An albumin enhancer located 10 kb upstream functions along with its promoter to direct efficient, liver-specific expression in transgenic mice
Liver-specific control elements for albumin had not been mapped, despite known enhancers in the related alpha-fetoprotein gene. Transgenic mice with albumin-growth hormone fusions located an enhancer 8.5 to 10.4 kb upstream, boosting hepatic mRNA about 50-fold.
Parts used
Key findings
- Pinkert et al. mapped cis-acting elements directing liver-specific albumin gene expression in transgenic mice using albumin–hGH fusion constructs spanning 0.3–12 kb of 5'-flanking sequence. A far-upstream region located 8.5–10.4 kb upstream of the albumin promoter was essential for high-level hepatic expression; the 12-kb construct produced ~5,000 hGH mRNA molecules per hepatocyte, approaching the ~10,000 molecules/cell of endogenous albumin mRNA.
- The active enhancer was localized to a ~1.9-kb NheI-BamHI fragment: placed upstream of the 0.3-kb albumin promoter, it drove hepatic hGH mRNA averaging ~1,200 molecules/cell in 9 of 12 transgenic founders with no ectopic kidney expression. The element functioned equally in reverse orientation but failed to activate the heterologous hGH promoter, indicating the albumin enhancer requires a liver-specific promoter context.
- DNase hypersensitivity mapping identified four sites in the albumin 5'-flanking region: HS1 at −10.5 kb within the active enhancer, HS2 at −7.5 kb, HS3 at −3.1 kb, and HS4 near the promoter. HS1 and HS4 were present exclusively in liver nuclei and absent in kidney nuclei, demonstrating that open chromatin at the promoter and far-upstream enhancer correlates with tissue-specific transcriptional activity.
Abstract
Transgenic mice were used to locate the cis-acting DNA elements that are important for efficient, tissue-specific expression of the mouse albumin gene in the adult. Chimeric genes with up to 12 kb of mouse albumin 5'-flanking region fused to a human growth hormone (hGH) reporter gene were tested. Remarkably, a region located 8.5-10.4 kb upstream of the albumin promoter was essential for high-level expression in adult liver and the region in between -8.5 and -0.3 kb was dispensable. The far-upstream region behaved like an enhancer in that its position and orientation relative to the albumin promoter were not critical; however, it did not function well with a heterologous promoter. Two of four DNase hypersensitive sites found in the 5'-flanking region of the albumin gene map to the far-upstream and promoter regions; the others may reflect regions involved in developmental or environmental control of this gene.