Fig. 2. The histone demethylases, LSD1 and PLU-1, and the LSD1 partner CoREST, together mediate hypoxia-induced H3K4 demethylation at the MLH1 promoter.

SW480 cells with LSD1 knockdown, with PLU-1 knockdown, or with double knockdown of both LSD1 and PLU-1 were established using lentiviral shRNAs constructs targeting LSD1 or PLU-1. Control cells were transduced with a lentiviral expression construct for a GFP shRNA. (A) Western blot analyses to determine LSD1 and PLU-1 expression levels in the SW480-GFPsh, SW480-LSD1sh, SW480-PLU-1sh and double knockdown SW480-LSD1-PLU-1sh cells under normoxic and hypoxic conditions. (B) qChIP analyses of H3K4 methylation levels at the MLH1 promoter following 48 h exposure to normoxia or hypoxia in SW480-GFPsh cells; SW480-LSD1sh cells; SW480-PLU-1sh cells; and double knockdown SW480-LSD1-PLU-1sh cells (indicated as SW-L1P2). Promoter occupancy levels are expressed as the fold change relative to the normoxic control SW480 GFPsh cells. Standard errors are indicated. (C). Quantitative real-time PCR analysis of MLH1 mRNA levels in SW480 GFPsh, SW480 LSD1sh, SW480 PLU-1sh and SW480 LSD1-PLU-1sh cells after 48 h of normoxic or hypoxic exposure. mRNA levels are expressed as the fold change relative to normoxic control SW480 GFPsh cells. (D). Western blot analysis of MLH1 protein levels in the same cell lines as in (C), above, after 48 h of normoxic or hypoxic exposure (E). CoREST also plays a role in hypoxia-induced H3K4 demethylation at the MLH1 promoter in SW480 cells. Quantification of ChIP analyses of H3K4 methylation levels at the MLH1 promoter following 48 h exposure to normoxia or hypoxia in the SW480 GFPsh cells compared to the SW480 CoRESTsh cells. Promoter occupancy levels are expressed as the fold change relative to the normoxic SW480 GFPsh cells, based on three independent ChIP assays with error bars based on SEs.