Extended Data Fig. 4: DNMT3A1 N-terminal domain is an intrinsically disordered region.
a, Prediction of intrinsic disorder for DNMT3A1 by PONDR (Predictor of Natural Disordered Regions) online. Amino acid positions are shown on the x axis. The cyan bars designate the N-terminal regions investigated. b, Fluorescence images of living Hela cells expressing Cry2-mCherry or N-terminal IDR fusion proteins (optoN219 and optoN278). All cells were subjected to 488 nm blue light stimulation under identical conditions. The experiment was repeated three times independently with similar results. Representative images after 80-second stimulation are presented. Scale bar = 10 μm. c, Purification of 6×His tagged GFP and N-terminal IDR-GFP fusion proteins for in vitro droplet formation assay. d, Visualization of turbidity of indicated protein solutions (20 µM) in droplet formation buffer in the absence (–) or presence (+) of 8% PEG-8000. e, Representative images of GFP-positive spherical protein droplets formed at concentrations of 5 µM and 20 µM. Proteins were diluted to the final concentrations with droplet formation buffer in the presence of 8% PEG-8000. The experiment was repeated four times with similar results. Scale bar = 20 μm. f, Fusion events between proximal droplets of N219-GFP (top) or N278-GFP (bottom). g, Live-cell images of FRAP analysis on GFP-DNMT3A1 expressed in NIH 3T3 cells. Scale bar = 10 μm. h, Average fluorescence recovery trace in GFP-DNMT3A1 FRAP experiments (n = 12 cells). All data are presented as mean ± s.d. i, Live-cell images of FRAP analysis on GFP-MeCP2. Scale bar = 10 μm. j, Average fluorescence recovery trace in GFP-MeCP2 FRAP experiments (n = 6 cells).
