Molecular cloning and functional analysis of a factor that binds to the proximal promoter of human angiotensinogen.
A significant association has been reported between a common variant in the angiotensinogen gene (AGT), allele T235, and essential hypertension. In subsequent work, it was found that another variant, the presence of an adenine instead of a guanine 6bp upstream from the initiation site of transcription, was in absolute linkage disequilibrium with T235. The nucleotide substitution at the -6 position affected the formation of DNA-protein complexes in gel mobility shift assays and the basal transcription of AGT in transactivation experiments. We have further examined the potential impact of this polymorphism on AGT promoter function. In ultraviolet cross-linking analysis. 150- and 75-kDa proteins bound to the AGT proximal promoter. The possible involvement of factors that bind to GC-rich domains, including Sp1, Sp3, and AP2, was not supported by gel mobility shift assays. Screening an expression library with a double-stranded DNA segment centered on -6 led to the isolation of cDNA clones encoding the YB1 protein. The specificity of the interaction of YB1 with the proximal promoter of AGT was verified by Southwestern blotting and gel mobility shift assays. In cotransfection experiments, YB1 reduced basal AGT promoter activity in a dose-dependent manner. Although these observations suggest a possible role for YB1 in modulating AGT expression, this function is likely to occur in the context of complex interactions involving other nuclear factors. The work illustrates the challenge of developing a molecular understanding of the relationship between common genetic variants and conditions that are only partly caused by them.
Nakajima
Toshiaki
T
Department of Human Genetics, University of Utah, Salt Lake City 84112-5331, USA.
Inoue
Ituro
I
Cheng
Tong
T
Lalouel
Jean-Marc
JM
D000808:Angiotensinogen / Q000235:genetics* / Q000378:metabolism; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins; D024202:Electrophoretic Mobility Shift Assay; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020080:Tandem Repeat Sequences; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014466:Ultraviolet Rays; D051840:Y-Box-Binding Protein 1
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D000808:Angiotensinogen; D008156:Luciferases; D005982:Glutathione Transferase