H3K9me2/3 Binding of the MBT Domain Protein LIN-61 Is Essential for Caenorhabditis elegans Vulva Development

MBT domain proteins are involved in developmental processes and tumorigenesis. In vitro binding and mutagenesis studies have shown that individual MBT domains within clustered MBT repeat regions bind mono- and dimethylated histone lysine residues with little to no sequence specificity but discriminate against the tri- and unmethylated states. However, the exact function of promiscuous histone methyl-lysine binding in the biology of MBT domain proteins has not been elucidated. Here, we show that the Caenorhabditis elegans four MBT domain protein LIN-61, in contrast to other MBT repeat factors, specifically interacts with histone H3 when methylated on lysine 9, displaying a strong preference for di- and trimethylated states (H3K9me2/3). Although the fourth MBT repeat is implicated in this interaction, H3K9me2/3 binding minimally requires MBT repeats two to four. Further, mutagenesis of residues conserved with other methyl-lysine binding MBT regions in the fourth MBT repeat does not abolish interaction, implicating a distinct binding mode. In vivo, H3K9me2/3 interaction of LIN-61 is required for C. elegans vulva development within the synMuvB pathway. Mutant LIN-61 proteins deficient in H3K9me2/3 binding fail to rescue lin-61 synMuvB function. Also, previously identified point mutant synMuvB alleles are deficient in H3K9me2/3 interaction although these target residues that are outside of the fourth MBT repeat. Interestingly, lin-61 genetically interacts with two other synMuvB genes, hpl-2, an HP1 homologous H3K9me2/3 binding factor, and met-2, a SETDB1 homologous H3K9 methyl transferase (H3K9MT), in determining C. elegans vulva development and fertility. Besides identifying the first sequence specific and di-/trimethylation binding MBT domain protein, our studies imply complex multi-domain regulation of ligand interaction of MBT domains. Our results also introduce a mechanistic link between LIN-61 function and biology, and they establish interplay of the H3K9me2/3 binding proteins, LIN-61 and HPL-2, as well as the H3K9MT MET-2 in distinct developmental pathways

Etude du rôle des modifications post-traductionnelles des histones dans l’acclimatation embryonnaire à la chaleur chez le poulet

L’environnement précoce des individus est capable d’impacter l’expression des gènes à long terme via des altérations de leur épigénome. Par exemple chez Drosophila melanogaster l’exposition à un stress thermique au cours de l’embryogenèse induit des modifications épigénétiques visibles à l’âge adulte via le complexe PRC2 (Polycomb Repressive Complex 2) (pour revue Steffen and Ringrose 2014). Une marque d’histones modulée par PRC2 est la tri-méthylation de la lysine 27 sur l’histone H3 (H3K27Me3). Cette marque est décrite comme ayant un rôle dans la mémoire d’un état répressif sous l’influence de l’environnement. Notamment chez Arabidopsis thaliana, suite à l’exposition au froid de l’hiver cette marque maintient la répression d’un répresseur floral pour permettre la floraison (Coustham et al. 2012). Dans un contexte de réchauffement climatique, notre équipe a étudié les effets d’un protocole expérimental permettant d’acclimater les poulets à la chaleur pendant l’embryogénèse (Piestun et al., 2008 ; Loyau et al., 2013). En effet, les animaux sélectionnés pour leurs performances de croissance sont peu résistants aux variations de températures. Le traitement d’acclimatation embryonnaire à la chaleur (TAEC) correspond à une augmentation cyclique de la température et de l’humidité relative (HR) pendant l’incubation des jours E7 à E16 de l’embryogénèse (de 37,8°C et 55% HR à 39,5°C et 65% HR pendant 12h). Cette modification de l’environnement précoce des individus altère peu l’éclosabilité et améliore le taux de survie lors d’un coup de chaleur survenant à l’âge d’abattage (J35). Cette thermotolérance accrue est associée de changements physiologiques, métaboliques et d’expressions de gènes à 5 semaines (Piestun et al., 2011, et Loyau et al., 2013, 2014). Nos recherches reposent sur l’hypothèse que les différences d’expressions de gènes observées chez les individus acclimatés peuvent être induites par des modifications épigénétiques survenant durant le TAEC et qui perdurent au cours du développement. Les travaux présentés porteront sur la mise au point de l’immunoprécipitation de la chromatine (ChIP) sur deux tissus de poulet afin de cartographier H3K27Me3

A simple PCR method for sexing Japanese quail <i>Coturnix japonica</i> at hatching

<p>1. The quail is a potentially important avian model for molecular studies; a major drawback is the inability to sex visually before 3 weeks of age. Molecular sexing is therefore an absolute requirement when animals are sampled before that age.</p> <p>2. A low-cost method using common laboratory equipment based on Allele-Specific Multiplex-Polymerase Chain Reaction was developed to undertake reliable molecular identification of the sex of <i>Coturnix japonica</i> directly at hatching.</p> <p>3. This simple method works with down feathers collected from behind the neck of the newly hatched quail and includes internal controls during the PCR to limit risks of error. Males and females can be discriminated on the basis of the presence of one or two amplicons, respectively.</p

Variations épigénétiques en réponse à la chaleur chez le poulet de chair

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The C. elegans HP1 homologue HPL-2 and the LIN-13 zinc finger protein form a complex implicated in vulval development

HP1 proteins are essential components of heterochromatin and contribute to the transcriptional repression of euchromatic genes via the recruitment to specific promoters by corepressor proteins including TIF1 and Rb. The Caenorhabditis elegans HP1 homologue HPL-2 acts in the "synMuv" (synthetic multivulval) pathway, which defines redundant negative regulators of a Ras signaling cascade required for vulval induction. Several synMuv genes encode for chromatin-associated proteins involved in transcriptional regulation, including Rb and components of the Mi-2/NuRD and TIP60/NuA4 chromatin remodeling complexes. Here, we show that HPL-2 physically interacts in vitro and in vivo with the multiple zinc finger protein LIN-13, another member of the synMuv pathway. A variant of the conserved PXVXL motif found in many HP1-interacting proteins mediates LIN-13 binding to the CSD of HPL-2. We further show by in vivo localization studies that LIN-13 is required for HPL-2 recruitment in nuclear foci. Our data suggest that the LIN-13/HPL-2 complex may physically link a subset of the Rb related synMuv proteins to chromatin

Optimization of embryonic thermal programming confirms increased liver fattening in mule ducks and changes in lipid metabolism

Introduction: The embryonic thermal programming (TM) in birds has been shown to impact several physiological parameters such as resistance to thermal stress, muscle growth or immunity. In mule ducks, it has recently been shown that TM can induce metabolic programming resulting in increased liver weight and fat storage after overfeeding. However, a decrease in hatchability and foie gras quality was also observed, suggesting that this technique needs to be optimized. Here, we tested a new thermal manipulation condition determined with the objective of avoiding negative impacts while maintaining or improving liver properties.Methods: The eggs of the control group were incubated at 37.6°C during the whole incubation period while those of the experimental group (TM group) were incubated at 39.3°C 16 h/24 h from the 11th day of incubation to the 21st. After hatching, all the animals were fed and raised under the same conditions until the age of 12 weeks. At this stage, one part of the animals was overfed and then slaughtered 2 h (to measure rapid changes in metabolism) or 10 h after the last meal (to obtain the best technological yields), while the other part was ration-fed and slaughtered 2 h after the last meal, at the same age.Results: An 8% increase in foie gras production was measured in the TM group compared to the control group without altering the quality of the final product (nor hatchability), confirming the successful optimization of the metabolic programming. Interestingly, these results allowed us not to reject the previously suggested hypothesis of a potential delay in metabolic processes involved in liver fattening in programmed animals, in particular by measuring a trend reversal regarding the amount of total hepatic lipids in both groups at 2 h and then 10 h after the last meal.Discussion: This study therefore validates the optimization of metabolic programming by embryonic thermal manipulation for duck liver fattening. The understanding of the mechanisms of embryonic thermal programming in birds remains today very incomplete and the search for epigenetic marks (main hypothesis of the concept of programming) at the origin of the observed phenotypes could be the next step of this work

Mise au point de l’immunoprécipitation de la chromatine (ChIP) pour une étude en séquençage haut-débit sur tissus de poulet

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Cyclic variations in incubation conditions induce adaptive responses to later heat exposure in chickens: a review

peer reviewedSelection programs have enabled broiler chickens to gain muscle mass without similar enlargement of the cardiovascular and respiratory systems that are essential for thermoregulatory efficiency. Meat-type chickens cope with high ambient temperature by reducing feed intake and growth during chronic and moderate heat exposure. In case of acute heat exposure, a dramatic increase in morbidity and mortality can occur. In order to alleviate heat stress in the long term, research has recently focused on early thermal manipulation. Aimed at stimulation of long-term thermotolerance, the thermal manipulation of embryos is a method based on fine tuning of incubation conditions, taking into account the level and duration of increases in temperature and relative humidity during a critical period of embryogenesis. The consequences of thermal manipulation on the performance and meat quality of broiler chickens have been explored to ensure the potential application of this strategy. The physiological basis of the method is the induction of epigenetic and metabolic mechanisms that control body temperature in the long term. Early thermal manipulation can enhance poultry resistance to environmental changes without much effect on growth performance. This review presents the main strategies of early heat exposure and the physiological concepts on which these methods were based. The cellular mechanisms potentially underlying the adaptive response are discussed as well as the potential interest of thermal manipulation of embryos for poultry production. © The Animal Consortium 2014