MS22. REGULATION OF IMPRINTING AT THE H19/IGF2 LOCUS. Bartolomei, Marisa¹, Fedoriw, Andrew ¹, Engel, Nora ¹, Wan, Le-Ben¹, Schultz, Richard ², ¹ University of Pennsylvania School of Medicine, Philadelphia, PA² University of Pennsylvania, Philadelphia, PA
     The opposite imprinting of H19 and Igf2 is mediated through shared enhancers and a 2 kb differentially methylated domain (DMD). The DMD is hypermethylated on the repressed paternal H19 allele and hypersensitive to nucleases on the active maternal allele and is required for H19 and Igf2 imprinted expression on both chromosomes. It is postulated that the DMD acts as a methylation-sensitive insulator and hypermethylated mediator of H19 repression on the maternal and paternal alleles, respectively. The insulator activity is mediated, at least in part, by CTCF. Mutation of 9 CpG dinucleotides within the CTCF binding sites disrupts repression and hypermethylation of the paternal H19 allele while maintaining CTCF binding. Because methylation is acquired on the paternal allele during spermatogenesis but lost early in development on this mutant allele, we hypothesize that the binding of CTCF in the preimplantation embryo may contribute to methylation loss on the mutant paternal allele and may also protect the expressed allele(s) from methylation after implantation. The protective role of CTCF at the DMD also appears to extend to the time when maternal methylation is established at other imprinted loci. A transgenic RNAi approach that targets CTCF in the growing oocyte shows that transgenic lines with significant CTCF depletion in the growing oocyte exhibit DMD methylation. Interestingly, the methylation is incomplete in the GV-stage oocyte, with the paternal alleles exhibiting extensive hypermethylation and the maternal alleles remaining unmethylated. Together, these experiments indicate that CTCF binding protects the DMD from DNA methylation. These experiments also suggest that the two roles of the DMD, insulation and repression, are antagonistic.

MS23. DYNAMIC CHROMATIN CHANGES DURING THE FIRST CELL CYCLE. Dean, Wendy ¹, Reik, Wolf¹, Santos, Fatima¹, ¹ The Babraham Institute, Cambridge, UK
     On fertilisation, gametes undergo epigenetic reorganisation and re-establish totipotency. We have investigated links between chromatin remodelling and asymmetric maintenance of DNA methylation during the first cell cycle in the mouse. Using antibodies against a broad repertoire of methylated lysine residues on H3 in the core nucleosome reveals that the male and female pronuclei are organised into chromatin that is modified in very different ways. As an example, the male pronucleus is negative for di- and trimethyl H3K9, modifications associated to inactive euchromatin and heterochromatin respectively, yet the female is positive for these residues. Despite these differences, both pronuclei are transcriptionally inactive at this stage. However, the male is positive for monomethyl H3K9 and H3K27 and these signals increase during pronuclear maturation. Non-histone chromatin proteins of the Polycomb group, Eed and the histone methyl transferase (HMT) activity Ezh2, are also found in the paternal compartment as early as sperm decondensation. However, despite the presence of the HMT usually responsible for trimethyl of H3K27, this residue is not trimethylated in the male pronucleus until the completion of DNA replication. Additional significant chromatin proteins such as Heterochromatin protein 1 beta (HP1) is abundant in the male pronucleus, despite the absence of di- and trimethyl H3K9 the preferred binding partners, but instead HP1 apparently co-localises with monomethyl H3K9. Recent evidence identifies monomethyl H3K9 as the preferred substrate of Suvar39h, the histone methyl transferase (HMT) responsible for heterochromatic H3K9 trimethylation. This association of monomethyl H3K9 and HP1 may assist in preventing further modification of H3K9. Association of dimethylation but not trimethylation of H3K9 with DNA methylation, in the female pronucleus, suggests a mechanistically significant link. These differences begin to provide a chromatin based explanation for paternal-specific active DNA demethylation and maternal specific protection in the mouse.

MS24. SETTING AND PERTURBING GAMETIC IMPRINTS. Trasler, Jacquetta¹, Toppings, Marc¹, Reinhart, Bonnie², Lucifero, Diana¹, La Salle, Sophie¹, Chaillet, Richard², ¹ McGill University-Montreal Childrens Hospital Research Institute, Montreal, QC, Canada² University of Pittsburgh, Pittsburgh, PA
     Genomic imprinting involves the formation of an epigenetic mark on a subset of mammalian genes in a parent-of-origin-specific manner, such that the genes affected are expressed monoallelically in the resulting offspring. The process is initiated in the germline and persists through preimplantation development. DNA methylation, catalyzed by the DNA methyltransferase (Dnmt) enzymes,is one of the best characterized epigenetic modifications of imprinted genes. Examination of the methylation status of Snrpn, Igf2r, Peg1 and Peg3 during oocyte development identified the postnatal growth phase as the critical period when maternal methylation takes place. Dnmt3a and Dnmt3l have been shown to be important for the acquisition of maternal methylation imprints whereas Dnmt1o plays a role in the maintenance of gamete-derived imprints in the preimplantation embryo. Expression studies and gene-targeting have been used to examine the regulation and functions of the Dnmts. Real-time RT-PCR showed that Dnmt3a, Dnmt3b and Dnmt3l levels increase 47-, 16-, and 9600-fold during oocyte growth; in Dnmt1o-deficient oocytes, Dnmt3l levels were unchanged, whereas both Dnmt3a and Dnmt3b were upregulated. The consequences of perturbing the maintenance of methylation imprints was examined in 7.5 dpc embryos derived from Dnmt1o-deficient oocytes. Embryos that develop in the absence of Dnmt1o demonstrate striking phenotypic variation between embryos as well as evidence of developmental abnormalities. Loss of methylation at imprinted genes was similar in extraembryonic tissue and different regions of the embryos. Studies are underway to better understand the variable phenotypes and nature of the epigenetic mosaicism in the Dnmt1o-deficient embryos. Supported by NIH, CIHR, FRSQ