Minimal sequences necessary for imprinted expression of the Prader-Willi locus.

摘要

Prader-Willi (PWS) ands Angelman (AS) syndromes are neurodevelopmenal disorders arising from the improper expression of oppositely imprinted genes located on human chromosome 15 q11-q13. Imprint regulation of this region is under the control of a bi-partite imprinting center consisting of an Angelman Imprinting Center (AS-IC) located approximately 35kb upstream of the paternally expressed Snrpn exon 1 and a Prader-Willi Imprinting Center (PWS-IC) located 5' to and including Snrpn exon 1. The PWS-IC has been shown to be a positive element promoting expression of a set of genes on the paternal allele, while the AS-IC provides suppression of the PWS-IC on the maternal allele thereby suppressing expression of the same set of genes and allowing expression of the maternal program. Both are required for proper establishment and/or maintenance of the imprint in the germline. In the mouse, both gene order and the imprinted expression pattern have been conserved, with the syntenic region being located on murine chromosome 7C. While the location of the PWS-IC has also been conserved, the position of the murine AS-IC remains unknown.;We have taken a transgenic approach to locating the AS-IC and further dissecting out components of the PWS-IC. Using a recombineering method that utilizes the lambda phage Red genes, we have created a series of deletions within a Snrpn containing bacterial artificial chromosome (BAC) that we have shown recapitulates the imprinted expression of the endogenous locus. First we have created a deletion of 30 kb just 5' to Snrpn exon 1 and show that imprinted expression is retained. This shows that the components required for establishment and/or maintenance of the imprint on Snrpn are located within the retained sequence. Second, we have created a series of deletions that remove several alternative upstream exons of Snrpn in various combinations and show that they contain the AS-IC element and each can act as an independent AS-IC when the other are deleted. These findings are significant because they further restrict the minimal necessary imprinting center functional unit and may suggest a possible mechanism of action.