ABCG2 regulatory single-nucleotide polymorphisms alter in vivo enhancer activity and expression

Abstract

Objectives The expression and activity of the breast cancer resistance protein (ABCG2) contributes to the pharmacokinetics of endogenous and xenobiotic substrates. The effect of genetic variation on the activity of cis-regulatory elements and nuclear response elements in the ABCG2 locus and their contribution to ABCG2 expression has not been systematically investigated. In this study, the effect of genetic variation on the in vitro and in vivo enhancer activity of six previously identified liver enhancers in the ABCG2 locus was examined. Methods Reference and variant liver enhancers were tested for their ability to alter luciferase activity in vitro in HepG2 and HEK293T cell lines and in vivo using a hydrodynamic tail vein assay. Positive in vivo SNPs were tested for association with gene expression and for altered protein binding in electrophoretic mobility shift assays (EMSA). Results Multiple SNPs were found to alter enhancer activity in vitro. Four of these variants (rs9999111, rs12508471, ABCG2RE1*2 and rs149713212) decreased and one (rs2725263) increased enhancer activity in vivo. Additionally, rs9999111 and rs12508471 were associated with ABCG2 expression in lymphoblastoid cell lines, lymphocytes and T cells, and showed increased HepG2 nuclear protein binding. Conclusion This study identifies SNPs within regulatory regions of the ABCG2 locus that alter enhancer activity in vitro and in vivo. Several of these SNPs correlate with tissue-specific ABCG2 expression and alter DNA/protein binding. These SNPs could contribute to reported tissue-specific variability in ABCG2 expression and may influence the correlation between ABCG2 expression and disease risk or the pharmacokinetics and pharmacodynamics of BCRP substrates.

Toward the Identification of a Genetic Risk Signature for Pulmonary Aspergillosis in Chronic Obstructive Pulmonary Disease

“…Genetic variation in PTX3 has already been recognized as a major determinant of susceptibility to aspergillosis in stemcell [2,3] and solid organ transplant recipients [4,5]. By validating these findings in COPD patients, He et al further highlight the potential applicability of PTX3-based genetic markers in predicting infection across patients with intrinsically different predisposing conditions and disease courses.…”

Pharmacogenetics of hepatocellular carcinoma and cholangiocarcinoma

“…In healthy liver tissue, the expression of c.421C>A (p.Gln141Lys) variant correlates with low BCRP protein levels [ 77 ] . In addition, several SNPs that modify enhancer activity at the ABCG2 gene locus have been reported [ 64 ] . Four of these variants (g.89073197A>G, g.88924371A>G, g.89189602G>A and ABCG2RE1*2, which is a combination of g.88923906G>A, g.88924176C>T and g.88924371A>G) decreased the promoter activity and hence reduced gene expression, contrary to g.89026428A>C that is associated with increased BCRP activity.…”

“…Four of these variants (g.89073197A>G, g.88924371A>G, g.89189602G>A and ABCG2RE1*2, which is a combination of g.88923906G>A, g.88924176C>T and g.88924371A>G) decreased the promoter activity and hence reduced gene expression, contrary to g.89026428A>C that is associated with increased BCRP activity. Moreover, other genomic variants (g.89073197A>G and g.88924371A>G) increase the ability of ABCG2 gene to bind to nuclear proteins in human hepatoma HepG2 cells [ 64 ] .…”

Rare Variants in theABCG2Promoter Modulate In Vivo Activity

“…Electrophoretic Mobility Shift Assay. EMSAs were performed using a 2.5 nM 59 IRDye 700 (LI-COR)-labeled probe, incubated with 5 mg of HepG2 nuclear extract using the Odyssey EMSA Buffer Kit (LI-COR) as previously described (Eclov et al, 2017a). Competition assays were performed by adding a 40-fold molar excess of unlabeled reference oligonucleotide.…”

“…Additionally, genetic variation in promoters for transporters and enzymes have been linked to adverse drug reactions (Innocenti et al, 2004;Wang et al, 2008a;Kenna et al, 2009;McLeod et al, 2010;Toffoli et al, 2010). Previous studies of the effect of regulatory variants on ABCG2 expression (Zamber et al, 2003;Poonkuzhali et al, 2008;Eclov et al, 2017a) have only considered regions outside the primary promoter. In the present study, the basal activity of the major ABCG2 promoter (2499 to +21 bp relative to the TSS) was investigated in transiently transfected kidney, liver, intestine, and breast cell lines.…”