Silencing of transposons in mammalian germ cells

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DESCRIPTION (provided by applicant): Ectopic gene silencing can contribute to disease. Human cancer cells frequently contain genomes that have undergone a net loss of m5C and there are many reports of tumor suppressor genes that are normal in sequence but not expressed; such genes have methylated promoters. Many associations between abnormalities of genomic methylation patterns and cancer have been reported The nature of the alterations that lead to global demethylation with focal hypermethylation in many common tumors is completely unknown, but must involve the gain or loss of regulatory factors; none of the DNA methyltransferases has been shown to be mutated or silenced in cancer. Methylation and ectopic gene silencing have also been attributed a role in genotype- independent phenotypic diversity, as in discordance in monozygotic twins. This suggests errors in targeting of gene silencing during gametogenesis or early in development, again by unknown regulatory factors. The identification of Dnmt3L as an essential factor in the establishment of genomic methylation patterns in germ cells provides the first opportunity to isolate factors that regulate de novo methylation and to define the cues that direct methylation to specific sequences. We will use a new and minimally perturbed interaction screen and state-of-the-art protein identification by mass spectrometry to identify factors that complex with Dnmt3L, and we will apply new genetic methods in the functional analysis of Ecat11, an especially interesting factor found to interact with Dnmt3L in preliminary studies. Ectopic silencing or activation in tumors of factors that control de novo methylation is a likely cause of the disrupted methylation patterns often seen in cancer genomes. The identification of factors that regulate de novo methylation is an issue of immediate importance in the field of mammalian (and human) epigenetics. The function of the mammalian genome depends on the accurate establishment and maintenance of genomic methylation patterns during gametogenesis and early development. Abnormal methylation patterns are common in many malignant tumors; it is possible that re-expression of germ-cell or embryonic methylation regulators is responsible for tumor-specific methylation abnormalities. Errors in the establishment of methylation imprints in germ cells can cause Angelman syndrome, Prader-Willi syndrome, or Beckwith-Wiedeman syndrome, and mutations in genes for imprinting factors cause recurrent familial biparental hydatidiform moles, in which paternal genomic imprints are imposed on the maternal genome (Judson et al., 2002; Bestor and Bourc'his, 2006). Errors in the establishment or maintenance of genomic methylation patterns are currently believed to contribute to genotype-independent phenotypic variation, especially in psychiatric illness where rates of discordance in monozygotic twins often exceeds 40% (Meissner, 1965). The biological importance of genomic methylation patterns has only recently become clear, but at this time almost nothing is known of the mechanisms by which they are established. The studies described here are almost certain to identify factors that regulate de novo methylation in the male germ line.