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

Abstract

The gene encoding the small nuclear ribonucleoprotein-associated polypeptide N (SNRPN) maps to the Prader-Willi syndrome critical region on chromosome 15 and is expressed preferentially from the paternal allele. A CpG island encompassing the first exon of SNRPN is methylated on the inactive maternal allele. DNA sequence was determined for a cosmid containing the first three exons of SNRPN and extending 20 kb upstream and 15 kb downstream from the CpG island. This region is extremely rich in Alu elements and other repetitive sequences and contains a single CpG island, which includes numerous short direct repeat sequences. Functional analysis of the first exon revealed strong promoter activity for a 260-bp fragment extending 207 bp upstream from the exon. In vitro methylation of this 260-bp fragment abolished promoter activity completely, suggesting that the silencing of the maternal SNRPN allele may be a direct consequence of methylation of the promoter region.

Figure 1

Sequence analysis of cosmid c102. The frequency of CpG dinucleotides and Alu elements are shown with the locations of exons α, β, and 1. Potential additional exons predicted from the GRAIL program are shown as in the same or opposite orientation to the transcription of SNRPN.

Figure 2

Promoter analysis for exons α and 1. CAT activity is presented relative to β-galactosidase activity for all transfections. The pCAT Control plasmid (pCATC), which includes the SV40 promoter and enhancer, is shown as 100%. The promoterless plasmid containing the SV40 enhancer is designated pCATE. Other constructs are described in the text. Separate transfection experiments (n) were performed with multiple constructs.