Sequencing and functional analysis of the SNRPN promoter: in vitro methylation abolishes promoter activity.

The gene encoding the small ribonucleoprotein-associated polypeptide N (SNRPN) is located within chromosome 15q11–q13 in the region associated with Prader–Willi syndrome (PWS) and Angelman syndrome (AS). SNRPN is imprinted with preferential expression from the paternal chromosome (Glenn et al. 1993; Nakao et al. 1994; Reed and Leff 1994). The gene was initially described as having eight exons (Schmauss et al. 1992), but two additional upstream exons were described subsequently and designated as exons α and β (Sutcliffe et al. 1994) or −1 and 0 (Glenn et al. 1996). The imprinted expression of SNRPN is associated with preferential methylation of the maternal allele in the region of exon α, but there is a reciprocal methylation difference between the maternal and paternal alleles in intron 5 (Sutcliffe et al. 1994; Glenn et al. 1993, 1996). PWS is believed to be associated with deficiency of paternally expressed genes, with most patients having large deletions or maternal uniparental disomy (UPD) for chromosome 15 (Nicholls 1994; Ledbetter and Ballabio 1995). AS is believed to be caused by deficiency of a maternally expressed gene, and most AS patients have large deletions or paternal UPD; a significant fraction have no molecular abnormality identified to date (Nicholls 1994; Ledbetter and Ballabio 1995). Rare PWS and AS patients have “imprinting mutations” that are associated with smaller deletions upstream of SNRPN (Sutcliffe et al. 1994; Buiting et al. 1995). These deletions prevent the appropriate erasure and reestablishment of methylation patterns and are interpreted as evidence for the existence of an imprinting center that acts to establish the methylation and expression pattern appropriate for either a paternal or maternal chromosome. Although SNRPN is the only paternally expressed gene in this region known to encode a protein, other paternally expressed transcripts have been identified including PAR-1 (Prader–Willi Angelman region) (Sutcliffe et al. 1994), PAR-5 (Sutcliffe et al. 1994), and IPW (imprinted in Prader–Willi) (Wevrick et al. 1994). The IPW gene is comprised of three exons and may encode a functional RNA, as there is no substantial open reading frame within the 2.2-kb transcript. The relatively long transcripts of PAR-5 and PAR-1 are only partially characterized with no evidence of open reading frames (Sutcliffe et al. 1994). Prior to our identification of exon α as the most upstream exon and presumptive 5′ end of SNRPN, exon 1 was believed to be the most upstream exon. Previous analysis of exon 1 revealed detectable promoter activity, and this was interpreted as evidence that exon 1 had a natural promoter and was the first exon of SNRPN (Schmauss et al. 1992). If exon α is the true first exon, it should be associated with promoter activity, and this promoter should display some properties (e.g., response to methylation) related to the imprinted regulation of expression. To analyze the regulation of gene expression for SNRPN, a cosmid containing exons α, β, and 1, and extending 20 kb upstream, was sequenced, and the promoter activity for exons α and 1 was compared with and without in vitro methylation.