Identification of strong intron enhancer in the heparanase gene: effect of functional rs4693608 variant on HPSE enhancer activity in hematological and solid malignancies

摘要

Heparanase is an endo-β-glucuronidase that specifically cleaves the saccharide chains of heparan sulfate (HS) proteoglycans and releases HS-bound cytokines, chemokines, and bioactive growth-promoting factors. Heparanase plays an important role in the nucleus as part of an active chromatin complex. Our previous studies revealed that rs4693608 correlates with heparanase levels and increased risk of acute and extensive chronic graft vs. host disease (GVHD). Discrepancy between recipient and donor in this SNP significantly affected the risk of acute GVHD. In the present study, we analyzed the HPSE gene region, including rs4693608, and demonstrated that this region exhibits SNPs-dependent enhancer activity. Analysis of nuclear proteins from normal leukocytes revealed their binding to DNA probe of both alleles with higher affinity to allele G. All malignant cell lines and leukemia samples disclosed a shift of the main bands in comparison to normal leukocytes. At least five additional shifted bands were bound to allele A while allele G probe was bound to only one main DNA/protein complex. Additional SNPs rs4693083, rs4693084, and rs4693609 were found in strong linkage disequilibrium (LD) with rs11099592 (exon 7). Only rs4693084 affected protein binding to DNA in cell lines and leukemia samples. As a result of the short distance between rs4693608 and rs4693084, both SNPs may be included in a common DNA/protein complex. DNA pull-down assay revealed that heparanase is involved in self-regulation by negative feedback in rs4693608-dependent manner. During carcinogenesis, heparanase self-regulation is discontinued and the helicase-like transcription factor begins to regulate this enhancer region. Altogether, our study elucidates conceivable mechanism(s) by which rs4693608 SNP regulates HPSE gene expression and the associated disease outcome.