Active CHD3.1 opposes SNF2H chromatin remodeling activity in heterochromatin, which requires RNF20 to localize to heterochromatic DSBs. (A) Schematic of the site-specific heterochromatin relaxation assay. (B) Representative images of cells containing a LacO array integrated within a region of heterochromatin and transfected with LacR-GFP, LacR-GFP-SNF2H (wild type or K211R), LacR-mCherry, or LacR-mCherry-CHD3.1 (wild type or K767Q), as indicated. Bars, 5 µm. (C) Cells from B were transfected with LacR-GFP-SNF2H wild type and either LacR-mCherry or LacR-mCherry-CHD3.1 (wild type or K767Q), as indicated. The nuclear volume of the LacR-GFP signal, as a percentage of the overall nuclear volume was plotted for each condition (from B and C), either as a mean of three independent experiments (∼75 cells for each condition). P-values (standard two-tailed Student’s t test) are indicated. Bars, 5 µm. Error bars show SD. (D) Schematic of the site-specific heterochromatin DSB recruitment assay. (E) U2OS 2-6-3 cells treated with either RNF20 (siRNF20) or luciferase (siLuc) siRNA and stably expressing ER-Fok1-mCherry-LacR-DD were induced with 300 nM 4-OHT and 1 µM Shield-I for 5 h. Subsequently, cells were preextracted using 0.25% Triton X-100 in CSK buffer for 10 min, fixed with formaldehyde, and immunostained with SNF2H (green) and γ-H2AX (greyscale). Boxes are enlarged in the right images. Arrows point to the site of DSB induction at the array. Bars, 5 µm. (F) The mean percentages of cells with SNF2H foci present at Fok1/γ-H2AX foci were quantified (130 cells for each condition from two independent experiments). siRNA efficiency was assessed by immunoblotting.