A COMPARISON OF GLOBAL DNA METHYLATION PATTERNS IN DEVELOPMENTALLY-COMPETENT VS -INCOMPETENT NUCLEAR TRANSFER DONOR CELLS (2007)

The production of pigs by nuclear transfer (NT) is currently still a very inefficient process. It has been firmly established by our previous research, and by others that different cell types can vary widely in their ability to successfully direct embryo development after nuclear transplantation. One of the primary goals of this line of research is to identify and characterize specific patterns of DNA methylation in donor cells and NT embryos that are conducive to successful embryo development, the establishment and maintenance of pregnancy, and ultimately, the birth of viable offspring. Here we detail results from our experiments comparing global DNA methylation patterns in two different donor cell types: one that resulted in the birth of live, healthy piglets, and one that did not (Enos2 and Enos1, respectively). The two cell types were from a very similar genetic background (full siblings), were treated similarly in their culture, and were expected to have performed similarly as donor cells for the nuclear transfer procedure. In order to probe the basis for the discrepancy in the performance of the two cell types as nuclear transfer donor cells, we assessed the methylation patterns of the Enos1 and Enos2 cell types by Differential Methylation Hybridization (DMH). The DMH procedure was carried out as outlined previously, with the genomic DNA of each cell type being compared directly to an identical internal control sample of liver genomic DNA. Three independent replicates of the entire DMH procedure were carried out, albeit with the source of the genomic DNA for each sample being identical for each replicate. Of the 5280 spots on the DMH array, 1364 (25.8%) provided sufficient data for comparison. In all, 271 genomic regions (271/1364 = 19.9%) were shown by DMH to be differentially methylated in Enos1 cells as compared to Enos2 cells, with a false discovery rate of 0.05. Overall, the Enos2 cell line showed a methylation pattern that was very similar to that of the reference sample (liver), while the genomic DNA from the Enos1 cells had acquired a methylation pattern that was very divergent from that of the liver sample and the Enos2 cell line. Fifteen of the most highly significant differentially methylated clones were sequenced; 11/15 clones came back with useful annotation: five sequences showed significant similarity to ribosomal RNA (three clones = 28S rRNA; two clones = 18S rRNA), one was similar to a viral oncogene-like sequence (MYBL1), and five were similar to coding sequences of known proteins (RPS19, BAD, FEZF2, NDUFS8, and ARF4); four of the clones were not significantly identifiable with any known sequence. Bisulfite sequencing of a number of these clones in all three samples (Liver, Enos1, and Enos2) is currently in progress, with preliminary results confirming the data from the hybridizations. Results from these experiments further validate our previous findings that the success of a particular cell type as a NT donor cell may depend, at least in part, on the pattern of genomic DNA methylation in those cells. Also, these results provide additional encouragement that global DNA methylation profiling by DMH may ultimately serve as a pre hoc diagnostic tool to assess the suitability of individual cell types for use as donor cells for NT experiments. Funding derived from NIH Grant # RR13438. (poster)