GENOMIC IMPRINTING ON DISTAL MOUSE CHROMOSOME 7

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Normal mammalian development requires the contribution of haploid genomes from both parents, indicating that the two genomes are not functionally equivalent. This non-equivalence is the result of gamete-specific epigenetic modifications of a number of genes that lead to the unequal expression of the two parental alleles during development, a phenomenon known as imprinting. In humans, several developmental disorders are associated with disruption of the normal patterns of genomic imprinting. One specific example is Beckwith-Wiedemann Syndrome which is associated with disruption of imprinting at 11p15.5. Our group is investigating molecular mechanisms for gene regulation on mouse distal chromosome 7, a region syntenic with human 11p15.5, in order to understand how the cell distinguishes parental origins of its chromosomes and how this distinction is manifested in unequal expression of the two chromosomes. Mouse distal 7 contains at least 5 imprinted genes: p57KIP2, Mash-2, Ins-2, Igf-2, and H19. Our approach toward understanding regulation in this region takes three directions. First, we are investigating the molecular mechanisms for imprinting of the H19 gene. We have introduced a 16 kb DNA fragment carrying the Mus spretus H19 gene into heterologous locations in the genome and demonstrated that multiple copies are sufficient for paternal silencing and DNA methylation. Replacing the H19 structural gene with a luciferase reporter gene results in loss of imprinting of the transgene. The removal of 701 base pairs at the 5' end of the structural gene results in a similar loss of parental-specific DNA methylation demonstrating that these sequences are required for both the full establishment and maintenance of the sperm-specific gametic mark. Expression of two additional transgenic constructs demonstrates that sequences in the H19 structural gene further upstream are not absolutely required for imprinting. Current experiments are designed to distinguish between a role for the 5' sequences in cis or in trans for transgene imprinting. Previous work has demonstrated that H19 is a regulatory locus, required for silencing of the neighboring insulin genes on the maternal chromosome. We have generated knockout mice that will distinguish between two possible models explaining this requirement for H19: competition between the promoters of the insulin genes and of H19 versus the action of a methylation sensitive boundary element. Finally, we are generating a physical map and contig of the region between p57KIP2 and H19. We will use this DNA to search for other imprinted genes in the region. In addition, we will generate transgenic mice to investigate the mechanisms for imprinting of p57KIP2 in order to ascertain the generality of the mechanisms noted at the H19 locus.