Functional analysis of a Sp1 binding site polymorphism in the collagen type α gene (COL1A1)
The exact role of the COL1A1 first intron in gene transcription has not been characterized. To study its role in bone metabolism I examined a transgenic mouse with a targeted deletion of the col1A1 first intron (col-Int). I measured, by peripheral quantitative computed tomography, bone mineral density (BMD), geometry and predicted biomechanical properties in wild-type, heterozygous col-Int and homozygous col-Int mice. First intron deletion affected bone geometry independently of BMD. Homozygous col-Int bones had significantly reduced cross sectional area and bone mineral content compared with wild-type bones, however there was no significant difference in total BMD between the genotypes. Examination of heterozygous col-Int mice demonstrated that the genotype affects were allele-dose and age dependent. The COL1A1 Spl polymorphism is associated with reduced BMD and an increased fracture risk. The polymorphism is situated in COL1A1 intron 1 within a Spl exacting element. Using electrophoretic mobility shift assays I have shown that the unfavourable "s" allele binds Spl with significantly greater affinity than the "S" allele. A fluorescent semi-nested RT-PCR assay demonstrated that in bone RNA from heterozygous subjects (Ss) the transcript abundance from the "s" allele is 2.5-fold greater than from the "S" allele. I examined the effects of the polymorphism on transcription using a reporter fusion gene construct (pCOL-hGH). The gene consisted of a 2.3 kb promoter, exon 1 and intron 1 from COL1A1 fused to the 3' section of the hGHgene. pCOL-hGH contained the "S" variant of the COL1A1 Spl polymorphism, pCOL-hGH-"s" was created by site directed mutagenesis. The pCOL-hGH variants were transfected into a human osteoblast-like cell line (MG63) and incubated for 48 hours. Transfection efficiency was monitored by co-transfection with a luciferase plasmid (pGL3). pCOL-hGH expression was detected by RT-PCR assays, however transcripts were of too low abundance to be detected by RNA hybridization.