An upstream polymorphism associated with lactase persistence has increased enhancer activity  Jesper T Troelsen, Jørgen Olsen, Jette Møller, Hans SjÖstrÖm  Gastroenterology  Volume 125, Issue 6, Pages 1686-1694 (December 2003) DOI: 10.1053/j.gastro.2003.09.031

Figure 1 (A) Sequences of positions −13915 to −13905 and −22023 to −22013 from the LPH start codon from a lactase-persistent person (Lac. per.) and from a lactase-nonpersistent person (Lac. non.). (B) Transfection analysis of the gene regulatory effect of the −13910 region and the −22018 region on SI promoter-driven transcription. A 455-bp fragment surrounding the −13910 position and a 440-bp fragment surrounding the −22018 region from a lactase-persistent and a -nonpersistent person were cloned immediately in front of the human SI promoter. Caco-2 cells were transfected and analyzed 2 days after transfection (■, Undif. Caco-2) or 9 days after transfection (□, Dif. Caco-2). The luciferase activities were corrected for transfection efficiency and normalized according to the expression of pGL3 SI257 (n = 8). Gastroenterology 2003 125, 1686-1694DOI: (10.1053/j.gastro.2003.09.031)

Figure 2 Transfection analysis of the transcriptional importance of the T/C polymorphism at position −13910 on LPH promoter-driven transcription. The −13910T and the −13910C regions from a lactase-persistent and a -nonpersistent person were cloned in a LPH promoter/luciferase construct 3960 bp upstream of the start codon of the reporter gene. The −13910 region was analyzed in both orientations as indicated by arrows. Caco-2 cells were transfected and analyzed (A) 2 days after transfection or (B) 9 days after transfection. The luciferase activities were corrected for transfection efficiency and normalized according to the expression of pGL3 LPH1085. The statistical significance of the differences between the reporter gene levels was tested using the Student t test. pGL3 LPH1085-13910T was tested against pGL3 LPH1085-13910C. pGL3 LPH1085-13910Trev was tested against pGL3 LPH1085-13910Crev. ∗P < 0.05; ∗∗∗P < 0.01; n = 6. ■, Undif Caco-2; □, Dif. Caco-2. Gastroenterology 2003 125, 1686-1694DOI: (10.1053/j.gastro.2003.09.031)

Figure 3 Transfection analysis of the transcriptional importance of the A/G polymorphism at position −22018. The −22018A and the −22018G variants were cloned in a LPH promoter/luciferase construct 3960 bp upstream of the start codon of the reporter gene. Promoter constructs were generated containing both the −13910 and −22018 regions upstream of the LPH promoter. The 14T/22A combination has been reported to be associated with the lactase-persistent phenotype and the 14C/22G combination with the lactase-nonpersistent phenotype.18 Caco-2 cells were transfected and analyzed 2 days after transfection (■, Undif. Caco-2) or 9 days after transfection (□, Dif. Caco-2). The luciferase activities were corrected for transfection efficiency and normalized according to the expression of pGL3 LPH1085. The statistical significance of the differences between the reporter gene levels was tested using the Student t test. pGL3 LPH1085-14T/22A was tested against pGL3 LPH1085-13910T. pGL3 LPH1085-14C/22G was tested against pGL3 LPH1085-13910C. pGL3 LPH1085-22018A and pGL3 LPH1085-22018G were both tested against pGL3 LPH1085 ∗P < 0.05; ∗∗∗P < 0.01; n = 4. Gastroenterology 2003 125, 1686-1694DOI: (10.1053/j.gastro.2003.09.031)

Figure 4 Nuclear extracts from differentiated (A) Caco-2 cells and (B) HeLa cells were assayed for binding activity to P32-labeled −13910T and −13910C-oligonucleotides by EMSA. A specific complex (Comp) was formed using −13910T as probe (lane 1), and a weaker band was seen using −13910C as probe (lane 6). Competition of binding was examined by 20-fold excess (0.5 pmol) of unlabeled −13910T and −13910C oligonucleotides (lanes 2, 3, 7, and 8), 20-fold excess of an AP2-oligonucleotide (lanes 4 and 9), or an unspecific oligonucleotide (unspec) (lanes 5 and 10). (C) Southwestern blot with Caco-2 nuclear extracts using 32P-labeled −13910T and −13910C oligonucleotides as probes. Unlabeled oligonucleotides were used as competitors to identify specific bands (arrows). Unspecific bands are indicated by us. Gastroenterology 2003 125, 1686-1694DOI: (10.1053/j.gastro.2003.09.031)

Figure 5 Schematic model showing the interaction between the promoter of LPH gene and polymorphic −13910 and −22018 regions in the MCM6 gene in children and lactase-persistent and -nonpersistent adults. During childhood and before weaning in other mammals the level of LPH expression is high because the transcription factors (HNF1α, GATA factors, Cdx-2) known to regulate LPH expression are available in excess. The expression of LPH is therefore not dependent on the −13910 enhancer activity. In adulthood the accessibility to the transcription factors is reduced. The strong enhancer activity of the −13910T variant ensures an active LPH gene throughout life (lactase persistence). The lower activity of the −13910C variant fails to activate/recruit the transcription factors, which results in a low LPH gene activity in nonpersistent adults. Although the −22018 region represses LPH transcription the role of the −22018 region is unclear, but the repression does not seem to be related to the A/G polymorphism. Gastroenterology 2003 125, 1686-1694DOI: (10.1053/j.gastro.2003.09.031)