Use of adenoviral E1A protein to analyze K18 promoter deregulation in colon carcinoma cells discloses a role for CtBP1 and BRCA1

BACKGROUND: The promoter of the keratin 18 (K18) gene is 5- to 10-fold more active in tumorigenic (T-type) cell clones derived from the SW613-S human colon carcinoma cell line than in non-tumorigenic (NT-type) clones. We have reported previously that the mechanism responsible for this differential activity is acting on the minimal K18 promoter (TATA box and initiation site). This mechanism does not require the binding of a factor to a specific site on the DNA but involves the acetylation of a non-histone substrate. To get further insight into this mechanism, we investigated the effect of the adenovirus E1A protein on the activity of the K18 promoter, both in T and NT cells. RESULTS: Wild type adenovirus E1A protein and C-terminal deletion mutants inhibit the K18 promoter, specifically in T-type cells. The domain responsible for this inhibitory effect is located in the 12–25 region of the viral protein. E1A mutants that have lost this region but retain the PLDLS motif (the C-terminal binding site for CtBP1) stimulate the K18 promoter, specifically in NT cells. The inhibitory or stimulatory effects of the different E1A mutants are not dependent on a particular sequence of the promoter. An E1A N-terminal deletion mutant carrying point mutations in the PLDLS motif cannot stimulate the K18 promoter. CtBP1 interacts with CtIP, which is a known partner of BRCA1, itself a component of the RNA polymerase II holoenzyme. The stimulatory effect of two BRCA1 mutants, specifically in NT cells, implicates a tripartite BRCA1-CtIP-CtBP1 complex in the regulation of the K18 promoter. CONCLUSION: Since we have shown previously that the K18 promoter is stimulated by deacetylase inhibitors, specifically in NT cells, we conclude that the activity of the promoter is repressed in NT cells by a mechanism involving the recruitment, by a BRCA1/CtIP complex, of CtBP1 and associated deacetylases to the preinitiation complex. We propose a model depicting the mechanism responsible for the differential activity of the K18 promoter between T and NT cells of the SW613-S cell line

Sequence-based GWAS, network and pathway analyses reveal genes co-associated with milk cheese-making properties and milk composition in Montbéliarde cows

Background Milk quality in dairy cattle is routinely assessed via analysis of mid-infrared (MIR) spectra; this approach can also be used to predict the milk’s cheese-making properties (CMP) and composition. When this method of high-throughput phenotyping is combined with efficient imputations of whole-genome sequence data from cows’ genotyping data, it provides a unique and powerful framework with which to carry out genomic analyses. The goal of this study was to use this approach to identify genes and gene networks associated with milk CMP and composition in the Montbéliarde breed. Results Milk cheese yields, coagulation traits, milk pH and contents of proteins, fatty acids, minerals, citrate, and lactose were predicted from MIR spectra. Thirty-six phenotypes from primiparous Montbéliarde cows (1,442,371 test-day records from 189,817 cows) were adjusted for non-genetic effects and averaged per cow. 50 K genotypes, which were available for a subset of 19,586 cows, were imputed at the sequence level using Run6 of the 1000 Bull Genomes Project (comprising 2333 animals). The individual effects of 8.5 million variants were evaluated in a genome-wide association study (GWAS) which led to the detection of 59 QTL regions, most of which had highly significant effects on CMP and milk composition. The results of the GWAS were further subjected to an association weight matrix and the partial correlation and information theory approach and we identified a set of 736 co-associated genes. Among these, the well-known caseins, PAEP and DGAT1, together with dozens of other genes such as SLC37A1, ALPL, MGST1, SEL1L3, GPT, BRI3BP, SCD, GPAT4, FASN, and ANKH, explained from 12 to 30% of the phenotypic variance of CMP traits. We were further able to identify metabolic pathways (e.g., phosphate and phospholipid metabolism and inorganic anion transport) and key regulator genes, such as PPARA, ASXL3, and bta-mir-200c that are functionally linked to milk composition. Conclusions By using an approach that integrated GWAS with network and pathway analyses at the whole-genome sequence level, we propose candidate variants that explain a substantial proportion of the phenotypic variance of CMP traits and could thus be included in genomic evaluation models to improve milk CMP in Montbéliarde cows.info:eu-repo/semantics/publishedVersio

Sequence-based GWAS, network and pathway analyses reveal genes co-associated with milk cheese-making properties and milk composition in Montbéliarde cows

International audienceAbstractBackgroundMilk quality in dairy cattle is routinely assessed via analysis of mid-infrared (MIR) spectra; this approach can also be used to predict the milk’s cheese-making properties (CMP) and composition. When this method of high-throughput phenotyping is combined with efficient imputations of whole-genome sequence data from cows’ genotyping data, it provides a unique and powerful framework with which to carry out genomic analyses. The goal of this study was to use this approach to identify genes and gene networks associated with milk CMP and composition in the Montbéliarde breed.ResultsMilk cheese yields, coagulation traits, milk pH and contents of proteins, fatty acids, minerals, citrate, and lactose were predicted from MIR spectra. Thirty-six phenotypes from primiparous Montbéliarde cows (1,442,371 test-day records from 189,817 cows) were adjusted for non-genetic effects and averaged per cow. 50 K genotypes, which were available for a subset of 19,586 cows, were imputed at the sequence level using Run6 of the 1000 Bull Genomes Project (comprising 2333 animals). The individual effects of 8.5 million variants were evaluated in a genome-wide association study (GWAS) which led to the detection of 59 QTL regions, most of which had highly significant effects on CMP and milk composition. The results of the GWAS were further subjected to an association weight matrix and the partial correlation and information theory approach and we identified a set of 736 co-associated genes. Among these, the well-known caseins, PAEP and DGAT1, together with dozens of other genes such as SLC37A1, ALPL, MGST1, SEL1L3, GPT, BRI3BP, SCD, GPAT4, FASN, and ANKH, explained from 12 to 30% of the phenotypic variance of CMP traits. We were further able to identify metabolic pathways (e.g., phosphate and phospholipid metabolism and inorganic anion transport) and key regulator genes, such as PPARA, ASXL3, and bta-mir-200c that are functionally linked to milk composition.ConclusionsBy using an approach that integrated GWAS with network and pathway analyses at the whole-genome sequence level, we propose candidate variants that explain a substantial proportion of the phenotypic variance of CMP traits and could thus be included in genomic evaluation models to improve milk CMP in Montbéliarde cows

FROM’MIR : Développer des outils de prédiction et de conseil pour maîtriser la fromageabilité des laits destinés à la fabrication des fromages traditionnels franc-comtois

Ce volume regroupe les textes issus du programme Casdar "Innovation et Partenariat" et "Recherche finalisée et innovation" de 2014. Il a été réalisé sous l’égide du GIS Relance Agronomique.Mid-infrared spectroscopy prediction equations of the cheese-making properties of milk, established inthe Franche-Comté PDO/PGI context, exist for the first time in France. Laboratory curd yield in DryMatter was consistent with the yields observed in mini-manufactures of soft and pressed cookedcheeses and it is the best predicted parameter. Under our conditions, some coagulation properties suchas curd firmness could be estimated. The acidification properties, which heavily depend on themicrobiological component of milk, are poorly estimated. The best prediction performances wereobtained on individual cow milks. The performances were poorer on the scale of bulk milks, herd tankmilk but especially dairy vat milk. The study of variation factors made it possible to highlight theimportant weight of genetics with a high level of heritability and strong effects of the genome regionsinvolved. The quality and quantity of fodder and the distribution of calves were influential in the contextstudied. In this same context, few factors of variation have been identified at the scale of dairy vat milks,as the practices were very much governed by the PDO specifications. At the end of this project, anobservatory, from the quality of the milk to the quality of the cheese, will be set up in Franche Comté.Studies will also be carried out at the national level to consolidate and improve the equations in othercontexts.Des équations MIR (spectrométrie moyen infrarouge) d'estimation de la fromageabilité des laits,établies en contexte AOP/IGP franc-comtois, existent pour la première fois en France. Le rendementlaboratoire extrait sec (ES), cohérent avec les rendements observés en mini-fabrications de fromages àpâte molle et à pâte pressée cuite, est le paramètre le mieux prédit. Dans nos conditions, certainsaspects de l'aptitude à la coagulation enzymatique, comme la fermeté des gels, peuvent être estimés.L’aptitude à l’acidification, dépendant fortement de la composante microbiologique des laits, est quant àelle mal estimée. Les meilleures performances de prédiction sont obtenues sur les laits individuels devaches. Les performances sont moins bonnes à l’échelle des laits de mélange, des laits de troupeauxmais surtout des laits de cuves de fromagerie. L'étude des facteurs de variation a permis de mettre enévidence le poids important de la génétique avec un niveau d’héritabilité élevé et des effets forts desrégions du génome impliquées. La qualité et la quantité de fourrages ainsi que la répartition desvêlages sont influents dans le contexte étudié. Dans ce même contexte, peu de facteurs de variationont été mis en évidence à l’échelle des laits de cuves, les pratiques étant très encadrées par le cahierdes charges AOP. A l’issue de ce projet, un observatoire, depuis la qualité des laits jusqu’à celle desfromages, va être mis en place en Franche Comté. Des études seront aussi mises en œuvre au niveaunational pour permettre notamment une consolidation et une amélioration des équations dans d'autrescontextes

The INTAQT project’s stakeholders’ involvement: impact on the research work?

International audienceMulti-actor research projects are still a recent trend, putting at work together researchers with non-academic partners,like representants of food chain actors. The INTAQT project (INnovative Tools for Assessment and Authenticationof chicken meat, beef and dairy products’ QualiTies) has chosen to play the game fully: the project was set up withstakeholders’ consultations at the heart of the project and with their recommendations implemented in the research.Stakeholders from the beef, dairy or poultry sectors in seven European countries are involved from producers toretailers, including processors and other relevant actors in each context. They were consulted at three levels: firstthrough individual qualitative interviews then invited to national group discussions and some of them to Europeangroup meetings. Their role was to give their opinions and suggestions for the choice of farming systems to be studiedin the project and on the choice of quality criteria to take into account and on analysis to be conducted. They will beinvolved all along the project life with their contribution next year to the co-construction of the multi-criteria scoringtool. This presentation is based on analysis of interviews with the project’s Work Package leaders and Task Leadersin order to confirm or refute the following hypotheses: (1) there was an a priori reticence of some of the researchersabout the importance and relevance of including stakeholders’ voices in a research project; (2) after almost of 2 yearsin the process, there is an interest in the world of research, but also difficulties in effectively integrating positionsthat are difficult to reconcile (temporality, decision-making methods, governance, etc.). Globally we reflect on howmulti-actor approach like the one in INTAQT questions a research project

SW613-3 (T) or SW613-B3 (NT) cells were transfected with plasmid pK18(80)luc alone (none) or in the presence of 24 μg of plasmids pCBRCA1C (BRCA1wt), pcDNA3-BRCA1(Y1853→STOP) or pcDNA3-BRCT, as indicated

<p><b>Copyright information:</b></p><p>Taken from "Use of adenoviral E1A protein to analyze K18 promoter deregulation in colon carcinoma cells discloses a role for CtBP1 and BRCA1"</p><p>BMC Molecular Biology 2005;6():8-8.</p><p>Published online 14 Apr 2005</p><p>PMCID:PMC1087485.</p><p></p> Results are presented as in figure 3. Error bars represent standard deviation of the mean of three experiments. and Expression level of the BRCA1 constructs. SW613-3 (3) or SW613-B3 (B3) cells were untransfected (mock) or transfected with plasmid pCBRCA1C (wt), pcDNA3-BRCA1(Y1853→STOP) (mut) or pcDNA3-BRCT, as indicated. Cellular extracts were analyzed by Western blotting with an anti-HA antibody

Use of adenoviral E1A protein to analyze K18 promoter deregulation in colon carcinoma cells discloses a role for CtBP1 and BRCA1-8

<p><b>Copyright information:</b></p><p>Taken from "Use of adenoviral E1A protein to analyze K18 promoter deregulation in colon carcinoma cells discloses a role for CtBP1 and BRCA1"</p><p>BMC Molecular Biology 2005;6():8-8.</p><p>Published online 14 Apr 2005</p><p>PMCID:PMC1087485.</p><p></p>if. The horizontal line represents the DNA and the initiation site is symbolized by an arrow, whose thickness is an indication of the activity of the promoter. Functioning of the K18 promoter in T and NT cells. The F factor is an as yet unidentified protein which has a HAT/FAT activity and is supposedly more abundant or more active in T cells (bold) than in NT cells. F acetylates (Ac) the substrate protein S, a non-histone protein whose acetylation level controls the activity of the K18 promoter. The acetylation level of S is also under the control of HDAC proteins. The CtBP1 protein interacts with the PLDLS motif of the CtIP protein. CtBP1 associates with some HDAC proteins by a domain that is different from the one recognizing the PLDLS motif [4, 48]. The recruited HDAC proteins deacetylate the S substrate in NT cells where the acetylase activity is already weak and this down-regulates the promoter. CtIP is an interaction partner of BRCA1 which is represented here as part of the preinitiation complex (PIC). Effects of C-terminal deletion mutants of E1A on the activity of the K18 promoter in T and NT cells. The binding of these mutants to the F factor through the 12–25 domain results in a low HAT/FAT activity, deacetylation of S and inhibition of the promoter in T cells. These mutants are expected to have little effect in NT cells since the level of acetylation of S is already low in these cells. Effects of N-terminal deletion mutants of E1A on the activity of the K18 promoter. The C-terminal part of E1A contains the PLDLS motif which permits its interaction with CtBP1. The displacement of CtBP1 from CtIP prevents the recruitment of HDAC proteins to the preinitiation complex. This is of no consequence in T cells where the S protein is maintained in an hyperacetylated state by the high HAT/FAT activity. In contrast, in NT cells removal of HDAC proteins allows the weak HAT/FAT activity to acetylate S to a high level which results in a stimulation of the promoter

Hepatic global DNA Hypomethylation Phenotype in Rainbow Trout Fed Diets Varying in Carbohydrate to Protein Ratio

International audienceAbstract Background A high carbohydrate-low protein diet can induce hepatic global DNA hypomethylation in trout. The mechanisms remain unclear. Objective We aimed to investigate whether increase in dietary carbohydrates (dHC) or decrease in dietary proteins (dLP) can cause hepatic global DNA hypomethylation, and to explore the underlying mechanisms in trout. Methods Two feeding trials were conducted on juvenile males, both of which involved a 4-day fasting and 4-day refeeding protocol. In Trial 1, trout were fed either a high protein-no carbohydrate (HP-NC, protein 60% dry matter (DM), carbohydrates 0% DM) or a moderate protein-high carbohydrate (MP-HC, protein 40% DM, carbohydrates 30% DM) diet. In Trial 2, fish were fed either a moderate protein-no carbohydrate (MP-NC, protein 40% DM, carbohydrates 0% DM), a MP-HC (protein 40% DM, carbohydrates 30% DM), or a low protein-no carbohydrate (LP-NC, protein 20% DM, carbohydrates 0% DM) diet to separate the effects of dHC and dLP on the hepatic methylome. Global CmCGG methylation, DNA demethylation derivative levels, and mRNA expression of DNA (de)methylation-related genes were measured. Differences were tested by one-way ANOVA when data were normally distributed or by Kruskal-Wallis non-parametric test if not. Results In both trails, global CmCGG methylation levels remained unaffected, but the hepatic 5-mdC content decreased after refeeding (1–3%). The MP-HC group had 3.4-fold higher hepatic 5-hmdC and a similar 5-mdC level compared to the HP-NC group in Trial 1. Both MP-HC and LP-NC diets lowered the hepatic 5-mdC content (1–2%), but only the LP-NC group had a significantly lower 5-hmdC level (P &lt; 0.01) compared with MP-NC group in Trail 2. Conclusions dHC and dLP independently induced hepatic global DNA demethylation in trout. The alterations in other methylation derivatives levels indicated the demethylation process was achieved through an active demethylation pathway and probably occurred at non-CmCGG sites

Towards a genomic evaluation of cheese-making traits including candidate SNP in Montbéliarde cows

International audienc

Implementation of husbandry practices improving quality and sustainability: a living lab approach

International audienceThe living lab approach to innovation is receiving increasing attention also in the agricultural sector in view of thecurrent environmental, economic, and social challenges. This contribution presents some preliminary results ofINTAQT project (EU Horizon 2020), which aims to perform an in- depth multi-criteria assessment of the relationshipsbetween animal husbandry and qualities of products. In specific, this research aims to identify and implement on-farmchanges in the production processes (e.g. feeding regimes, outdoor access, herd management), which are expectedto improve intrinsic quality traits of the products and/or sustainability traits of the farms. A participatory approachwas used to establish farmer field-groups (living labs) representative of the different geographic regions and of themain production systems involved in the project. Each farmer field group involves from 5 to 8 farms. The groups areestablished considering different husbandry systems according to a gradient of intensification (extensive vs intensivesystems): 3 groups for dairy farms (Ireland, northern Italy and France); two groups for beef farms (Switzerland andnorthern Italy); two groups for poultry (France and Italy). The methodological approach is based on 5 steps: (1) tartinganalytical phase: a critical analysis of trade-offs / synergies between sustainability and quality traits for each farmfield group; (2) decision phase: development of practices to improve the identified synergies / mitigate trade-offs; (3)implementation phase: implementation of practices for at least one year. During this time, 2-3 meetings of the wholefarmers group on farms allow farmers discussions about their experiences, successes and drawbacks; (4) concludinganalytical phase: the aim is to analyse the effects of the implementation of the practices during a last meeting in thefarmer’s groups and presentation of the analysis results; (5) scientific data analysis and interpretation. The first resultsof this approach will be presented and discussed. The ambition is to establish a network of living labs usable as pilotand demonstration enterprises regarding practice improvements for better food quality and sustainabilit