Identification and characterization of cis-acting elements in the regulation of imprinted gene expression

The Prader-Willi and Angelman syndromes (PWS-AS) associated region includes a cluster of imprinted genes that are coordinately regulated by an imprinting center (IC) spanning the 5' region of the SNURF-SNRPN gene. The IC has a bipartite structure where the PWS-IC is postulated to create an active domain on the paternal allele, while the AS-IC is believed to silence the maternal allele. Also, alternative upstream SNURF-SNRPN promoters have been implicated in the AS-IC function. This project focused on identifying and characterizing cis-acting elements within the IC that may mediate IC function and/or SNURF-SNRPN promoter activity. The PWS-IC contains major nuclease hypersensitive sites (NHS1 and NSH2) associated with the SNURF-SNRPN promoter region and the 1st intron (adjacent to the IC), both specific to the paternal allele. In vivo footprint analysis of the promoter region has identified multiple factor binding sites, 4 specific to the paternal allele and 1 specific to the maternal allele, some of which affected promoter function in transient expression assays. Transient expression assays also helped to define other promoter elements in the SNURF-SNRPN 5' region and to identify a novel position-dependent and orientation-independent activator associated with NHS2. This element(s) preferentially activates the SNURF-SNRPN main and upstream promoters relative to the UBE3A and MKRN3 promoters. It appears to be a complex regulatory unit from which a subregion was mapped that sustains high levels of SNURF-SNRPN promoter activity. This subregion was also identified by sequence comparison of the intronic activator with the homologous region in mouse and contains several highly conserved sites including potential binding sites for SP1, NRF1 and YY1. In vivo and in vitro studies showed binding of YY1 to the intronic activator and its involvement in the activator function. Furthermore, recruitment of the non-processive form of RNA polymerase II to the activator was shown suggesting a possible role for the activator in the IC function by recruiting pol II and transferring it to the SNURF-SNRPN upstream promoters in the female germline and to the main promoter in the male germline/paternal allele. This transfer may be mediated by NRF-1 that was shown to bind in vivo to both the SNURF-SNRPN promoter and NHS2. Additionally YY1 may target the paternal allele to the nuclear matrix, which would account for the nuclear localization and early replication of that allele.