Final Activity Report Summary - HARUCHAN (Development and application of novel LD-based approaches for the positional cloning of quantitative trait loci in livestock) The vast majority of agronomically important traits are complex, multifactorial phenotypes influenced by multiple polygenes as well as by environmental factors. Identifying the genes and mutations that underlie the genetic variation for multifactorial traits is one of the most important objectives of modern genetics. During the two year Marie Curie fellowship supervised by Prof. Michel Georges, University of Liege in Belgium, I was involved in two studies to dissect mechanisms underlying a multifactorial trait, i.e. muscular hypertrophies, in two distinct sheep breeds, the Texel and callipyge sheep. In the first study, we applied a state-of-art methodology of genetics, namely the positional cloning of Quantitative trait loci (QTL), to identify genes responsible for the hyper-muscularity in the Belgian Texel sheep. This study led to the discovery of a novel class of mutation in the myostatin gene on sheep chromosome-2 that encoded a muscle-specific chalone, a guard of overgrowth. We demonstrated that the myostatin allele of Texel sheep created a target site for micro ribonucleic acids (microRNAs), a novel class of small regulatory RNA acting as posttranscriptional gene suppressors. This caused translational inhibition of the myostatin gene to protein and hence allowed for the overgrowth of skeletal muscle in the Texel sheep. Analysis of deoxyribonucleic acid (DNA) polymorphism databases for human and mice demonstrated that mutations creating or destroying putative microRNA target sites were abundant and might be important effectors of phenotypic variation. In the second study, I was involved in the functional analyses of the callipyge (CLPG) mutation influencing the other muscular hypertrophy in the callipyge sheep. The CLPG mutation was located in the middle of a 90 Kb intergenic region separating the imprinted DLK1 and GTL2 genes on sheep chromosome 18 and caused ectopic expression of a core cluster of neighbouring genes in cis along 350 Kb in postnatal skeletal muscle, a tissue in which these genes were normally silenced. To elucidate the mechanisms underlying the long range cis-effect of the CLPG mutation, we studied its effect on epigenetic features involving chemical modifications to DNA, structural changes of DNA strands and a prominent role for intergenic RNA expression. The results allowed us to propose the following working model. Intergenic RNA expression would function as activator of gene expression by promoting permissive epigenotypes through the 350 Kb region. According to development, however, a muscle-specific silencer in the CLPG mutation locus would suppress the intergenic RNA expression that led passive epigenotypes to spread through the region followed by an overall silencing of the neighbouring gene expressions. Somehow the CLPG mutation inactivated a muscle-specific silencer function and allowed a high level of gene expressions to persist after birth. One of the affecting genes, DLK1, was proposed as a possible growth promoter that could cause the muscular hypertrophy in the callipyge sheep. We could herein address mechanisms underlying multifactorial traits, i.e. two distinct muscular hypertrophies in sheep. The results obtained from the works would be useful for a DNA-based selection to improve productivities of sheep breeds. Moreover, they generated novel insights in the biochemical pathways of the genes as well as the physiology of the corresponding traits and would help to identify targets for drug development and genetic engineering with potential applications not only in agriculture but also in human medicine.
