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. 2007 Jun 18;27(17):6229–6242. doi: 10.1128/MCB.02246-06

FIG. 3.

FIG. 3.

The BNIP3 promoter contains a functional E2F binding site that interacts functionally with the HRE. (A) Transcriptional initiation sites in the mouse BNIP3 promoter were determined by RNase protection assays using RNA from untreated (lane 1) or DFO-treated (lane 2) MEFs and wild-type (lane 3) FL (Wt), Rb null (lane 4) FL, or control yeast (lane 5) RNA. A diagrammatic representation of the two major initiation sites in the mouse BNIP3 promoter is shown. (B) Sequence alignment of the proximal regions of the mouse and human BNIP3 promoters highlighting conserved regulatory elements that include an HRE (blue) at −94 bp relative to the start site of transcription and an E2F site (pink) at −142 bp (mouse) and −155 bp (human). (C) The proximal 500 bp of the mouse BNIP3 promoter was cloned upstream of the luciferase reporter gene and tested for its activity in U2OS cells in response to overexpression of E2F-1, and its activity then was compared to that of the control reporter construct pGL2 lacking a promoter and that of the characterized E2F-responsive mouse dhfr promoter. (D) The binding activity of the BNIP3 promoter E2F site was tested by EMSA using nuclear lysates from untreated (lanes 1 to 7) or DFO-treated (lanes 8 to 14) U2OS cells in the presence or absence of excess cold oligonucleotide competitor encoding either the consensus E2F recognition sequence (lanes 2 and 9) or the BNip3 E2F site (lanes 3 and 10). The effect of supershifting antibodies to E2F-1 (α-E2F1) (lanes 4 and 11), E2F-3 (α-E2F3) (lanes 5 and 12), pRB (α-pRB) (lanes 6 and 13), or E2F-4 (α-E2F4) (lanes 7 and 14) on DNA-protein complex migration also was examined. (E) The identity of E2Fs bound to the endogenous BNIP3 promoter in untreated (top panel) or DFO-treated (bottom panel) U2OS cells was examined by ChIPs using antibodies to E2F-1 (α-F1), E2F-3 (α-F3), E2F-4 (α-F4), pRB (α-pRB), or HIF-1α (α-HIF-1α) and compared to results with no-antibody negative control samples (No Ab) or to those with input positive control samples (Input). (F) Diagrammatic representation of mutations introduced into the mouse BNIP3 promoter to test the functional interaction between E2F and HIF in regulating BNip3 transcription. (G) Reporter (luciferase) gene assay for BNIP3 promoter activity in U2OS cells following DFO treatment, E2F-1 overexpression, or mutation of either the E2F site, the HRE, or both sites in the context of the proximal 500 bp of the mouse BNIP3 promoter. UT, untreated. (H) Schematic representation of the proposed interaction between pRB, E2F family members, and HIF at the BNIP3 promoter.