Fig. 3.

Hypoxia increases H3K9 methylation and decreases H3K9 acetylation at the BRCA1 and RAD51 promoters. ChIP assays were performed on MCF-7 or RKO cells following exposure to normoxia (N) or hypoxia (H; 0.01% O₂) for 48 h using antibodies to the indicated H3K9 modifications at the BRCA1, RAD51, and VEGF promoters. (A) ChIP analysis of H3K9 methylation levels at the BRCA1 (left), RAD51 (middle), and VEGF (right) promoters as quantified by real-time PCR in MCF-7 cells. Specific antibodies that recognize either the dimethylated (H3K9me2) or trimethylated (H3K9me3) form of H3K9 were used. Relative promoter occupancies (% input) are shown with error bars based on standard errors calculated from at least three replicates. (B) ChIP analysis of H3K9 methylation levels at the BRCA1 (left), RAD51 (middle), and VEGF (right) promoters as quantified by real-time PCR in RKO cells. Specific antibodies that recognize H3K9me3 were used. Relative promoter occupancies (% input) by trimethylated H3K9 are shown with error bars based on standard errors calculated from at least three replicates. (C) Time course of hypoxia-induced H3K9 methylation at the BRCA1 (left) or RAD51 (right) promoter regions by ChIP analysis. MCF-7 cells were exposed to normoxia (N) or hypoxia (H) for the indicated times, and H3K9 methylation levels at the promoters were analyzed by quantification of real-time PCR of ChIP samples corresponding to the BRCA1 or RAD51 promoters. Promoter occupancy levels were expressed as the fold change relative to promoter occupancy levels for normoxia, based on three independent ChIP assays with error bars based on SEs. Significant differences were identified as P < 0.05 (indicated by *) or P < 0.01 (indicated by **) compared to normoxia control levels. (D) ChIP analysis of H3K9 acetylation levels at the BRCA1 (left), RAD51 (middle), and VEGF (right) promoters as quantified by real-time PCR in MCF-7 cells. Relative promoter occupancies (% input) by acetylated H3K9 are shown with error bars based on SEs calculated from at least three replicates.