−13915*G DNA polymorphism associated with lactase persistence in Africa interacts with Oct-1
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- Olds, L.C., Ahn, J.K. & Sibley, E. Hum Genet (2011) 129: 111. doi:10.1007/s00439-010-0898-0
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Lactase gene expression declines with aging (lactase non-persistence) in the majority of humans worldwide. Lactase persistence is a heritable autosomal dominant condition and has been strongly correlated with several single nucleotide polymorphisms (SNPs) located ~14-kb upstream (−13907, −13910 and −13915) of the lactase gene in different ethnic populations. In contrast to the −13907*G and −13910*T SNPs, the −13915*G SNP was previously believed not to interact with Oct-1. In the present study, however, Oct-1 is shown to interact with the −13915*G SNP region DNA sequence by EMSAs and gel supershift. In addition, Oct-1 is capable of enhancing promoter activity of a lactase promoter–reporter construct harboring the 13915*G SNP sequence in cell culture. Oct-1 binding to the −13907 to −13915 SNP region therefore remains a candidate interaction involved in lactase persistence.
Relative light units
Single nucleotide polymorphism
Electrophoretic mobility shift assay
Lactase-phlorizin hydrolase, lactase, is the intestinal enzyme responsible for the digestion of the milk sugar lactose. The majority of the world’s human population experiences a decline in expression of the lactase gene by late childhood (lactase non-persistence). Individuals with lactase persistence, however, continue to express high levels of the lactase gene throughout adulthood. Lactase persistence is a heritable autosomal dominant condition and has been strongly correlated with several single nucleotide polymorphisms (SNPs) located ~14-kb upstream of the lactase gene in different ethnic populations: −13910*T in Europeans and −13907*G, −13915*G, and −14010*C in several African ethnic pastoral populations (Enattah et al. 2002, 2008; Ingram et al. 2007; Tishkoff et al. 2007). Though strongly correlated with the lactase persistence phenotypes, a functional mechanism for the SNPs in mediating lactase persistence has not been clearly defined. Transfection experiments have shown that the lactase persistence SNP region DNA can function as a cis element capable of enhancing differential transcriptional activation of the lactase promoter in cell culture (Olds and Sibley 2003; Troelsen et al. 2003; Lewinsky et al. 2005). While previous reports have indicated that the −13907*G and −13910*T SNP region sequences can interact with the Oct-1 transcription factor (Lewinsky et al. 2005; Ingram et al. 2007), Oct-1 binding to the −13915*G SNP was minimal or not detected (Ingram et al. 2007; Enattah et al. 2008). In the present study, however, we demonstrate that the −13915*G region can interact with the Oct-1 protein.
Results and discussion
The lactase persistence SNP region −13915*G interacts with the Oct-1 transcription factor
To confirm that Oct-1 interacts with the −13915*G SNP region, gel supershifts were performed in the presence of Oct-1 specific antibody or non-specific antibody control. Incubation of the −13915*G probe with Caco-2 cell nuclear extract in the presence of control non-specific IgM antibody results in the specific DNA/protein complex band (lane 7, arrow). Incubation in the presence of Oct-1 specific antibody, however, results in decreased abundance of the specific DNA/protein complex band (lane 6). This result is consistent with Oct-1 interaction to form the −13915*G DNA/Protein complex. The presence of a faint −13915*G band after Oct-1 competition (Fig. 1a, lane 4) or supershift (Fig. 1b, lane 2) suggests that additional nuclear protein(s) may also interact with the −13915*G probe.
In order to determine whether the DNA sequence surrounding the −13915*G human lactase persistence variant is capable of regulating lactase promoter activity, Caco-2 cells were transfected with lactase promoter–reporter constructs harboring the −13915*G or ancestral −13915*T sequences. The constructs were generated by cloning the G/T-13915 SNP region of the human lactase promoter upstream of the 2.0-kb rat lactase promoter in the luciferase reporter plasmid pGL3Basic. Transfected Caco-2 cell extracts were assayed for relative luciferase activity 48 h after transfection as shown in Fig. 1c. Transfection with the lactase persistence associated −13915*G SNP construct, p2K.Lac-13915G, results in significantly greater promoter activity than the ancestral p2K.Lac-13915T lactase promoter construct. Differential enhancement of lactase promoter activity mediated by the G/T-13915 SNP variant region suggests a functional role for the DNA variant in specifying the lactase persistence/non-persistence phenotypes. Co-transfection with an Oct-1 expression construct results in a ~2-fold increase in promoter activity driven by both promoter–reporter constructs, consistent with reports (Lewinsky et al. 2005; Ingram et al. 2007) that Oct-1 can function to regulate the human lactase promoter. Similar fold increase of both promoter–reporter constructs by the Oct-1 expression construct, however, suggests that excess Oct-1 does not result in the differential lactase promoter activity associated with the G/T-13915 SNP region but rather that Oct-1 general upregulation may be mediated by an invariant region of the promoter constructs.
In summary, we have demonstrated that the −13915*G SNP region (associated with lactase persistence) of the lactase gene interacts with the Oct-1 transcription factor in in vitro binding reactions. Such interaction is contrary to previous reports in which Oct-1 binding to the −13915*G SNP was not detected suggesting that the SNP mutation abolishes Oct-1 binding. With the current report, Oct-1 has now been shown to interact with the sequence surrounding each of the currently identified SNPs located between −13907 and −13915. Oct-1 binding to the SNP region therefore remains a candidate interaction involved in lactase persistence.
This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grants DK60074 and DK60715.