Fig. 7: OVOL2 competes with RCOR1 for binding to LSD1 and inhibits LSD1 demethylase activity in vitro.

A OVOL2 competes with RCOR1-HA for binding to LSD1-Flag. Increasing concentrations of purified OVOL2 were incubated overnight with pre-formed LSD1-Flag-RCOR1-HA complexes. Protein mixtures were immunoprecipitated using anti-FLAG M2 agarose and supernatants were immunoblotted with antibodies against HA (RCOR1). Immunoprecipitates were immunoblotted with OVOL2 antibody or FLAG antibody. B Size exclusion chromatography of pre-formed 7 μM LSD1-RCOR1 complex mixed with 7 μM OVOL2. (Inset) Coomassie blue staining of the peak fractions from 10 to 19 mL elution volume. C LSD1 demethylation activity assay. Demethylation activity of purified LSD1 (300 nM) towards 60 μM diMeH3_1-21K4 substrate was tested in the presence of OVOL2^WT and RCOR1 added at the indicated concentration ratios [RCOR1: OVOL2^WT = 6:1, 3:1, 1(6 μM):1(6 μM), 1:2, 1:3, 1:6, 1:9] (n = 3 wells per condition). D EMSA analysis of the effect of the C120Y mutation on OVOL2-DNA binding. 53 ng of purified OVOL2^WT or OVOL2^C120Y was incubated with OVOL2 probe (AACCGTTACC) (1 nM) with or without specific competitor (4.5 μM) for 30 min before being harvested for EMSA analysis. E EMSA analysis of the effect of RCOR1 on LSD1-OVOL2-DNA complex formation. 2 μM OVOL2^WT and 1 or 2 μM LSD1 were incubated overnight to form a complex. Pre-formed LSD1-OVOL2 complexes were incubated with 2 μM (1:1:1 OVOL2:LSD1:RCOR1) or 4 μM (1:1:2) RCOR1 and OVOL2 probe (1 nM) for 1 h before being harvested for EMSA analysis. F LSD1 demethylation activity assay. Demethylation activity of purified LSD1 (300 nM) towards 60 μM diMeH3_1-21K4 substrate was tested in the presence or absence of OVOL2^WT (1.5 μM) and increasing concentrations of the DNA motif bound by OVOL2 (n = 3 wells per condition). Data are representative of two (A, B, E, F) or four independent experiments (C, D). Data points represent individual wells and means ± SD are indicated (C, F). P-values were determined by two-tailed Student’s t-test (C, F).