Comparative genomic analysis reveals a distant liver enhancer upstream of the COUP-TFII gene

COUP-TFII is a central nuclear hormone receptor that tightly regulates the expression of numerous target lipid metabolism genes in vertebrates. However, it remains unclear how COUP-TFII itself is transcriptionally controlled since studies with its promoter and upstream region fail to recapitulate the gene's liver expression. In an attempt to identify liver enhancers in the vicinity of COUP-TFII, we employed a comparative genomic approach. Initial comparisons between humans and mice of the 3470-kb gene-poor region surrounding COUP-TFII revealed 2023 conserved noncoding elements. To prioritize a subset of these elements for functional studies, we performed further genomic comparisons with the orthologous pufferfish (Fugu rubripes) locus and uncovered two anciently conserved noncoding sequences (CNS) upstream of COUP-TFII (CNS-62kb and CNS-66kb). Testing these two elements using reporter constructs in liver cells (HepG2) revealed that CNS-66kb, but not CNS-62kb, yielded robust in vitro enhancer activity. In addition, an in vivo reporter assay using naked DNA transfer with CNS-66kb linked to luciferase displayed strong reproducible liver expression in adult mice, further supporting its role as a liver enhancer. Together, these studies further support the utility of comparative genomics to uncover gene regulatory sequences based on evolutionary conservation and provide the substrates to better understand the regulation and expression of COUP-TFII.