The overall goals of this project are 1) to determine genotype/phenotype relationships across the chromosome 15q11-q13 imprinted domain and its mouse equivalent to include Angelman syndrome (AS), Prader-Willi syndrome (PWS), and other phenotypes; 2) to explore the molecular genetic and biochemical bases for regulation of genomic imprinting in mice and humans; 3) to expand the understanding of the role of genomic imprinting and epigenetics more generally in human disease; and 4) to explore therapeutic strategies for AS in mice and in cultured human cells as a model for epigenetic therapies. Initial gene trap experiments identified retinoblastoma binding protein 1 (Rbbp1) and Rbbp1-like-1 (Rbbp111) as having effects on imprinting; in addition, cultured cells lacking expression of the retinoblastoma (Rb) protein show altered DNA methylation and gene expression in both the Snrpn and H19 imprinted domains.
The aims related to studies of regulation of genomic imprinting include the following: a) Continue genetic analysis of human chromosome 15q11-q13 and mouse 7C; b) Extend genetic screens in mice using ENU mutagenesis and perhaps sleeping beauty mutagenesis with new """"""""yellow"""""""" Snrpn-agouti allele and in ES cells using gene-trap mutagenesis; c) Prepare null mutations in mice for Rbbp1 and Rbbp111 and characterize the phenotypes; d) Genetic analysis in mice using Mecp2, eed, Rbbp1, Rbbp111, Rb and other candidate genes and genes isolated in screens; and e) Biochemical and microscopy studies to include co-immunoprecipitation (IP) studies to identify protein-protein interactions, chromatin immunoprecipitation (CHIP) studies, immuno-histochemistry, immuno-FISH, and analysis of gene expression and DNA methylation. One goal is to validate whether five proteins identified by 2D gels of Purkinje cells from Ube3a mutant and wild-type mice are increased in abundance in vivo in mutant mice using immuno-histochemistry and western blots. Treatment for AS will be explored attempting to increase leaky expression of the silenced paternal allele of UBE3A. Studies will test the effect of various drugs on human cultured fibroblasts and in mice, and an ongoing clinical trial of folate and betaine will be modified based on these results. The hypothesis that intracytoplasmic sperm injection (ICSI) may cause imprinting defects will be tested in mice using a gene fusion method which allows detection of these defects based on coat color.
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