Functional characterization of a genetic polymorphism in the promoter of the ESR2 gene

Abstract

The ESR2 gene encodes the estrogen receptor beta protein. Several studies have shown that genetic variants in the ESR2 gene are associated with a variety of clinical phenotypes. However, very little is known about the functional significance of ESR2 genetic variants. We used a bioinformatics approach to identify regions of the ESR2 promoter that is evolutionarily conserved across the genomes of several species. We resequenced 1.6 kb of the ESR2 gene which included 0.8 kb of the promoter, 0.3 kb of exon ON, and 0.5 kb of the following intron. We identified five single-nucleotide polymorphisms (SNPs) in the ESR2 promoter and one SNP in the intron. Phase analysis indicated that the SNPs likely exist in 11 different haplotypes. Three of the SNPs (rs8008187, rs3829768, rs35036378) were predicted to alter transcription factor binding sites in the ESR2 promoter. All three were detected only in African American subjects. The rs35036378 SNP was in the TATA box and was highly conserved across species. ESR2 promoter reporter assays in LNCaP and SKBR3 cell lines showed that the variant construct containing the rs35036378 SNP allele had approximately 50% less activity relative to the wild-type construct. We conclude that the rs35036378 SNP appears to cause a reduced promoter activity of the ESR2 gene.

Fig. 1

Schematic representation of the positions of the SNPs in the ESR2 gene. The designated position of each SNP is relative to the first nucleotide of Exon ON, which is nucleotide no. 45761078 of accession no. NT_026437. The black boxes represent the transcription factor binding sites in which the SNP exists. TSS transcription start site

Fig. 2

Conservation of the TATA box region of the ESR2 gene. The alignment was constructed using ClustalW. The asterisk below the sequence indicates conservation across all four species. The bold nucleotides indicate the TATA box and the arrow above indicates the position of the SNP

Fig. 3

Effect of the TATA box SNP on ESR2 promoter activity. Wild-type (WT) and variant (VAR; -43(T/G)) promoters were cloned into the pGL3 plasmid and expressed in LNCaP (A) and SKBR3 (B) cells. Values are means and standard errors of the firefly luciferase activity normalized to the renilla luciferase activity of three experiments performed on three different days (p value 0.009 (LNCaP), 0.0002 (SKBR3))