Integrated methodologies to study human transcriptional regulation from functional genomics data

Abstract : The human body is composed of a huge number of cells acting together in a concerted manner. The current understanding is that proteins perform most of the necessary activities in keeping a cell alive. The DNA, on the other hand, stores the information on how to produce the different proteins in the genome. Regulating gene transcription is the first important step that can thus affect the life of a cell, modify its functions and its responses to the environment. Regulation is a complex operation that involves specialized proteins, the transcription factors. Transcription factors (TFs) can bind to DNA and activate the processes leading to the expression of genes into new proteins. Errors in this process may lead to diseases. In particular, some transcription factors have been associated with a lethal pathological state, commonly known as cancer, associated with uncontrolled cellular proliferation, invasiveness of healthy tissues and abnormal responses to stimuli. Understanding cancer-related regulatory programs is a difficult task, often involving several TFs interacting together and influencing each other's activity. This Thesis presents new computational methodologies to study gene regulation. In addition we present applications of our methods to the understanding of cancer-related regulatory programs. The understanding of transcriptional regulation is a major challenge. We address this difficult question combining computational approaches with large collections of heterogeneous experimental data. In detail, we design signal processing tools to recover transcription factors binding sites on the DNA from genome-wide surveys like chromatin immunoprecipitation assays on tiling arrays (ChIP-chip). We then use the localization about the binding of TFs to explain expression levels of regulated genes. In this way we identify a regulatory synergy between two TFs, the oncogene C-MYC and SP1. C-MYC and SP1 bind preferentially at promoters and when SP1 binds next to C-NIYC on the DNA, the nearby gene is strongly expressed. The association between the two TFs at promoters is reflected by the binding sites conservation across mammals, by the permissive underlying chromatin states 'it represents an important control mechanism involved in cellular proliferation, thereby involved in cancer. Secondly, we identify the characteristics of TF estrogen receptor alpha (hERa) target genes and we study the influence of hERa in regulating transcription. hERa, upon hormone estrogen signaling, binds to DNA to regulate transcription of its targets in concert with its co-factors. To overcome the scarce experimental data about the binding sites of other TFs that may interact with hERa, we conduct in silico analysis of the sequences underlying the ChIP sites using the collection of position weight matrices (PWMs) of hERa partners, TFs FOXA1 and SP1. We combine ChIP-chip and ChIP-paired-end-diTags (ChIP-pet) data about hERa binding on DNA with the sequence information to explain gene expression levels in a large collection of cancer tissue samples and also on studies about the response of cells to estrogen. We confirm that hERa binding sites are distributed anywhere on the genome. However, we distinguish between binding sites near promoters and binding sites along the transcripts. The first group shows weak binding of hERa and high occurrence of SP1 motifs, in particular near estrogen responsive genes. The second group shows strong binding of hERa and significant correlation between the number of binding sites along a gene and the strength of gene induction in presence of estrogen. Some binding sites of the second group also show presence of FOXA1, but the role of this TF still needs to be investigated. Different mechanisms have been proposed to explain hERa-mediated induction of gene expression. Our work supports the model of hERa activating gene expression from distal binding sites by interacting with promoter bound TFs, like SP1. hERa has been associated with survival rates of breast cancer patients, though explanatory models are still incomplete: this result is important to better understand how hERa can control gene expression. Thirdly, we address the difficult question of regulatory network inference. We tackle this problem analyzing time-series of biological measurements such as quantification of mRNA levels or protein concentrations. Our approach uses the well-established penalized linear regression models where we impose sparseness on the connectivity of the regulatory network. We extend this method enforcing the coherence of the regulatory dependencies: a TF must coherently behave as an activator, or a repressor on all its targets. This requirement is implemented as constraints on the signs of the regressed coefficients in the penalized linear regression model. Our approach is better at reconstructing meaningful biological networks than previous methods based on penalized regression. The method is tested on the DREAM2 challenge of reconstructing a five-genes/TFs regulatory network obtaining the best performance in the "undirected signed excitatory" category. Thus, these bioinformatics methods, which are reliable, interpretable and fast enough to cover large biological dataset, have enabled us to better understand gene regulation in humans.

Spatial weights : constructing weight-compatible exchange matrices from proximity matrices

Exchange matrices represent spatial weights as symmetric probability distributions on pairs of regions, whose margins yield regional weights, generally well-specified and known in most contexts. This contribution proposes a mechanism for constructing exchange matrices, derived from quite general symmetric proximity matrices, in such a way that the margin of the exchange matrix coincides with the regional weights. Exchange matrices generate in turn diffusive squared Euclidean dissimilarities, measuring spatial remoteness between pairs of regions. Unweighted and weighted spatial frameworks are reviewed and compared, regarding in particular their impact on permutation and normal tests of spatial autocorrelation. Applications include tests of spatial autocorrelation with diagonal weights, factorial visualization of the network of regions, multivariate generalizations of Moran's I, as well as "landscape clustering", aimed at creating regional aggregates both spatially contiguous and endowed with similar features.

The joint influence of gender and amount of smoking on weight gain one year after smoking cessation.

Weight gain is often associated with smoking cessation and may discourage smokers from quitting. This study estimated the weight gained one year after smoking cessation and examined the risk factors associated with weight gain in order to identify socio-demographic groups at higher risk of increased weight after quitting. We analyzed data from 750 adults in two randomized controlled studies that included smokers motivated to quit and found a gradient in weight gain according to the actual duration of abstinence during follow-up. Subjects who were abstinent for at least 40 weeks gained 4.6 kg (SD = 3.8) on average, compared to 1.2 kg (SD = 2.6) for those who were abstinent less than 20 weeks during the 1-year follow-up. Considering the duration of abstinence as an exposure variable, we found an age effect and a significant interaction between sex and the amount of smoking before quitting: younger subjects gained more weight than older subjects; among light smokers, men gained more weight on average than women one year after quitting, while the opposite was observed among heavy smokers. Young women smoking heavily at baseline had the highest risk of weight gain after quitting.

Cost-effectiveness of low-molecular-weight heparin for secondary prophylaxis of cancer-related venous thromboembolism.

Although extended secondary prophylaxis with low-molecular-weight heparin was recently shown to be more effective than warfarin for cancer-related venous thromboembolism, its cost-effectiveness compared to traditional prophylaxis with warfarin is uncertain. We built a decision analytic model to evaluate the clinical and economic outcomes of a 6-month course of low-molecular-weight heparin or warfarin therapy in 65-year-old patients with cancer-related venous thromboembolism. We used probability estimates and utilities reported in the literature and published cost data. Using a US societal perspective, we compared strategies based on quality-adjusted life-years (QALYs) and lifetime costs. The incremental cost-effectiveness ratio of low-molecular-weight heparin compared with warfarin was 149,865 dollars/QALY. Low-molecular-weight heparin yielded a quality-adjusted life expectancy of 1.097 QALYs at the cost of 15,329 dollars. Overall, 46% (7108 dollars) of the total costs associated with low-molecular-weight heparin were attributable to pharmacy costs. Although the low-molecular-weigh heparin strategy achieved a higher incremental quality-adjusted life expectancy than the warfarin strategy (difference of 0.051 QALYs), this clinical benefit was offset by a substantial cost increment of 7,609 dollars. Cost-effectiveness results were sensitive to variation of the early mortality risks associated with low-molecular-weight heparin and warfarin and the pharmacy costs for low-molecular-weight heparin. Based on the best available evidence, secondary prophylaxis with low-molecular-weight heparin is more effective than warfarin for cancer-related venous thromboembolism. However, because of the substantial pharmacy costs of extended low-molecular-weight heparin prophylaxis in the US, this treatment is relatively expensive compared with warfarin.

Consequences of smoking for body weight, body fat distribution, and insulin resistance

Our aim was to critically evaluate the relations among smoking, body weight, body fat distribution, and insulin resistance as reported in the literature. In the short term, nicotine increases energy expenditure and could reduce appetite, which may explain why smokers tend to have lower body weight than do nonsmokers and why smoking cessation is frequently followed by weight gain. In contrast, heavy smokers tend to have greater body weight than do light smokers or nonsmokers, which likely reflects a clustering of risky behaviors (eg, low degree of physical activity, poor diet, and smoking) that is conducive to weight gain. Other factors, such as weight cycling, could also be involved. In addition, smoking increases insulin resistance and is associated with central fat accumulation. As a result, smoking increases the risk of metabolic syndrome and diabetes, and these factors increase risk of cardiovascular disease. In the context of the worldwide obesity epidemic and a high prevalence of smoking, the greater risk of (central) obesity and insulin resistance among smokers is a matter of major concern

ADOS: an educational primary prevention programme for preventing excess body weight in adolescents.

OBJECTIVE: The prevalence of adolescent obesity has increased considerably over the past decade in Switzerland and has become a serious public health problem in Europe. Prevention of obesity using various comprehensive programmes appears to be very promising, although we must admit that several interventions had generally disappointing results compared with the objectives and target initially fixed. Holistic programmes including nutritional education combined with promotion of physical activity and behaviour modification constitute the key factors in the prevention of childhood and adolescent obesity. The purpose of this programme was to incorporate nutrition/physical education as well as psychological aspects in selected secondary schools (9th grade, 14-17 years). METHODS: The educational strategy was based on the development of a series of 13 practical workshops covering wide areas such as physical inactivity, body composition, sugar, energy density, invisible lipids, how to read food labels, is meal duration important? Do you eat with pleasure or not? Do you eat because you are hungry? Emotional eating. For teachers continuing education, a basic highly illustrated guide was developed as a companion booklet to the workshops. These materials were first validated by biology, physical education, dietician and psychologist teachers as well as school medical officers. RESULTS: Teachers considered the practical educational materials innovative and useful, motivational and easy to understand. Up to now (early 2008), the programme has been implemented in 50 classes or more from schools originating from three areas in the French part of Switzerland. Based on the 1-week pedometer value assessed before and after the 1 school-year programme, an initial evaluation indicated that overall physical placidity was significantly decreased as evidenced by a significant rise in the number of steps per day. CONCLUSION: Future evaluation will provide more information on the effectiveness of the ADOS programme.

Impact of the C2/C6 ratio of high-molecular-weight hydroxyethyl starch on pharmacokinetics and blood coagulation in pigs

Rapport de synthèse : Contexte: l'hydroxyéthylamidon (HEA) est largement utilisé comme expanseur volémique en anesthésiologie et réanimation. Cependant, cette classe de produits perturbe le system de la coagulation. Des améliorations restent possibles dans le choix de la combinaison optimale de poids moléculaire, de degré de substitution en radicaux éthyle et de localisation de ces radicaux sur le squelette glucidique des polymères, afin d'optimiser leur efficacité et leur tolérance. L'HEA de poids moléculaire élevé et faiblement substitué n'affecte pas plus la coagulation sanguine que de l'HEA de bas poids moléculaire faiblement substitué. Nous examinons in vivo l'effet d'un abaissement du rapport C2/C6 sur les caractéristiques pharmacocinétiques et l'impact sur la coagulation sanguine d'un HEA de haut poids moléculaire faiblement substitué. Matériels et méthode: nous comparons dans une étude prospective, randomisée et parallèle l'HEA 650/0.42/2.8 avec l'HEA 650/0.42/5.6 auprès de 30 cochons. Avant, pendant et jusqu'à 630 minutes après une perfusion de 30 ml/kg d'HEA, des échantillons sanguins ont été collectés pour mesurer les concentrations d'HEA, les tests de coagulation plasmatique classiques et la coagulation sanguine par thrombélastographie (TEG®, Haemoscope Corporation, Niles, II, U.S.). Les paramètres pharmacocinétiques ont été estimés en adaptant un modèle à deux compartiments. Résultats: la constante d'élimination est de 0.009 ± 0.001 (min-1) pour l'HEA 650/0.42/2.8 et 0.007 ± 0.001 (min-1) pour l'HEA 650/0.42/5.6 (p<0.001); la surface sous la courbe de concentration est de 1374 ± 340 min*g/L pour l'HEA 650/0.42/2.8 et 1697 ± 411 min*g/L pour l'HEA 650/0.42/5.6 (p=0.026). Les concentrations mesurées d'HEA ne montrent pas de différence entre l'HEA 650/0.42/2.8 et l'HEA 650/0.42/5.6. Les deux solutions d'HEA affectent de façon identique la coagulation sanguine: l'index de coagulation thrombélastographique diminue pareillement à ta fin de la perfusion d'HEA 650/0.42/2.8 et d'HEA 650/0.42/5.6 (p=0.29). De même, le temps de thromboplastine partielle activée et le temps de prothrombine augmentent de manière similaire pour l'HEA 650/0.42/2.8 et l'HEA 650/0.42/5.6 (p=0.83). Conclusion: la réduction du rapport C2/C6 de l'HEA de poids moléculaire élevé et faiblement substitué aboutit à une élimination légèrement accélérée d'HEA. Cependant, elle ne modifie pas l'effet perturbateur sur la coagulation.

Maximal Physiological Parameters during Partial Body-Weight Support Treadmill Testing.

PURPOSE: This study investigated maximal cardiometabolic response while running in a lower body positive pressure treadmill (antigravity treadmill (AG)), which reduces body weight (BW) and impact. The AG is used in rehabilitation of injuries but could have potential for high-speed running, if workload is maximally elevated. METHODS: Fourteen trained (nine male) runners (age 27 ± 5 yr; 10-km personal best, 38.1 ± 1.1 min) completed a treadmill incremental test (CON) to measure aerobic capacity and heart rate (V˙O2max and HRmax). They completed four identical tests (48 h apart, randomized order) on the AG at BW of 100%, 95%, 90%, and 85% (AG100 to AG85). Stride length and rate were measured at peak velocities (Vpeak). RESULTS: V˙O2max (mL·kg·min) was similar across all conditions (men: CON = 66.6 (3.0), AG100 = 65.6 (3.8), AG95 = 65.0 (5.4), AG90 = 65.6 (4.5), and AG85 = 65.0 (4.8); women: CON = 63.0 (4.6), AG100 = 61.4 (4.3), AG95 = 60.7 (4.8), AG90 = 61.4 (3.3), and AG85 = 62.8 (3.9)). Similar results were found for HRmax, except for AG85 in men and AG100 and AG90 in women, which were lower than CON. Vpeak (km·h) in men was 19.7 (0.9) in CON, which was lower than every other condition: AG100 = 21.0 (1.9) (P < 0.05), AG95 = 21.4 (1.8) (P < 0.01), AG90 = 22.3 (2.1) (P < 0.01), and AG85 = 22.6 (1.6) (P < 0.001). In women, Vpeak (km·h) was similar between CON (17.8 (1.1) ) and AG100 (19.3 (1.0)) but higher at AG95 = 19.5 (0.4) (P < 0.05), AG90 = 19.5 (0.8) (P < 0.05), and AG85 = 21.2 (0.9) (P < 0.01). CONCLUSIONS: The AG can be used at maximal exercise intensities at BW of 85% to 95%, reaching faster running speeds than normally feasible. The AG could be used for overspeed running programs at the highest metabolic response levels.

Nuclear Factor I genomic binding associates with chromatin boundaries.

BACKGROUND: The Nuclear Factor I (NFI) family of DNA binding proteins (also called CCAAT box transcription factors or CTF) is involved in both DNA replication and gene expression regulation. Using chromatin immuno-precipitation and high throughput sequencing (ChIP-Seq), we performed a genome-wide mapping of NFI DNA binding sites in primary mouse embryonic fibroblasts. RESULTS: We found that in vivo and in vitro NFI DNA binding specificities are indistinguishable, as in vivo ChIP-Seq NFI binding sites matched predictions based on previously established position weight matrix models of its in vitro binding specificity. Combining ChIP-Seq with mRNA profiling data, we found that NFI preferentially associates with highly expressed genes that it up-regulates, while binding sites were under-represented at expressed but unregulated genes. Genomic binding also correlated with markers of transcribed genes such as histone modifications H3K4me3 and H3K36me3, even outside of annotated transcribed loci, implying NFI in the control of the deposition of these modifications. Positional correlation between + and - strand ChIP-Seq tags revealed that, in contrast to other transcription factors, NFI associates with a nucleosomal length of cleavage-resistant DNA, suggesting an interaction with positioned nucleosomes. In addition, NFI binding prominently occurred at boundaries displaying discontinuities in histone modifications specific of expressed and silent chromatin, such as loci submitted to parental allele-specific imprinted expression. CONCLUSIONS: Our data thus suggest that NFI nucleosomal interaction may contribute to the partitioning of distinct chromatin domains and to epigenetic gene expression regulation.NFI ChIP-Seq and input control DNA data were deposited at Gene Expression Omnibus (GEO) repository under accession number GSE15844. Gene expression microarray data for mouse embryonic fibroblasts are on GEO accession number GSE15871.

Predictors of weight change in sedentary smokers receiving a standard smoking cessation intervention

L'arrêt de la cigarette est généralement associé à une prise de poids. Celle-ci peut menacer la motivation des fumeurs à s'engager dans un processus d'arrêt du tabac et constitue un motif de rechute. L'ordre de grandeur et la cinétique de la prise de poids liée à une tentative d'arrêt chez les fumeurs pris en charge selon les recommandations cliniques actuelles est peu décrite dans la littérature médicale. Le but de cette étude était de quantifier cette prise de poids, d'en déterminer la cinétique ainsi que les facteurs qui l'influencent, chez des fumeurs sédentaires bénéficiant d'une intervention d'aide à l'arrêt du tabac individualisée, composée de conseils individuels et d'une substitution nicotinique associant plusieurs modes d'administration. Nous avons analysé des données récoltées durant un essai clinique randomisé contrôlé au cours duquel était étudié l'impact d'une activité physique modérée sur les taux d'arrêt du tabac après un an chez des fumeurs sédentaires. Nous avons modélisé l'évolution du poids de l'ensemble des participants au cours du temps, selon la technique statistique des « modèles mixtes longitudinaux ». En séparant les périodes d'abstinence de la cigarette de celles de rechute et de l'utilisation reportée de substituts nicotiniques. Cette approche nous a permis de prendre en compte chaque participant à l'étude, par opposition à un modèle plus simple qui séparerait les sujets abstinents de ceux qui rechutent à n'importe quel moment de la période de suivi. Nous avons également ajusté ces modèles pour l'âge, le sexe, le niveau de dépendance à la nicotine et le niveau de formation des participants. Parmi l'ensemble des participants, nous avons noté une augmentation du poids durant les trois premiers mois de l'intervention, suivie d'une stabilisation. Au total, la prise de poids moyenne s'est élevée à 3.3 kg pour les femmes et 3.9 kg pour les hommes. Durant les périodes d'abstinence, les caractéristiques suivantes étaient associées à la prise de poids : sexe masculin et forte dépendance nicotinique. Un âge supérieur à 43 ans était associé à une prise de poids également durant les périodes de rechute. Nous avons observé une tendance, non statistiquement significative, vers une réduction de la prise des poids avec l'utilisation de substituts nicotiniques. Notre étude apporte de nouvelles données sur l'évolution du poids chez les fumeurs sédentaires qui bénéficient d'une intervention d'aide à l'arrêt du tabac. Ils prennent donc du poids, de manière modérée et limitée aux premiers mois. Parmi eux, les hommes, les individus les plus dépendants à la nicotine et les plus âgés doivent s'attendre à une prise de poids supérieure à la moyenne.

The complement inhibitor low molecular weight dextran sulfate prevents TLR4-induced phenotypic and functional maturation of human dendritic cells.

Low molecular weight dextran sulfate (DXS) has been reported to inhibit the classical, alternative pathway as well as the mannan-binding lectin pathway of the complement system. Furthermore, it acts as an endothelial cell protectant inhibiting complement-mediated endothelial cell damage. Endothelial cells are covered with a layer of heparan sulfate (HS), which is rapidly released under conditions of inflammation and tissue injury. Soluble HS induces maturation of dendritic cells (DC) via TLR4. In this study, we show the inhibitory effect of DXS on human DC maturation. DXS significantly prevents phenotypic maturation of monocyte-derived DC and peripheral myeloid DC by inhibiting the up-regulation of CD40, CD80, CD83, CD86, ICAM-1, and HLA-DR and down-regulates DC-SIGN in response to HS or exogenous TLR ligands. DXS also inhibits the functional maturation of DC as demonstrated by reduced T cell proliferation, and strongly impairs secretion of the proinflammatory mediators IL-1beta, IL-6, IL-12p70, and TNF-alpha. Exposure to DXS leads to a reduced production of the complement component C1q and a decreased phagocytic activity, whereas C3 secretion is increased. Moreover, DXS was found to inhibit phosphorylation of IkappaB-alpha and activation of NF-kappaB. These findings suggest that DXS prevents TLR-induced maturation of human DC and may therefore be a useful reagent to impede the link between innate and adaptive immunity.

Prévenir la prise de poids à l'arrêt du tabac: pas de solution miracle: revue Cochrane pour le praticien. [Preventing weight gain in smoking cessation: there is no miracle solution]

http://resfranco.cochrane.org/sites/resfranco.cochrane.org/files/uploads/Arrettabac2009.pdf