Genotype-dependent regulation at the Autoimmune Hepatitis-associated ALDH2 locus

Abstract

By Amber Sauder, Health Sciences

Advisor: Rachel Gleason

Presentation ID: AM_D13

Abstract: Autoimmune Hepatitis (AIH) is an inflammatory disease of the liver affecting both children and adults. Genome-wide association studies (GWAS) has identified regions in the genome associated with AIH and this project focused on the region containing the ALDH2 gene. In a known risk locus for AIH, the most highly associated genetic variant in this region is rs3184504, along with 10 other variants in linkage disequilibrium. Rs3184504 is the top functional candidate among these variants because its genomic coordinates coincide with the largest number of transcription factor binding events and histone modifications in disease-relevant cell types. We predict that the region containing rs3184504 loops around and acts as an enhancer for ALDH2 in a genotype-dependent manner, which will alter binding of transcription factor YBX1 and ultimately ALDH2 expression. This was demonstrated through an Electrophoresis Mobility Shift Assay (EMSA) and a luciferase assay. Using nuclear lysate extracted from phenotype-relevant cell lines, an EMSA was performed to determine whether genotype-dependent binding is present. Recombinant protein was then be used to perform an EMSA supershift. Expression constructs that contain an enhancer region around the risk and non-risk versions of rs3184504 have been previously cloned, and were used for quantitative measurements of ALDH2 expression. They were transfected into liver cells to measure the amount of luciferase produced to quantify genotype-dependent ALDH2 expression. We expect to see genotype-dependent binding of YBX1 to the rs3184504, and differential luciferase reporter activity with the ALDH2 promoter and two different versions of the genetic variant (risk and non-risk).