The differential modification in expression of certain mammalian genes dependent upon parental origin is known as genomic imprinting. The Angelman (AS) and Prader-Willi (PWS) syndromes are clinically distinct neurobehavioral disorders that represent the best example of this phenomenon in humans. They provide an ideal system in which to study the mechanisms of genomic imprinting. Loss of paternally active 15q11-q13 gene(s) results in PWS, whether by paternal deletion or maternal uniparental disomy, whereas loss of a maternally active 15q11-q13 gene(s) leads to AS, whether by maternal deletion, paternal uniparental disomy, or presumed point mutations. Our strategy to identify imprinted genes in 15q11-q13, which may thus be involved in AS and PWS, is to screen 15q11-q13 sequences for regions that display features of genomic imprinting. We have already identified one gene, ZNF127, that has a striking parental DNA methylation imprint. Two other loci in 15q11-q13 that may be imprinted have subsequently been identified by us and other workers. Furthermore, we have shown by replication timing studies that the entire 15q11-q13 region composes an imprinted domain. The three specific aims of our proposal are to: (1) Clone and characterize the complete ZNF127 gene (the model locus for our studies). We have isolated and sequenced a large part of the human and mouse ZNF127 genes, including the sites that show a methylation imprint in the CpG- island spanning the putative promotor, and will complete this work. The 5' start site of transcription will be determined by primer extension, 5' RACE and RNase protection. Promoter elements will be further defined by DNase I hypersensitivity, in vivo footprinting and deletion mapping. (2) Correlate the differential methylation patterns of ZNF127 with expression and function by examining expression via Northern analysis and RT-PCR on various tissues, including brain samples from AS and PWS patients, hydatidiform moles/ovarian teratomas, mouse uniparental disomies, and mouse parthenogenetic/androgenetic embryonic stem cells. Using the same strategies, we will also examine expression of another candidate human imprinted gene, SNRPN. (3) Assess replication timing in 15q11-q13 by FISH in unique AS and PWS patients with alterations in the DNA methylation imprint at ZNF127 as a consequence of distant chromosomal rearrangements. These studies will address the inter-relationships between DNA methylation, replication, chromatin structure, and DNA binding proteins in the mechanism of genomic imprinting.
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