The goals of this revised application are to use genetically altered mice to investigate the molecular basis for imprinting and the developmental consequences of imprinting imbalances. These experiments are based on MTase deficient mice produced through gene targeting. Since the previous submission the applicant has made important progress. He has created a new MTase knock-out mouse that is fully deficient in MTase activity. Moreover he showed that the knock-out that he made some time ago is also a null mutation. Both mutant alleles result in embryonic lethality at E10. As Li recognizes, these MTase deficient mice make it possible to answer many important questions about methylation and imprinting: Is there a second MTase gene?, How do MTase deficient embryos survive, and what is the cause of their death?, and How is gene expression affected in deficient mice? Two fundamentally important observations were recently made. The first was, as mentioned above, the creation of a new MTase deficient mouse line and the demonstration that another line made previously is in fact also deficient. The second observation is that methylation controls H19 expression in an allele-specific manner. A hybrid ES cell line was made from MTase mutant mice and wild-type ES cells derived from these show that expression and methylation of H19 is allele-specific, thereby demonstrating that methylation and imprinting operate in ES cells and that methylation is required for repression of the paternal allele. Experiments are now proposed to address the sequence specificity of methylation. Recent results for two different imprinted genes suggest that demethylation shows sequence specificity. MTase deficient mice may provide a system for characterizing the sequence requirements controlling the loss of methylation.
Specific Aim 1 Examination of methylation patterns and gene expression in MTase mutants including characterization of DNA methylation patterns in mutant embryos and ES cells, and examination of gene expression patterns. Genes to be studied include Hbb, Pgk2, Apoa1, Alb, H19, Igf2, Snrnp, Xist, Pgk1, Rps4, Hprt and others. (The applicant recently showed that Xist, which is normally paternally imprinted, is expressed in male mutant embryos, arguing that methylation is required for Xist repression.) Specific Aim 2 Characterization of tissue and cellular defects of MTase mutants and examination of the manner in which MTase deficiency affects cell proliferation and differentiation, including histological analyses, cell proliferation rates, differentiation potential of mutant ES cells in vivo and in vitro, and characterization of cell death patterns in MTase mutants. Studies are also proposed to determine the nature of the cellular and tissue defect that compromises viability in MTase deficient embryos and to examine the way in which the MTase mutation affects cell proliferation and differentiation. These studies will involve histological analyses of mutant embryos, tests of cell proliferation rates, examination of differentiation potential for ES cells in vitro and in vivo in aggregation chimeras (using a lacZ marker), and characterization of the nature of cell death in mutant mice.
Specific Aim 3 Determination of the relationship between methylation, chromatin conformation and expression of imprinted genes, including tests for expression of imprinted genes in MTase mutants, expression of wild- type MTase in mutant ES cells and its effects on allele-specific methylation and expression of the H19 gene, and examination of the chromatin structure of the H19 gene in wild-type and MTase mutants.
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