FIG. 3.

Transcriptional regulation of RTP801. (A) Northern blot analysis of RTP801 transcription in wild-type mouse ES cells (ES+/+) and in HIF-1α null mouse ES cells (ES−/−) cultured under normoxic (N) or hypoxic conditions (H) for 16 h. Total RNA (15 μg) was loaded in each slot. (B) Nucleotide sequences of immediate upstream genomic regions of mouse and human RTP801 orthologues. The initiation ATG codon is in boldface, and the position of T is counted as +1. The TATA box is shaded gray. White letters in black background, putative HRE; dashed line, putative Egr-1 binding site. (C) EMSA and supershift analysis of mouse RTP801 promoter region. All the binding reactions except for those whose mixtures are loaded in lanes 2, 4, and 5 were performed with nuclear extracts prepared from wild-type ES cells cultured under hypoxic conditions for 16 h. The reaction mixture loaded in lane 2 contains nuclear extract prepared from wild-type ES cells cultured in normoxia, whereas reaction mixtures loaded in lanes 4 and 5 contain nuclear extracts from HIF-1α^−/− ES cells maintained in normoxic and hypoxic conditions, respectively. Lane 1, ³²P-TR-HRE oligonucleotide; lanes 2 to 5, ³²P-RTP801-HRE oligonucleotide; lane 6, ³²P-RTP801-HRE oligonucleotide and the excess of nonlabeled RTP801-HRE oligonucleotide; lane 7, ³²P-RTP801-HRE oligonucleotide and the excess of nonlabeled TR-HRE oligonucleotide; lane 8, ³²P-RTP801-HRE oligonucleotide and anti-HIF-1α antibodies; lane 9, ³²P-RTP801-HRE oligonucleotide and anti-Flag antibodies; lane 10, ³²P-RTP801-MHRE oligonucleotide. For details see Results and Materials and Methods. (D) Northern blot analysis demonstrating the p53 independence of hypoxic transactivation of RTP801. H1299 is a human lung carcinoma p53-negative cell line that was engineered to express the wild-type p53 under the control of a tetracycline-repressible promoter. The cells were cultured either in the absence (left) or presence (right) of tetracycline to induce (left) or to suppress (right) p53 expression, respectively. Both p53-positive and p53-negative H1299 cells were maintained either under normal (N) or hypoxic (H) conditions or in the presence of doxorubicin (D). Total RNA (15 μg) derived from each experiment was analyzed by Northern blotting using the probes for human RTP801 and for Waf1 (as a positive control for p53-dependent transactivation).