with response to antidepressants treatment that might partly be mediated by genetic factors. In this study, we examined whether polygenic scores (PGSs) derived from the Big Five personality traits predict treatment response and remission in patients with MDD who were prescribed selective serotonin reuptake inhibitors (SSRIs). In addition, we performed meta-analyses of genome-wide association studies (GWASs) on these traits to identify genetic variants underpinning the cross-trait polygenic association. The PGS analysis was performed using data from two cohorts: the Pharmacogenomics Research Network Antidepressant Medication Pharmacogenomic Study (PGRN-AMPS, n = 529) and the International SSRI Pharmacogenomics Consortium (ISPC, n = 865). The cross-trait GWAS meta-analyses were conducted by combining GWAS summary statistics on SSRIs treatment outcome and on the personality traits. The results showed that the PGS for openness and neuroticism were associated with SSRIs treatment outcomes at p < 0.05 across PT thresholds in both cohorts. A significant association was also found between the PGS for conscientiousness and SSRIs treatment response in the PGRN-AMPS sample. In the cross-trait GWAS meta-analyses, we identified eight loci associated with (a) SSRIs response and conscientiousness near YEATS4 gene and (b) SSRI remission and neuroticism eight loci near PRAG1, MSRA, XKR6, ELAVL2, PLXNC1, PLEKHM1, and BRUNOL4 genes. An assessment of a polygenic load for personality traits may assist in conjunction with clinical data to predict whether MDD patients might respond favorably to SSRIs.Azmeraw T. Amare, Klaus Oliver Schubert, Fasil Tekola-Ayele, Yi-Hsiang Hsu, Katrin Sangkuhl … Bernhard T. Baune … et al

TPH2 Gene Polymorphisms and Major Depression – A Meta-Analysis

BACKGROUND: Tryptophan hydroxylase-2 (TPH2) is the rate-limiting enzyme in the synthetic pathway for brain serotonin and is considered key factor for maintaining normal serotonin transmission in the central neuron system (CNS). Gene-disease association studies have reported a relationship between TPH2 and major depressive disorder (MDD) in different populations, however subsequent studies have produced contradictory results. OBJECTIVES: We performed a systematic overview and a meta-analysis with all available data up-to-date. METHODS: We scrutinized PubMed, Embase, HuGNet and China National Knowledge Infrastructure (CNKI ) and last update was held on October 2011. We also searched the manuscripts and the supplementary documents of the published genome-wide association studies in the field. Effect sizes of independent loci that have been studied in more than 3 articles were synthesized using fixed and random effects models. RESULTS: We found 27 eligible articles that studied a total of 74 single nucleotide polymorphisms (SNPs). Finally, 12 independent loci were included in the meta-analysis. The synthesis of the data shown that two SNPs (rs4570625 and rs17110747) were associated with MDD using fixed effects models. SNP rs4570625 had low heterogeneity and remained significant using the more conservative random effects calculations with a summary OR = 0.83 (95% CI: 0.73-0.96). CONCLUSION: The current study identified a SNP (rs4570625) with strong epidemiological credibility; however more studies are required to provide robust evidence for other weak associations