66 DNA METHYLOME ANALYSIS IN NUCLEAR TRANSFER DONOR CELLS AND ITS RELATIONSHIP TO CLONING EFFICIENCY IN SWINE

Abstract

The success of cloning by somatic cell nuclear transfer is unpredictable due in part to abnormal patterns of DNA methylation and histone modifications in the donor genome. The state of these epigenetic markers appears to regulate the timing and degree of embryonic gene expression by altering chromatin structure. In this study, we sought to determine the pattern of DNA methylation in genetically distinct clonal populations of swine fetal fibroblast donor cells that have previously been demonstrated to have either consistently high or consistently low success at producing healthy cloned pigs. To accomplish this goal, we combined methylated DNA immunoprecipitation using antibodies to 5-methylcytosine with Illumina high throughput sequencing (MeDIP-seq) to detect broad regions of methylated and unmethylated DNA in the 2 populations of donor cells (termed high success v. low success). Size-selected immunoprecipitated DNA samples each had matching input controls. Model-based analysis of ChIP-Seq revealed a total of 272 598 and 261 339 genomic regions enriched for methylated cytosine in high v. low success donor cells, respectively. The majority of these enriched regions (peaks) in high and low success samples coincided in genomic position, but 12.9% of the peaks were unique in high success cells, while 20.4% were unique in low success cells. When intersected with 38 778 predicted CpG islands in the swine genome, 3129 of the total MeDIP peaks (1.1%) overlapped for high success cells as compared to 1836 peaks (0.7%) for the low success cells. Of these intersecting MeDIP-CpG peaks for both groups, 1505 peaks were located in unique locations for high success cells and 205 were unique for low success cells. The chromosomal location of the CpG intersections differed between each cell populations, with the greatest number of overlaps on chromosomes 1 and 3 for high and low success cells, respectively, and the least number of overlaps on chromosomes 9 and 13, respectively. Of the total MeDIP peaks, 6632 (2.4%) from the high success cell population overlapped with predicted swine genes (UCSC Genome Database/Ensembl) compared to 4041 (1.5%) from low success donor cells. Less similar was the percentage of the predicted genes in these methylated regions that were unique to each cell group: 2875 (43.4%) for high success cells v. 338 (8.3%) for low success cells. These results highlight differences in the degree of DNA methylation and the pattern of methylated genes in NT donor cells that produce healthy clones v. those that do not. Such comprehensive genomic analyses of epigenetic markers may provide predictive data to screen for optimal NT donor cells, thereby improving cloning efficiency.