Characterization of the murine Angelman syndrome imprinting center

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct neurological disorders resulting from improper gene expression from the imprinted domain on chromosome 15q11-q13, the PWS/AS locus. This locus is controlled by a bipartite imprinting center consisting of the PWS-IC and the AS-IC. Evidence suggests that the PWS-IC acts as a positive element to promote gene expression from the paternal allele. The AS-IC acts in the oocyte to inactivate the PWS-IC on the future maternal allele thus silencing the paternally expressed genes. The PWS-IC is located just 5' to and including exon one of SNRPN whereas the AS-IC is 35 kb upstream of SNRPN. Importantly, the AS-IC includes two of several SNRPN alternative upstream exons.;The PWS/AS locus is well conserved in the mouse but a murine AS-IC remains uncharacterized. As in humans, the mouse Snrpn locus includes several upstream exons postulated to function in silencing the maternal allele. We have taken a transgenic approach to study the potential regulatory role of these alternative exons. To do so, we utilized the bacterial artificial chromosome (BAC) 425D18, which contains Snrpn and approximately 120 kb of 5'sequence in which three alternative upstream exons reside. We first confirmed that this BAC transgene displayed proper imprinted expression in multiple transgenic lines thus demonstrating the presence of a functional AS-IC. Imprinting was further examined by analysis of the epigenetic status of the Snrpn differentially methylated region (DMR), which lies within the PWS-IC.;To determine whether the upstream exons on the 425D18 BAC confer silencing upon maternal transmission, we used recombineering techniques to create targeted deletions of these exons. Deletion of the three upstream exons resulted in robust Snrpn expression upon both maternal and paternal transmission of the transgene as well as a loss of the epigenetic imprint at the Snrpn DMR. These results indicate that the three upstream exons comprise the AS-IC on the 425D18 BAC. Our data support a model in which transcription arising from the AS-IC and continuing through the PWS-IC results in epigenetic modification of the PWS-IC. Further experiments utilized this BAC transgenic system to investigate mechanisms of AS-IC action.