Gene Trapping Uncovers Sex-Specific Mechanisms for Upstream Stimulatory Factors 1 and 2 in Angiotensinogen Expression

A single-nucleotide polymorphism (C/A) located within an E-box at the −20 position of the human angiotensinogen (AGT) promoter may regulate transcriptional activation through differential recruitment of the transcription factors Upstream Stimulatory Factor (USF) 1 and 2. To study the contribution of USF1 on AGT gene expression, mice carrying a (−20C) human AGT transgene were bred with mice harboring a USF1 gene trap allele designed to knock down USF1 expression. USF1 mRNA was reduced relative to controls in liver (9±1%), perigenital adipose (16±3%), kidney (17±1%), and brain (34±2%) in double-transgenic mice. This decrease was confirmed by electrophoretic mobility shift assay. Chromatin immunoprecipitation (ChIP) analyses revealed a decrease in USF1, with retention of USF2 binding at the human AGT promoter in the liver of male mice. Human AGT expression was reduced in the liver and other tissues of female but not male mice. The decrease in endogenous AGT expression was insufficient to alter systolic blood pressure at baseline, but caused reduced systolic blood pressure in female USF1 gene trap mice fed a high fat diet. Treatment of USF1 knockdown males with intravenous adenoviral shRNA targeting USF2 resulted in reduced expression of USF1, USF2 and human AGT protein. Our data from ChIP assays suggests that this decrease in human AGT is due to decreased USF2 binding to the human AGT promoter. In conclusion, both USF1 and USF2 are essential for AGT transcriptional regulation, and distinct gender-specific and tissue-specific mechanisms are involved in the activities of these transcription factors in vivo.