Beckwith-Wiedeman Syndrome (BWS), and Prader-Willi/Angelman Syndromes (PWS/AS) are rare developmental disorders with parent-of-origin effects that occur in 1/15,000 live births in the US. The molecular basis for these diseases is deregulation of imprinted gene clusters on 11p15 and 15q11 caused predominantly by either deletions within imprinting control centers (ICC) or by epigenetic DNA methylation changes in the ICC. This project tests the overall hypothesis that epigenetic alterations alone can cause developmental pathologies that model these syndromes as a result of global loss of imprinting (LOI). We developed a mouse model of global loss of imprinting by inducing transient DNA demethylation and observed spontaneous immortalization in embryonic fibroblasts, and tumor predisposition within 12 months (Holm et al, Cancer Cell, 2005). The specific hypothesis we are presently testing is that deregulation of growth pathways regulated by DNA methylation-dependent genomic imprinting leads to tumorigenesis in this model. We have examined phenotypic consequences on fetal growth that revealed mutant embryos display increased body weight and crown-rump length compared to controls. Elevated CTCF binding in the mutant ES cells at a site of DNA methylation-dependent regulation of insulin-like growth factor 2 (Igf2) was observed using chromatin immunoprecipitation. Loss of CTCF regulation has been implicated as a potential molecular mechanism in LOI. RT-PCR analysis of candidate imprinting pathways revealed variably elevated expression of the paternal growth factors delta-like kinase1, and insulin-like growth factor 2, and consistent loss of maternal gene expression for Igf2R, and p57 in LOI cells. We tested the role of aberrant Igf2-signaling as the driving mechanism for fetal growth deregulation by restoring monoallelic expression of Igf2r. To accomplish this we deleted a critical repression element from one Igf2R allele in the LOI mutants, which restored Igf2R expression to wildtype levels. By examining the fetal growth in the LOI mutants with reactivated Igf2R, we compared weight and size in mid-gestation chimeras and observed phenotypic rescue of the fetal overgrowth upon Igf2R re-expression. We further demonstrated that the size of each class of embryo corresponded to the degree of ES cell contribution in the chimeric embryos using Q-PCR to quantify chimerism. This study has allowed us to define a growth pathway that leads to quantitative differences in fetal growth and is set by epigenetic alterations within stem cells. We are examining the epigenomic state of these cells to uncover potential changes that may influence the developmental potency of these cells. Finally, by characterizing neuronal development and function in this model we hope to provide a new system for studying epigenetic control in the brain
