Epigenetic control of antioxidant gene expression

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 29-10-2015To respond to exogenous and endogenous stimuli, organisms have developed a variety of mechanisms to modulate the quantity, duration and the type of response to these stimuli. Of these mechanisms, one of the most important is the regulation of gene expression. This regulation of gene expression occurs at various levels but especially by the employment of different transcription factors and cofactors. Transcription factors interact with specific sequences within the regulated genes. The way in which co-factors act can be very diverse and are generally less known. The present work aims to analyse the mechanisms used by cells to regulate gene expression for reactive oxygen species (ROS) detoxification genes, and in particular, the mechanisms controlling the activity of the two co-factors SirT1 and PGC-1α on those genes. PGC-1α was first discovered as a regulator of biogenesis and mitochondrial activity. Later, it was demonstrated that PGC-1α operated as an expression coordinator of a group of genes controlling ROS levels and therefore prevented the accumulation of oxidative stress due to its binding to the transcription factor FoxO3a. Moreover, SirT1 was first discovered as a Histone deacetylase, involved in the regulation of genomic stability. Later on, SirT1 was shown to regulate activity of other proteins, specifically transcription factors or cofactors such as PGC-1α. The first aim of the work presented in this thesis was to determine if SirT1 collaborates with PGC-1α in regulating antioxidant genes and to find out through which mechanisms this regulation is mediated. The results demonstrate not only that SirT1 deacetylates PGC-1α and FoxO3a, but also that all three proteins together form an efficient ternary transcription elongation complex recruited to the regulating regions of the target antioxidant genes. After recruitment to the regulating regions, SirT1 deacetylates the nearby histones; this specific deacetylation does not lead to a gene silencing. However the exact mechanism of regulation is still not clear and remains to be elucidated. These results show for the first time that SirT1 can not only lead to a general silencing of chromatin, but can also induce specific gene expression when involved in a specific transcription complex. PGC-1α has a RNA recognition motif (RRM) whose deletion abolishes some of the transactivation capacity of PGC-1α. The second aim of this work was to study the functionality of this motif in the specific context of regulating antioxidant genes. The results showed that some genes are more sensitive to the absence of RRM than others. To have a global overview of the genomic functionality of the RRM, a complete genome microarray expression analysis comparing wild-type PGC-1α protein to the PGC-1α protein lacking the RRM was performed. The observation that the deletion of the PGC-1α RRM differentially affects genes regulated by PGC-1α suggests that the RRM could be involved in selection of 3’UTR and as a consequence modify RNA transcripts. The final aim of this study was to confirm and identify RNA species binding to PGC-1α RRM. The technique applied to confirm and identify these RNA species was high throughput cross-linked immuno-precipitation (HITS-CLIP). The use of HITS-CLIP confirmed binding of RNA to PGC-1α. The presence of RRM suggests preferential transcription of some protein isoforms. The combination of the obtained results from array and RNA sequencing support this hypothesis. In summary, this study not only gives detailed information about ROS detoxification gene regulation by PGC-1α but also allowed the identification of new mechanisms of action for SirT1 and PGC-1αPara responder de manera adecuada a estímulos exógenos y endógenos, los organismos han desarrollado diversos mecanismos los cuales permiten variar tanto la cantidad como el tipo de repuestas. Entre estos mecanismos se encuentran aquellos que permiten regular la expresión génica. Esta ocurre a varios niveles y a través de distintos tipos de factores, siendo los más importantes los factores de transcripción y los cofactores. Los factores de transcripción actúan reconociendo secuencias específicas en los genes regulados. Los cofactores operan a través de mecanismos muy variados y, en general, estos son más desconocidos. La presente Tesis analiza los mecanismos por los cuales la célula regula la expresión de los genes implicados en la destoxificación de especies reactivas de oxígeno (ROS). En primer lugar se determinaron los mecanismos que median la actividad de los cofactores SirT1 y PGC-1α sobre estos genes. Inicialmente el cofactor PGC-1α se describió como regulador de la biogénesis y de la actividad mitocondrial. Posteriormente, se demostró que PGC-1α regula de manera coordinada la expresión de un grupo de genes encargados de controlar los niveles de ROS y prevenir la aparición de estrés oxidativo a través de su interacción con el factor de transcripción FoxO3a. Por otra parte, el cofactor SirT1 se describió primeramente como una desacetilasa de histonas, responsable de la regulación de la estabilidad genómica, pero después se determinó que SirT1 puede también regular la actividad de otras proteínas, y en concreto, de factores de transcripción como PGC-1α. El primer objetivo de este trabajo fue determinar si SirT1 colaboraba con PGC-1α en la regulación de genes antioxidantes y a través de que mecanismo. Este estudio ha permitido demostrar que SirT1 no solo desacetila PGC-1α y FoxO3a, sino que forma con ellos un complejo ternario de elongación transcripcional más eficiente que es reclutado a las regiones reguladoras de los genes diana. Tras su reclutamiento, SirT1 desacetila las histonas próximas, pero esta desacetilación no va asociada a silenciamiento génico, el cual ocurre a través de un mecanismo desconocido. Estos resultados muestran por primera vez que más allá de la capacidad de SirT1 de inducir un silenciamiento general de la cromatina, puede además tener un efecto inductor en la expresión de genes específicos cuando forma parte de complejos de regulación transcripcional. PGC-1α contiene un dominio de unión a RNA (RNA recognition motif; RRM) cuya deleción compromete la capacidad transactivadora de PGC-1α. El segundo objetivo de este trabajo fue estudiar la funcionalidad de este dominio en el contexto específico de la regulación de genes antioxidantes. Se concluyó que algunos genes son más sensibles a la deleción de este dominio y que el coactivador TLS es capaz de activar PGC-1α solo en algunos de ellos. Con el fin de tener una visión global a nivel genómico de la funcionalidad del RRM, se realizó un análisis de expresión con microarrays de genoma completo, comparando la actividad de la proteína WT con el mutante en la region RRM. Se observó que la deleción del dominio afecta de forma diferencial a los genes regulados por PGC-1α, sugiriendo así que el dominio RRM podría afectar a la selección del 3‘UTR y alterar la estabilidad del transcrito. El último objetivo de esta Tesis fue el de confirmar e identificar qué especies de ARN se unen al RRM de PGC-1α. Para ello, se utilizó la inmunoprecipitación con entrecruzamiento covalente apareada de alto rendimiento (HITS-CLIP), la cual confirmó la unión del ARN a la proteína PGC-1α. La presencia del RRM sugiere la transcripción preferencial de ciertas isoformas de la proteína. La combinación de los resultados obtenidos mediante arrays de ARN y la secuenciación de ARN apoyan esta hipótesis. En conclusión, los resultados obtenidos en este trabajo no solo proporcionan información detallada sobre la regulación de la expresión de genes antioxidantes, sino que también han permitido identificar dos nuevos mecanismos de acción para los cofactores SirT1 y PGC-1

How do food businesses provide information on allergens in non-prepacked foods? A cross-sectional survey in Switzerland

Purpose This project aimed to investigate allergen information practices of food businesses selling nonprepacked foods after the implementation of the new Swiss food law in May 2017. Methods A cross-sectional telephone survey was conducted with food businesses selling non-prepacked foods in Switzerland. A short, standardised questionnaire was developed in German, based on previous research and literature. It was subsequently translated into French and Italian. Altogether, 882 businesses (restaurants, dairies, butcher shops and bakeries) were contacted, of which 387 were willing to participate. SPSS® (IBM, Armonk, NY, USA) was used for statistical analyses. Results The vast majority (86.0%) of food businesses provides oral allergen information. Only 14.0% currently provide written allergen information to the customer, either upfront or on request. The most frequently used labelling system in written allergen declaration was naming all ingredients (35.2%). A significant number (39.8%) do not place a notice on how to obtain allergen information, although this is a legal requirement in Switzerland when not providing written information upfront. Conclusion So far, not all food businesses have been complying with the new Swiss food law on allergen information of non-prepacked food. Therefore, awareness of the legal obligations around communicating allergen information as well as the verification of its implementation should be enhanced. To meet the needs of consumers and avoid reactions, some form of written allergen information should be promoted. Giving this information on request might encourage communication between customer and staff, thus providing an extra measure of verification

Immediate stabilization of pedicle screws: Preclinical pilot study of polymer-augmented pedicle screws

This study was designed as proof of principle and safety test of the novel technique, the Immediate Stabilization System (ISS). The technique is designed to immediately stabilize polymer-a ugmented pedicle screws (PAS) in deficient bone and avoid complications of loosening pedicle screws at the bone-screw interface, especially in osteoporotic patients. A polymer sleeve was designed as augmentation to improve screw anchorage after drilling the screw hole. By applying ultrasonic energy, the polymeric tube was moldedinto the pores of the host bone forming a strong and uniform bond with the adjacent bone. The original screw was then implanted into the denser bony environment leading to an enhanced immediate stability. The ISS-treated implants were compared to conventionally placed pedicle screws in ex-vivocadaver bones (2 sheep spines, n = 6 implants per spine, total 12 screws) and in-vivo in a spinal sheep model (Swiss alpine sheep, n = 5, 4 implants per animal, total 20 screws). The primary stability of ISS-treated pedicle screws was increased in ex-vivo bone ( +24% insertion torque (IT)) and in-vivo(+32.9% IT) in sheep spine. Removal torque (RT) was lower in the in PAS tested for 8 weeks in -vivo. The ISS technology demonstrated improved anchorage of pedicle screws in ex-vivocadaver bones as well as in-vivo studies in sheep spine

Diffuse reflection of a Bose-Einstein condensate from a rough evanescent wave mirror

We present experimental results showing the diffuse reflection of a Bose-Einstein condensate from a rough mirror, consisting of a dielectric substrate supporting a blue-detuned evanescent wave. The scattering is anisotropic, more pronounced in the direction of the surface propagation of the evanescent wave. These results agree very well with theoretical predictions.Comment: submitted to J Phys B, 10 pages, 6 figure

Cognitive Function of Children and Adolescents with Attention-Deficit/Hyperactivity Disorder in a 2-Year Open-Label Study of Lisdexamfetamine Dimesylate

BACKGROUND: SPD489-404 was the first 2-year safety study of lisdexamfetamine dimesylate in the treatment of attention-deficit/hyperactivity disorder in children and adolescents. In accordance with advice from the European Medicines Agency, assessment of cognitive function was a predefined safety outcome in SPD489-404. OBJECTIVE: The objective of this study was to assess cognitive function over 2 years in study SPD489-404, using the Cambridge Neuropsychological Test Automated Battery (CANTAB). METHODS: Participants aged 6-17 years received dose-optimised open-label lisdexamfetamine dimesylate (30, 50 or 70 mg/day) for 104 weeks. Cognition was assessed using four CANTAB tasks; Delayed Matching to Sample (DMS), Spatial Working Memory (SWM), Stop Signal Task (SST) and Reaction Time (RTI). Key and additional variables were pre-specified for each CANTAB task; groupwise mean percentage changes in key variables from baseline of > 5% were considered potentially clinically significant. RESULTS: All 314 enrolled participants received lisdexamfetamine dimesylate and were included in the safety population, and 191 (60.8%) completed the study. No potentially clinically significant deteriorations from baseline were observed in any key CANTAB variable over the 2 years of the study. Based on predefined thresholds, potentially clinically significant improvements from baseline were observed at 6 months (DMS median reaction time, mean per cent change, - 6.6%; SWM total between-search errors, - 22.8%; SST stop signal reaction time, -18.9%), and at the last on-treatment assessment (DMS median reaction time, - 6.5%; SWM total between-search errors, - 32.6%; SST stop signal reaction time, - 25.7%). CONCLUSIONS: Lisdexamfetamine dimesylate treatment for 2 years was not associated with deterioration of cognitive function in children and adolescents with attention-deficit/hyperactivity disorder. Although improvements in some cognitive measures were observed, lack of a control group makes interpretation of the findings difficult. Further studies of the impact of stimulants on cognition are required

Silencing Nuclear Pore Protein Tpr Elicits a Senescent-Like Phenotype in Cancer Cells

Background: Tpr is a large coiled-coil protein located in the nuclear basket of the nuclear pore complex for which many different functions were proposed from yeast to human. Methodology/Principal Findings: Here we show that depletion of Tpr by RNA interference triggers G0–G1 arrest and ultimately induces a senescent-like phenotype dependent on the presence of p53. We also found that Tpr depletion impairs the NES [nuclear export sequence]-dependent nuclear export of proteins and causes partial co-depletion of Nup153. In addition Tpr depletion impacts on level and function of the SUMO-protease SENP2 thus affecting SUMOylation regulation at the nuclear pore and overall SUMOylation in the cell. Conclusions: Our data for the first time provide evidence that a nuclear pore component plays a role in controlling cellular senescence. Our findings also point to new roles for Tpr in the regulation of SUMO-1 conjugation at the nuclear pore and directly confirm Tpr involvement in the nuclear export of NES-proteins

Genome-wide association study identifies six new loci influencing pulse pressure and mean arterial pressure.

Numerous genetic loci have been associated with systolic blood pressure (SBP) and diastolic blood pressure (DBP) in Europeans. We now report genome-wide association studies of pulse pressure (PP) and mean arterial pressure (MAP). In discovery (N = 74,064) and follow-up studies (N = 48,607), we identified at genome-wide significance (P = 2.7 × 10(-8) to P = 2.3 × 10(-13)) four new PP loci (at 4q12 near CHIC2, 7q22.3 near PIK3CG, 8q24.12 in NOV and 11q24.3 near ADAMTS8), two new MAP loci (3p21.31 in MAP4 and 10q25.3 near ADRB1) and one locus associated with both of these traits (2q24.3 near FIGN) that has also recently been associated with SBP in east Asians. For three of the new PP loci, the estimated effect for SBP was opposite of that for DBP, in contrast to the majority of common SBP- and DBP-associated variants, which show concordant effects on both traits. These findings suggest new genetic pathways underlying blood pressure variation, some of which may differentially influence SBP and DBP

Target genes, variants, tissues and transcriptional pathways influencing human serum urate levels.

Elevated serum urate levels cause gout and correlate with cardiometabolic diseases via poorly understood mechanisms. We performed a trans-ancestry genome-wide association study of serum urate in 457,690 individuals, identifying 183 loci (147 previously unknown) that improve the prediction of gout in an independent cohort of 334,880 individuals. Serum urate showed significant genetic correlations with many cardiometabolic traits, with genetic causality analyses supporting a substantial role for pleiotropy. Enrichment analysis, fine-mapping of urate-associated loci and colocalization with gene expression in 47 tissues implicated the kidney and liver as the main target organs and prioritized potentially causal genes and variants, including the transcriptional master regulators in the liver and kidney, HNF1A and HNF4A. Experimental validation showed that HNF4A transactivated the promoter of ABCG2, encoding a major urate transporter, in kidney cells, and that HNF4A p.Thr139Ile is a functional variant. Transcriptional coregulation within and across organs may be a general mechanism underlying the observed pleiotropy between urate and cardiometabolic traits.The Genotype-Tissue Expression (GTEx) Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health, and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS. Variant annotation was supported by software resources provided via the Caché Campus program of the InterSystems GmbH to Alexander Teumer

New loci for body fat percentage reveal link between adiposity and cardiometabolic disease risk

To increase our understanding of the genetic basis of adiposity and its links to cardiometabolic disease risk, we conducted a genome-wide association meta-analysis of body fat percentage (BF%) in up to 100,716 individuals. Twelve loci reached genome-wide significance (P<5 × 10−8), of which eight were previously associated with increased overall adiposity (BMI, BF%) and four (in or near COBLL1/GRB14, IGF2BP1, PLA2G6, CRTC1) were novel associations with BF%. Seven loci showed a larger effect on BF% than on BMI, suggestive of a primary association with adiposity, while five loci showed larger effects on BMI than on BF%, suggesting association with both fat and lean mass. In particular, the loci more strongly associated with BF% showed distinct cross-phenotype association signatures with a range of cardiometabolic traits revealing new insights in the link between adiposity and disease risk

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead