Skip to main content
. 2011 Aug;31(16):3339–3350. doi: 10.1128/MCB.01121-10

Fig. 2.

Fig. 2.

The histone demethylase LSD1 mediates hypoxia-induced H3K4 demethylation at the BRCA1 and RAD51 promoters. MCF-7 cells were transduced with lentiviral expression constructs for a GFP shRNA (control) or for one of three different shRNAs targeting LSD1, and stable shRNA-expressing cell lines were established: MCF-7 shGFP and MCF-7 shLSD1-1, MCF-7 shLSD1-2, and MCF-7 shLSD1-3. (A) Western blot analyses to determine LSD1 expression levels in MCF-7 shGFP, MCF-7 shLSD1-1, shLSD1-2, and shLSD1-3 cells. Substantially reduced expression of LSD1 was detected in all three stable LSD1 shRNA-expressing MCF-7 cell lines. (B, C, and D) ChIP analyses of H3K4 methylation and H3K9 acetylation levels at the BRCA1 (B), RAD51 (C), and VEGF (D) promoters following 48-h exposure to normoxia or hypoxia in MCF-7 shGFP cells compared to those for MCF-7 shLSD1-1 cells. Representative agarose gels showing PCR amplification products corresponding to the BRCA1 (B), RAD51 (C), or VEGF (D) promoter regions are shown. (E) Quantification of H3K4 methylation levels by real-time PCR at the BRCA1 (left), RAD51 (middle), and VEGF (right) promoters following 48-h exposure to normoxia or hypoxia in MCF-7 shGFP and MCF-7 shLSD1-1 cells. Promoter occupancy levels are expressed as the fold change relative to the promoter occupancy levels of the normoxic MCF-7 shGFP cells, based on three independent ChIP assays, with error bars based on SEs. (F) Quantification of H3K9 acetylation levels by real-time PCR at the BRCA1 (left), RAD51 (middle), and VEGF (right) promoters in MCF-7 shGFP and MCF-7 shLSD1-1 cells under the same normoxic or hypoxic treatment. Promoter occupancy levels are expressed as the fold change relative to the acetylation levels of the normoxic MCF-7 shGFP cells, based on three independent ChIP assays, with error bars based on SEs. (G) Quantitative real-time PCR analysis of endogenous BRCA1 (top) and RAD51 (bottom) mRNA expression levels in normoxic and hypoxic MCF-7 shGFP cells and in LSD1 knockdown MCF-7 shLSD1-1 cells, normalized to 18S rRNA expression. mRNA levels are expressed as the fold change relative to those of the corresponding normoxic control cells.

HHS Vulnerability Disclosure