Single-nucleotide polymorphisms interact to affect ADH7 transcription

Abstract

Background: The class IV alcohol dehydrogenase (ADH7, μ-ADH, σ-ADH) is important in the metabolism of ethanol and retinol. ADH7 is the only ADH not expressed in liver, instead being expressed mainly in the upper gastrointestinal tract. Genome-wide studies have identified significant associations between single-nucleotide polymorphisms in ADH7 and alcoholism and cancer, but the causative variants have not been identified.

Methods: In vitro studies of gene expression by transient transfection into cell lines that express endogenous ADH7 (CP-A cells) and that do not (HepG2 cells).

Results: We have identified transcriptional regulatory elements of ADH7 and observed differences in the effects of variants on gene expression in CP-A cells and HepG2 cells. Two haplotypes of the proximal promoter that differ in a single nucleotide at rs2851028, A7P-G and A7P-A, have different transcriptional activities. There is an interaction between variants farther upstream and these proximal variants: Upstream regulatory sequences generally showed a greater increase or smaller reduction in activity when combined with the A7P-A promoter than with the A7P-G promoter. A sequence located 12.5-kb upstream (7P10) can function as an enhancer. In CP-A cells, both haplotypes of 7P10 increased A7P-A activity by 2.5-fold while having only 1.2-fold effect on A7P-G. In HepG2 cells, the 7P10-TTT haplotype had no effect on the A7P-A promoter but decreased A7P-G promoter activity by 50%, whereas the CTT haplotype increased A7P-A activity by 50%, but had no effect on A7P-G.

Conclusions: These complex interactions indicate that the effects of variants in the ADH7 regulatory elements depend on both sequence and cellular context and should be considered in interpretation of the association of variants with alcoholism and cancer.

Keywords: Alcohol Dehydrogenase 7; Alcoholism; Class IV Alcohol Dehydrogenase; Gene Regulation; Genetic Association.

Figure 1

Map of the ADH7 promoter and upstream fragments in chromosomal orientation, drawn to scale, with promoter (black solid), monomorphic fragments (patterned) and polymorphic fragments (not shaded). Transcription occurs right to left (arrow) from the translation start site (TSS). SNPs are shown, with the three LD blocks represented as solid black circles, plain circles or grey circle.

Figure 2

ADH7 promoter haplotypes are active in both CP-A and HepG2 cells. The relative activity is the ratio of promoter activity of the G construct (A7P-G) to the A construct (A7P-A) within each cell line. Standard errors of mean are shown (n≥12). P-value was 3 × 10⁻⁵ (HepG2 cells) and 1 × 10⁻⁶ (CP-A cells).

Figure 3

Activity of monomorphic fragments on the two promoter haplotypes. Transient transfections of the monomorphic upstream sequences 7P2, 7P3, 7P4, 7P7 and 7P9 were done in A) CP-A cells and B) HepG2 cells. Relative activity of each construct represents the ratio of normalized luciferase activity of the test fragment to the A7P-A (black) promoter or A7P-G (grey shaded). Scales of vertical axes are different. Error bars represent standard errors of mean. * indicate p-values ≤ 0.01; ** indicate p-values ≤ 1 × 10⁻⁵.

Figure 4

Activity of polymorphic fragments on the two promoter haplotypes. Transient transfections of two naturally occurring haplotypes of each polymorphic fragment (7P5, 7P6, 7P8 and 7P10) were done in A) CP-A cells and B) HepG2 cells. Each sequence was tested for effect on both promoters A7P-G and A7P-A. Relative activity of each construct represents the ratio of normalized luciferase activity to the promoter on which it was tested, A7P-G (grey shaded) or A7P-A (black). Error bars represent standard errors of mean. Scales of vertical axes are different. Relative activities are shown with * indicating p-values ≤ 0.008; ** indicate p-values ≤ 1 × 10⁻⁴

Figure 5

Localization of regulatory elements in fragment 7P10. A) 7P10 sub-fragments approximately 500 bp in size are shown as oriented on the chromosome, with E1 being the farthest upstream from ADH7. B) Activity of sub-fragments of 7P10 in CP-A cells.

Figure 6

Fragment 7P10 acts as an enhancer. A) Map of the p×P2 vector construct with E2 or E2flip (E2 in reverse orientation) cloned into the HindIII and XhoI sites immediately upstream of A7P drawn to scale. E2far contains E2 cloned into the ApaI and SwaI sites 3.5 kb away from the promoter to test for position effects. B) 7P10-E2 tested for promoter activity (E2prom) and enhancer properties, orientation independence (E2flip) and position independence (E2far), by transient transfections in CP-A cells. Activity was measured relative to A7P-A. Error bars represent standard error of means. All p-values ≤ 1 × 10⁻⁸