Ets factors regulate the polycystic kidney disease-1 promoter.
The Ets family of transcription factors consists of a group of highly conserved sequence-specific DNA binding proteins that functionally cooperate with other transcription factors to regulate a number of diverse cellular processes including proliferation, differentiation, and apoptosis. We have analyzed a 3.3kb 5'-upstream region of the human PKD1 promoter, using transient transfection in HEK293T cells and Drosophila SL2 cells, to demonstrate that the PKD1 promoter is a target of Ets family transcription factors. Our studies showed that PKD1 promoter-luciferase reporter gene expression is downregulated by cotransfected Fli-1 and is upregulated by cotransfected Ets-1. Using deletion constructs, we demonstrated that the sequences responding to Fli-1 and Ets-1 lie within the -200 to +33bp proximal promoter. This region was found to contain two putative Ets response elements (EREs): an upstream (Ets-A) sequence 5'-CGGAA-3' (-181 to -185) and a downstream (Ets-B) sequence 5'-CGGAT-3' (-129 to -133). Site-directed mutagenesis indicated that both EREs are functional. A Fli-1 DNA binding domain mutant construct (W321R), which is incapable of binding DNA, was unable to inhibit basal promoter activity. In contrast, a Fli-1 DNA binding domain truncation mutant construct, which only contains the DNA binding domain and lacks the transactivation domain, was able to inhibit. These results suggest that the effect of Fli-1 is through direct binding to these EREs. Direct binding of Fli-1 and Ets-1 to the Ets-A and Ets-B sites was supported by electrophoretic mobility shift assays. Lastly, competition between Fli-1 and Ets-1 for the two EREs was demonstrated by showing that increasing amounts of Ets-1 could overcome Fli-1 repression of promoter activity. Taken together, these experiments define the proximal PKD1 promoter region as a potential target of Ets family transcription factors.
Puri
Sanjeev
S
Department of Biochemistry and Molecular Biology, and The Kidney Institute, University of Kansas Medical Center, Kansas City, KS 66160, USA.
Rodova
Marianna
M
Islam
M Rafiq
MR
Magenheimer
Brenda S
BS
Maser
Robin L
RL
Calvet
James P
JP
D000818:Animals; D002478:Cells, Cultured; D004330:Drosophila; D018260:Gelsolin / Q000235:genetics*; D005786:Gene Expression Regulation / Q000235:genetics; D008840:Microfilament Proteins; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011506:Proteins / Q000235:genetics*; D050783:Proto-Oncogene Proteins c-ets / Q000235:genetics*; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics*; D050396:TRPP Cation Channels; D015534:Trans-Activators; D015533:Transcriptional Activation / Q000235:genetics*
D018260:Gelsolin; D008840:Microfilament Proteins; D011506:Proteins; D050783:Proto-Oncogene Proteins c-ets; D018160:Receptors, Cytoplasmic and Nuclear; D050396:TRPP Cation Channels; D015534:Trans-Activators; C098179:FLII protein, human