Regulatory Elements Required for Human Angiotensinogen Expression in HepG2 Cells Are Dispensable in Transgenic Mice

Abstract

Abstract —Previous researchers have identified two sequences present upstream (angiotensinogen gene–activating element [AGE2]) and downstream (d61–2) of the human angiotensinogen gene that act as cell-specific enhancers of transcription in transiently transfected HepG2 cells. To examine the importance of these two sequences in regulating tissue- and cell-specific expression of the gene in vivo, we generated transgenic mice containing the mutations in the context of a genomic transgene previously shown to exhibit appropriate tissue and cell specificity. The ability of these sequences to enhance transcription of a basal human angiotensinogen promoter was confirmed in transient transfection assays in HepG2 cells, and mutations within the AGE2 and d61–2 sequences abolished transactivation of the promoter. Tissue- and cell-specific expression was examined in three lines of transgenic mice carrying the d61–2 mutation, two lines of transgenic mice carrying the AGE2 mutation, and three founder transgenic mice carrying a double-mutant construct. Although the absolute levels of expression varied among lines, the pattern of tissue-specific expression was essentially unaltered by the mutations. In situ hybridization confirmed that the mutations were also dispensable for proximal tubule-specific expression within the kidney. Finally, a comparison of transgene expression with transgene copy number revealed a direct proportionality in liver ( R =.77, P =.0014) and kidney ( R =.76, P =.0024). These results clearly demonstrate that these sites, which strongly induce promoter activity in cells in culture, are not required for appropriate expression of the gene when present in a genomic construct in vivo.