This Article Full Text Full Text (PDF) Data Supplement All Versions of this Article: 103/9/940    most recent CIRCRESAHA.108.180653v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Dickson, M. E. Articles by Sigmund, C. D. Search for Related Content PubMed PubMed Citation Articles by Dickson, M. E. Articles by Sigmund, C. D. Related Collections Animal models of human disease Other hypertension Genomics Hypertension - basic studies Genetics of cardiovascular disease

(Circulation Research. 2008;103:940.)
© 2008 American Heart Association, Inc.

Molecular Medicine

Upstream Stimulatory Factor Is Required for Human Angiotensinogen Expression and Differential Regulation by the A–20C Polymorphism

Matthew E. Dickson, Xin Tian, Xuebo Liu, Deborah R. Davis, Curt D. Sigmund

From the Genetics and Medical Scientist Training Programs (M.E.D.), Department of Internal Medicine (X.T., X.L., D.R.D., C.D.S.), Molecular Physiology and Biophysics (C.D.S.), Center on Functional Genomics of Hypertension (C.D.S.), Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City.

Correspondence to Curt D. Sigmund, PhD, Department of Internal Medicine, 3181B MERF, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242. E-mail curt-sigmund{at}uiowa.edu

Among naturally occurring polymorphisms in the 5' flanking region of the human angiotensinogen (AGT) gene, the –20 and –217 polymorphisms have the strongest effects on AGT regulation in AGT-expressing cells derived from liver, kidney, brain, and fat. These polymorphisms may affect allele-specific transcription factor binding, and the high-expressing alleles are both relatively common. We show herein that the –20C allele has higher transcriptional activity than –20A, and the –20A allele confers no additional transactivation potential beyond that of a mutated vector. Gel-shift assays show that upstream stimulatory factor (USF)1 and USF2 preferentially bind the –20C allele, whereas the –20A allele retains a low affinity USF binding site. Plasmid immunoprecipitation assays confirmed preferential association of USF1 with the –20C allele in transfected HepG2 cells. Chromatin immunoprecipitation confirmed that USF1 binds to the endogenous AGT –20C allele in CCF cells, the only cell line tested that carries the –20C allele, and to the human AGT promoter in liver and adipose tissue from transgenic mice. Transduction of AGT-expressing cells with short hairpin RNAs specifically targeting USF1 or USF2, resulted in cell- and allele-specific attenuation of AGT promoter activity. In vivo, knockdown of USF expression in the liver of transgenic mice expressing the –20C allele of AGT resulted in lower AGT expression, a decrease in circulating human AGT protein but no change in expression of GAPDH or hepatocyte nuclear factor-4. We conclude that USF1 functionally and differentially regulates AGT expression via the –20 polymorphism and that the differential expression exhibited by –20 can be accounted for by differential association with USF1.

Key Words: transcription • genetics • hypertension • chromatin