To identify distant regulatory elements that affect expression of the ADH4 gene, we took a comparative genomics approach. Using the UCSC Conservation track (Felsenstein and Churchill, 1996; Siepel et al., 2005; Yang, 1995), we identified conserved sequences in the intergenic regions between ADH4 and its flanking genes ADH5 and ADH6. We tested the effects of conserved regions on the activity of the ADH4 Basal promoter (4Basal) by transient transfection assays in HepG2 human hepatoma cells (data not shown). We identified a 1504 bp conserved region (4E), 13 kb upstream of the ADH4 translation start site (Figure 1B), that had significant effect on ADH4 promoter activity. Fragment 4E caused a 50-fold increase in activity (p = 6 × 10−15; Figure 2). In the absence of the promoter, the 4E fragment had weak activity, only 80% of the activity of the 4Basal promoter (p = 9 × 10−4; Figure 2)
