2.9. Promoter Analysis and Chromatin Immunoprecipitation (ChIP) Assays

The mouse (Ckb) and human (CKB) proximal promoters (−2000 to +500 bp) were scanned for putative functional hypoxic response elements (HREs) using the Transcription Factor Matrix (TFM) Explorer algorithm and weight matrices available from JASPER and TRANSFAC. PyMT HIF-1 WT and KO cells and MCF7 empty vector (pLKO.1-puro or EV) or shHIF1A transduced cells described in [34] were cultured at normoxia or 0.5% O₂ (hypoxia) for 6–24 h and fixed with 1% formaldehyde for 12 min. Positive controls included a previously validated functional HRE in the Vegf promoter for PyMT cells [35] and a previously validated functional HRE identified in the EPO promoter [36] for MCF-7 cells. Primers were designed to putative independent HRE sites, as well as to non-HRE sequences in the promoter regions (Supplementary Table S6). DNA was sheared to 500 bp and ChIP was performed using antibodies against HIF-1α or anti-rabbit IgG control, and raw data were analyzed as in [37].