Regulation of renin enhancer activity by nuclear factor I and Sp1/Sp3.
Transcription of the mouse Ren-1(c) gene in kidney tumor-derived As4.1 cells, which express high levels of renin mRNA, is dependent on a proximal promoter element and a 242-bp enhancer region located 2.6 kb upstream of the transcription start site. We showed previously that the enhancer contains a cAMP responsive element (CRE) and an E-box. Mutation of either element resulted in almost complete loss of the Ren-1(c) expression. In this report we show that there are additional transcription factor-binding sites within the Ren-1(c) enhancer contributing to the enhancer activity. Electrophoretic mobility shift and supershift assays have identified four nuclear factor I (NFI)-binding sites, an Sp1/Sp3 site and an unidentified transcription factor-binding site (Ei) located upstream of the CRE and E-box. Mutation of the Sp1/Sp3 site or Ei reduced Ren-1(c) expression by 40% or 30%, respectively, while mutations of four NFI-binding sites resulted in an 89% decrease in expression. Thus, these protein-DNA interaction sites are essential for transcription of mouse renin genes. There are four homologous NFI genes (NFI-A, -B, -C and -X) in vertebrates and multiple alternatively spliced isoforms from each gene. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assays have demonstrated that NFI-X is the predominant NFI mRNA expressed in As4.1 cells. Direct study of involvement of NFI-X in regulation of renin genes is underway.
Pan
Li
L
Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263-0001, USA.
Glenn
Sean T
ST
Jones
Craig A
CA
Gronostajski
Richard M
RM
Gross
Kenneth W
KW
D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins / Q000502:physiology*; D018797:Cell Cycle Proteins; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins; D024202:Electrophoretic Mobility Shift Assay; D004742:Enhancer Elements, Genetic / Q000502:physiology*; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D051839:NFI Transcription Factors; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics; D009687:Nuclear Proteins; D012333:RNA, Messenger / Q000032:analysis; D012083:Renin / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000502:physiology*; D014157:Transcription Factors; D014162:Transfection; D014166:Transferases; D014407:Tumor Cells, Cultured; D051840:Y-Box-Binding Protein 1
D022762:CCAAT-Enhancer-Binding Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D051839:NFI Transcription Factors; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; C466095:Kctd11 protein, mouse; D014166:Transferases; D012083:Renin