Figure 2

EZH2 direct binding to the KLF2 promoter mediates the transcriptional repression effect. (A) ChIP--qPCR of EZH2 occupancy and H3K27-3me marks in the KLF2 promoter in five cancer cell lines treated with EZH2 siRNA (72 h) or scrambled siRNA. Treatment with siRNA against EZH2 prevents EZH2 occupancy and the presence of the H3K27-3me mark, while enhanced RNAP binding is observed in the KLF2 promoter. ChIP was performed using polyclonal antibodies raised in rabbit against EZH2 (pAb-039-050, Diagenode, Liège, Belgium), RNAP-S2 (ab5095, Abcam, Cambridge, UK) and H3K27me3 (pAB-069-05, Diagenode), with rabbit IgG as a control (ab37415, Abcam, ChIP grade). The primers used for the ChIP--qPCR analysis of the KLF2 promoter were 5′-GAGACTCCAGACTTCCCATCC-3′ (sense) and 5′-CAGAGACTCTCAGGGGAGCAC-3′ (antisense). (B) qChIP for EZH2 occupancy and H3K27-3me presence for the KLF2 promoter in stable EZH2 knockdown clones (U2OS-shEZH2). (C) KLF2 promoter activities are analyzed by luciferase reporter assay in stable EZH2 knockdown clones. In each experiment, firefly luciferase activities are normalized against those of Renilla. n=3, mean±s.e.m. (error bars). We used a pGL3 Luciferase Reporter Vector (Promega, Madison, WI, USA) for the KLF2 promoter encompassing NheI/Hind III sites (from −916 to +129 bp). (D) Upregulation of KLF2 transcript (a) and protein (b) upon treatment with 5 μM DZNep for 72 h. (c) qChIP analysis shows how the treatment with DZNep decreased EZH2 occupancy and the H3K27-3me mark in the KLF2 promoter, while it enhanced RNAP-S2 occupancy. P-values obtained from Student’s t-test.