Extended Data Figure 5. Effect of SET8 and MOF depletion on TONSL chromatin binding.

a, Immunoprecipitation of GFP-TONSL from solubilized chromatin of GFP-TONSL U-2-OS cells (one representative experiment out of two is shown). Same exposures are shown for input and IP western blots of H3 and H4K16ac. b, TONSL ARD preference for H4K16ac could be mediated by I599 through hydrophobic association with the K16 acetyl group as I599E ARD mutation preferentially reduces binding to H4K16ac peptides as compared to the unmodified H4 tail. (Left panel) Pull-down of GFP-TONSL from cell extracts with biotinylated H4 tail peptides. (Right panel) Quantification of the western blot, GFP-TONSL binding to the H4K16ac peptide is shown relative to the unmodified peptide. Mean with individual data points are shown (n=2). c, High-content quantitative imaging of TONSL in pre-extracted U-2-OS cells. Plots show total chromatin-bound TONSL and DAPI intensities in cells treated with control or TONSL siRNA, confirming the specificity of TONSL antibody staining. Each dot represents one nucleus. d-f, Analysis of TONSL chromatin-binding in MOF-depleted (d), SET8-depleted (e) and IR-treated cells (f). Chromatin-bound TONSL was quantified by high content imaging of pre-extracted U-2-OS cells stained for endogenous TONSL. Mean TONSL intensity is shown. A.U., arbitrary units. (d, e) knock-down efficiency and expected effect on histone modification were confirmed by western blotting (representative of 2 experiments). (e, f) G1 cells were defined by gating on DAPI and EdU intensity. (f) Cells were irradiated (1.5 Gy) and analysed 1.5 hours later (representative of 2 experiments). (d, f) Error bars, SD; d from left, n=4920, 2341, 3608, 2917; f, n= 382 (-IR), 523(+IR).