51 INVESTIGATION OF INTER-INDIVIDUAL EPIGENETIC VARIABILITY IN BOVINE CLONES: A HIGH THROUGHPUT STUDY
H. Kiefer A B , L. Jouneau A B , M. L. Martin-Magniette C , S. Balzergue C , E. Campion A B , P. Chavatte-Palmer A B , Y. Heyman A B , C. Richard D , D. Le Bourhis A E , J. P. Renard A B and H. Jammes A BA Biology of Development and Reproduction, Institut National de Recherche Agronomique, Jouy-en-Josas, France;
B Ecole Nationale Vétérinaire de Maisons-Alfort, Maisons-Alfort, France;
C URGV Institut National de Recherche Agronomique, Evry, France;
D UCEA, Institut National de Recherche Agronomique, Leudeville, France;
E UNCEIA, Maisons-Alfort, France
Reproduction, Fertility and Development 25(1) 173-173 https://doi.org/10.1071/RDv25n1Ab51
Published: 4 December 2012
Abstract
Reprogramming the differentiated cell to totipotency can be achieved following the introduction of its nucleus into an enucleated oocyte, a procedure known as cloning. We used cattle clones as a pertinent model to assess the inter-individual epigenetic variability and its consequences on phenotypes, including agronomically relevant traits and developmental pathologies. Indeed, the developmental defects frequently associated with cloning could be related to the insufficient extent of reprogramming, leading to perturbations of the nuclear microenvironment of the early embryo, with long-term consequences on the phenotype. Immunoprecipitation of methylated DNA following hybridization on a new bovine-specific tiling array (MeDIP-chip) was used to describe the epigenomic patterns affected by incomplete reprogramming. We first focused on the liver, because overgrowth of this organ is, to a certain extent, bound to global foetal overgrowth, which is often observed in clones. The microarray represents the 21 416 bovine genes currently annotated (UMD3.1 genome assembly). Each gene was tiled by 34 probes, on average, spanning upstream regions from –2000 to +1360 bp relative to the transcription start site. This microarray was hybridized with MeDIP samples from livers of normal Holstein animals obtained by AI (4 perinatal controls and 8 adults: 2 males and 10 females) and livers of female Holstein clones (7 perinatal clones from 2 different genotypes, either stillborn or suffering from severe pathologies, and 7 adult clones from 3 different genotypes, with normal to pathological phenotypes). After normalization of the data, enriched probes were identified using the ChIPmix method (Martin-Magniette et al. 2008 Bioinformatics) and located on the Ensembl Genome Browser. Results of exploratory analysis, including correlation clustering, principal components analysis (PCA), and independent components analysis (ICA), will be presented. A statistical test based on differences in the spatial distribution of the enriched probes along promoters was applied to the data, to associate epigenetic signatures to specific parameters (cloning, phenotype, stage, and genotype). Most promoters with more than 5 enriched probes across individuals showed a clustered distribution of the enriched probes. This local enrichment was highly conserved among individuals for 96% of the promoters, suggesting that most of the methylated regions were common to all animals. More interestingly, the distributions of the enriched probes showed inter-individual variability for 4% of the promoters in all samples. The identification and validation of these promoters is currently in progress.
Funding was provided by ANR-09-GENM-012-01 and ACI-PHASE-INRA-2010.
