Identification of cis-Regulatory Sequences in the Human Angiotensinogen Gene by Transgene Coplacement and Site-Specific Recombination

Abstract

The function of putative regulatory sequences identified in cell transfection experiments can be elucidated only through in vivo experimentation. However, studies of gene regulation in transgenic mice (TgM) are often compromised by the position effects, in which independent transgene insertions differ in expression depending on their location in the genome. In order to overcome such a dilemma, a method called transgene coplacement has been developed in Drosophila melanogaster. In this method, any two sequences can be positioned at exactly the same genomic site by making use of Cre/loxP recombination. Here we applied this method to mouse genetics to characterize the function of direct repeat (DR) sequences in the promoter of the human angiotensinogen (hAGT) gene, the precursor of the vasoactive octapeptide angiotensin II. We modified a hAGT bacterial artificial chromosome to use Cre/loxP recombination in utero to generate TgM lines bearing a wild-type or a mutant promoter-driven hAGT locus integrated at a single chromosomal position. The expression analyses revealed that DR sequences contribute 50 or >95% to hAGT transcription in the liver and kidneys, respectively, whereas same sequences are not required in the heart and brain. This is the first in vivo dissection of DNA cis elements that are demonstrably indispensable for regulating both the level and cell type specificity of hAGT gene transcription.

ACKNOWLEDGMENTS

We thank Y. Tanimoto for technical assistance in preparing TgM. We also thank Kim-Chew Lim for help with the manuscript.

This study was supported by grants from the Yamanouchi Foundation for Research on Metabolic Disorders (K.T.); the Japan Heart Foundation (K.T.); the Uehara Memorial Foundation (K.T.); the Tokyo Biochemical Research Foundation (K.T.); the 21st Century COE Program (A.F.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT); a grant for the Support of Young Researchers (MEXT [K.T.]); and a Grant-in-Aid for Scientific Research (MEXT [A] to A.F. and [B] to K.T.).