DNA METHYLATION IDENTIFIES AND REGULATES IMPRINTS

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Imprinting is necessary for proper mammalian development and is defined as the epigenetic modification of chromosomes during gametogenesis, which limits expression of imprinted loci to either the maternal or paternal allele. Paternal-specific loss of imprinting (LOI) and loss of heterozygosity (LOH) are common occurrences in several types of childhood cancer (e.g. Wilms' tumor, retinoblastoma, rhabdomyosarcoma, neuroblastoma, and acute myelogenous leukemia) and in several genetic disorders (e.g. Prader-Willi and Beckwith-Wiedermann syndromes). Sixteen, of an estimated 100, imprinted genes have been identified in mice and regions of parental-specific DNA methylation have been identified at many of these loci. Inactivation of the DNA methyltransferase (Dnmt) disrupts imprinted gene regulations and re-introduction of the Dnmt cDNA into these ES cells (rescued cells) fails to restore parental-specific methylation. This suggests a great opportunity to search for new imprinted loci by screening DNA for methylation differences between normal and rescued ES cells. A more focused screen for parental-specific methylation on mouse chromosome 12, which has been identified as an imprinted region, will also be done using DNA from flow-sorted metaphase mouse chromosomes t(4:12), either paternally or maternally inherited. These DNAs will be digested and fractionated on a methyl-DNA binding domain column (MBD) to isolated highly methylated fragments which will then use used in PCR-select based subtraction protocols to yield imprint-associated fragments. Clones of interest will be systematically examined for sequence homologies, expression, and imprinting. Ultimately, identified loci will be tested for developmental function by homologous recombinant knockouts and gain of function insertions in transgenic mice. In addition, a reversibly repressible allele of Dnmt will be made and used to study the role of Dnmt in gene imprinting during gametogenesis.