In vitro functional assay of alleles and haplotypes of two COL1A1-promoter SNPs

Abstract

Osteoporosis is a common disease with a strong genetic component. We previously described two polymorphic sites in the COL1A1 gene promoter, −1997 G/T and −1663indelT, which have been associated with bone mineral density (BMD), a surrogate trait for osteoporosis. Here, we explore the molecular mechanisms underlying this association by performing transient transfections in MG-63 cells of constructs bearing different COL1A1 promoter regions, containing different alleles or haplotypes of the polymorphic sites. These promoter regions drove the transcription of a luciferase reporter gene. The main differences in transcriptional activity relied on an inhibitory region localized to the −1284 to −254 interval. Regarding the polymorphisms, reproducible differences were observed between the alleles of each of them: the G allele at −1997 showed a higher transcriptional activity than the T allele, as did the 7T allele of −1663 as compared with 8T. Accordingly, the T-8T haplotype was the weakest transcriber. A functional interaction was found between the −1997 and −1663 polymorphisms, in that the difference in transcriptional activity between the 7T and 8T alleles was dependent on the allele at −1997. This different transcriptional activity of the two −1663indelT alleles correlated with different binding capacities of the corresponding oligonucleotides to osteoblast nuclear proteins. Supershift assays allowed us to identify one of these proteins as the architectural transcription factor Nmp4/CIZ, a protein known to be an inhibitor of BMP/Smad signalling.

Introduction

Type I collagen is the most abundant component of the extracellular matrix of connective tissues and, in particular, of bone. Its transcriptional regulation is highly complex because of the influence of developmental, environmental and hormonal factors, and overall it plays an important role in many physiological as well as pathological events [13], [28]. The protein is encoded by two genes, COL1A1 and COL1A2, which, under physiological conditions, are expressed and regulated in a coordinated fashion to produce the α1 and α2 polypeptide chains, respectively. Two α1 chains interact with one α2 to form a characteristic triple-helix.

The control of collagen gene transcription is believed to involve complex interactions among DNA-binding proteins recognizing separate cis-acting elements in the 5′ regulatory and proximal promoter elements. Several studies of the promoter function of the mouse, rat, or human COL1A1 genes have been performed, both in vitro and using transgenic mice [2], [3], [14], [16], [21], [22]. A minimal promoter within the 220-bp region upstream of the transcriptional start site of COL1A1 contains sequences sufficient for its basal and tissue-specific transcription [7], [26]. In addition, Alvarez et al. [1] have characterized an architectural transcription factor that binds to regulatory regions located more than 1.5 kb upstream of the transcriptional start site.

The two type I collagen genes constitute good candidates to partly explain the genetic variability underlying osteoporosis or bone mineral density (BMD, a surrogate marker of the disease). A G to T polymorphism within an Sp1 binding element in intron 1 (position +1245) of the COL1A1 gene has been described as being associated with osteoporotic fracture [10].

In a previous study, we identified two single-nucleotide polymorphisms (SNPs) in the promoter of the COL1A1 gene: −1663indelT and −1997G/T [8]. The G to T transversion at −1997 was significantly associated with lumbar spine BMD in Spanish postmenopausal women, as was the interaction between the two polymorphisms. The association between the −1997G/T polymorphism and BMD was later replicated in a cohort of British women [19]. Interestingly, the −1663indelT polymorphism was in strong linkage disequilibrium with the +1245G/T (Sp1) polymorphism of intron 1. Gel retardation assays to test binding of osteoblast nuclear proteins to the PCOL1 and PCOL2 sites, which contained the two polymorphisms (−1663indelT and −1997G/T, respectively), revealed specific binding to single-stranded oligonucleotides harboring either of the sites [8]. In the case of the −1997G/T polymorphism, allelic differences in binding were observed, the G allele exhibiting stronger binding than the T.

To assess the involvement of these two COL1A1-promoter SNPs in transcriptional regulation, we have fused a 2.5-kb DNA fragment comprising bases −2483 to +40 of the human COL1A1 promoter to a luciferase reporter gene and assayed the four haplotypes in transiently transfected MG-63 osteosarcoma cells. We have also studied the influence of several hormones, cytokines, and vitamins on COL1A1 promoter function. Finally, we have identified one of the proteins binding to the PCOL1site.