Fig. 3
The CHD4/NuRD complex is a ZEB1 co-repressor. Chromatin immunoprecipitation (ChIP) for ZEB1, CHD4, H3K27ac, and control IgG was performed for known ZEB1 target genes (SEMA3F and miR-200c) and established CHD4/NuRD complex target (N-Myc) in the NSCLC cell line H1299. Primers corresponding to SEMA3F intron 13 (SEMA3F-neg) were included as a negative control for ZEB1 binding. When indicated, ChIP experiments were normalized to the non-specific IgG control. Graphs represent three experimental replicates; all asterisks indicate statistical significance by t-test (n ≥ 3, *p ≤ 0.05); error bars represent standard error mean. Transient knockdown of CHD4 by siRNA significantly reduced CHD4 binding at each of the queried promoters (a) but promoted ZEB1 binding (b). Despite increase ZEB1 binding, acetylation of H3K27 (c) increased at the miR-200c and SEMA3F promoter. d Relative CHD4 binding in the cell line H1299 after 24 h of treatment with the class I HDAC inhibitor, mocetinostat (1 μM). e Reduced CHD4 binding is observed upon treatment, corresponding with increased H3K27ac. f, g In the human NSCLC cell line H358, ZEB1 or GFP vector were overexpressed for 24 h prior to ChIP. ZEB1 and CHD4 concurrently bind to the promotor of miR-200 and SEMA3F, but not N-Myc. In addition, CHD4 binding to the promoter of miR-200c and SEMA3F occurs only upon ZEB1 overexpression. h Relative normalized luciferase activities from reporter constructs of either empty vector (pGL3), or a 321 base pair upstream fragment of the miR-200b-a-429 promoter29. Vectors were transfected in H358-GFP control or ZEB1-expressing cells, which were pre-transfected with siRNA control or siRNA targeting CHD4. Doxycycline induction was conducted prior to quantification of luciferase activity. Graphs represent normalization to control; all asterisks indicate statistical significance by t-test (n ≥ 3, *p ≤ 0.05); error bars represent standard error mean
