Upstream stimulatory factor is required for human angiotensinogen expression and differential regulation by the A-20C polymorphism.
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-4alpha. 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.
Dickson
Matthew E
ME
Genetics and Medical Scientist Training Programs, Roy J and Lucille A Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
Tian
Xin
X
Liu
Xuebo
X
Davis
Deborah R
DR
Sigmund
Curt D
CD
D041721:3T3-L1 Cells; D024506:5' Flanking Region; D000273:Adipose Tissue / Q000378:metabolism; D000808:Angiotensinogen / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D008099:Liver / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D011110:Polymorphism, Genetic; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D051720:Upstream Stimulatory Factors / Q000235:genetics / Q000378:metabolism*
D034741:RNA, Small Interfering; C497181:USF1 protein, human; C497184:USF2 protein, human; D051720:Upstream Stimulatory Factors; C497183:Usf1 protein, mouse; C497186:Usf2 protein, mouse; D000808:Angiotensinogen
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