Regulatory elements required for human angiotensinogen expression in HepG2 cells are dispensable in transgenic mice.
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.
Yang
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Department of Anatomy, The University of Iowa College of Medicine, Iowa City 52242, USA.
Sigmund
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D000808:Angiotensinogen / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D004742:Enhancer Elements, Genetic; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D006801:Humans; D017403:In Situ Hybridization; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic / Q000235:genetics*; D016296:Mutagenesis; D009154:Mutation; D012045:Regulatory Sequences, Nucleic Acid; D014407:Tumor Cells, Cultured
D000808:Angiotensinogen