Abstract
BackgroundWe have identified a cytosine-adenosine (CA) repeat length polymorphism in the 5′-regulatory region of the human integrin α2 gene ITGA2 that begins at −605. Our objective was to establish the contribution of this polymorphism to the regulation of integrin α2β1 expression, which is known to vary several-fold among normal individuals, and to investigate the underlying mechanism(s).Methodology/Principal FindingsIn combination with the SNP C-52T, previously identified by us as a binding site for the transcription factor Sp1, four ITGA2 haplotypes can be distinguished, in the order in which they enhance ITGA2 transcription: (CA)12/-52C>(CA)11/-52C>(CA)11/-52T>(CA)10/-52T. By DNA affinity chromatography and chromatin immunoprecipitation (ChIP) assays, we show that poly (ADP-ribose)polymerase-1 (PARP-1) and Ku80/70 bind specifically and with enhanced affinity to the longer (CA)12 repeat alleles.Conclusions/SignificanceThe increased binding of PARP-1 and Ku80/70, known components of transcription co-activator complexes, to the longer (CA)12 alleles of ITGA2 coincides with enhanced α2β1 expression. The most likely explanation for these findings is that PARP-1 and Ku80/70 contribute to the transcriptional regulation of ITGA2. These observations provide new insight into the mechanisms(s) underlying haplotype-dependent variability in integrin α2β1 expression in human platelets and other cells.
Highlights
The fact that cellular integrin a2b1 levels can vary up to tenfold among normal, healthy subjects was first discovered on blood platelets [1,2], where it leads to variation in adhesive function
We define the CA repeat length polymorphism as 10 to 12 repeats with the 39 sequence beginning at 2605, we show that it is in linkage disequilibrium with C-52T based on an analysis of 132 human chromosomes, and we analyze its contribution to transcription vis-a-vis C-52T in megakaryocytic (MK) and nonmegakaryocytic cell lines
We have observed that an increase in the length of a CA repeat polymorphism in the 59-regulatory region of ITGA2
Summary
The fact that cellular integrin a2b1 levels can vary up to tenfold among normal, healthy subjects was first discovered on blood platelets [1,2], where it leads to variation in adhesive function. The genetic basis for this heritable variation has been the subject of a comprehensive study by our lab, and progress has been made in understanding the variety of mechanisms, some species-dependent [8], that control expression of this integrin. A T at position -52 disrupts what is otherwise a highly favorable Sp1 binding site and decreases its binding by 8–10 fold [2] This SNP, in linkage disequilibrium with two coding region SNPs, C807T [10,11] and G1648A [12] defines five common and several rare ITGA2 haplotypes [13]. Our objective was to establish the contribution of this polymorphism to the regulation of integrin a2b1 expression, which is known to vary several-fold among normal individuals, and to investigate the underlying mechanism(s)
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