Monoallelic gene expression, resulting from genomic imprinting, appears to have important consequences both in normal development and in human genetic disease and cancer. However, to date there have been very few studies of monoallelic gene expression at defined loci. We have shown that the human H19 gene, at chromosome 11p15, is monoallelically expressed. The 11p15 region is of particular interest in that maternal alleles are commonly deleted in childhood tumors and the region is therefore predicted to contain one or more imprinted tumor suppressor genes. We plan to study the mechanism and consequences of monoallelic gene expression in humans, focusing on H19 and flanking genes, and to explore strategies for isolating additional imprinted genes. To determine whether 11p15 imprinting is regional or local we will clone the genes immediately flanking H19 and test them for monoallelic expression. We will test the possible role of DNA methylation in imprinting by analyzing allele-specific methylation in and around the H19 gene and by carrying out functional assays for the effect of methylation on H19 expression. We will search for candidate imprinting """"""""initiator"""""""" sequences in H19 and flanking DNA using a transfection assay in embryonal stem cells. We will characterize a novel phenomenon, tissue-specific somatic allele switching, which we have observed for human H19 and which suggests methylation threshold and feedback effects in maintenance of monoallelic gene expression. To explore the biological function of H19, and to determine whether this gene might have tumor suppressor activity, we will examine the ability of inducible H19 expression vectors to revert the phenotype of Wilms' tumor and embryonal rhabdomyosarcoma cells. Finally, to isolate additional imprinted genes, we will pursue a candidate gene approach in humans and, concurrently, develop a general strategy, based on arbitrarily-primed polymerase chain reaction (AP-RCR), to clone imprinted genes from interspecific mouse hybrids.
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