Fig 2: TDG forms condensates in cells.A, representative confocal fluorescent microscopy images of HeLa cells transfected with TDG fused to GFP. Red box indicates a larger TDG condensate in the nucleolus. Scale bars represent 5 µm. B, diameter of individual chromatin droplets formed by TDG in live HeLa cells. Data are mean ± SD (nucleoplasm: n = 10 cells, nucleolus: n = 3 cells). C, confocal fluorescent microscopy images and FRAP curve of GFP-TDG in HeLa cells. Data are mean ± SD (nucleoplasm: n = 10 cells, nucleolus: n = 3 cells). Scale bars represent 5 µm. D, representative fluorescent microscopy images of endogenous TDG showing loss of TDG foci upon 1,6-HD treatment. TDG was detected by immunostaining-fixed cells with TDG-specific antibody. Scale bars represent 5 µm. E, quantification of foci number and intensity upon of TDG with either 2,5-HD or 1,6-HD. Foci number are mean ± SD (n = 3 cells) for pre- and post-10 min treatment. Foci intensity is mean ± SD for 464, 428, and 335 individual foci pre-, post-2,5-HD, and post-1,6-HD treatment, respectively. **p < 0.01; ***p < 0.001; and ****p < 0.0001. 1,6-HD, 1,6-hexanediol; 2,5-HD, 2,5-hexanediol; FRAP, fluorescence recovery after photobleaching; TDG, thymine DNA glycosylase.

Fig 3: Full-length TDG induces phase separation of TFF1-derived chromatin.A, representative wide-field fluorescent microscopy images of TFF1e-Cy3 chromatin (100 nM) in the presence of the TDG. Scale bars represent 5 µm. B, Circularity and diameter of individual chromatin droplets formed by TDG. Data are mean ± SD (n > 600 droplets). C, representative wide-field fluorescent microscopy images demonstrating that Cy5-labled anti-TDG antibody (a-TDG360–410) penetrates into preformed TFF1e-Cy3-TDG droplets generated by mixing 1 µM TDG with 25 nM chromatin. Scale bars represent 5 µm. D, normalized CV analysis of TFF1e-Cy3 chromatin (25 nM) condensates formed by TDG (5 µM) following the addition of 1,6-HD. Data are mean ± SD (n = 10 images). **p < 0.01; ****p < 0.0001. E, phase diagrams of TFF1e-Cy3 chromatin under varying conditions. Red circles indicate phase separation. The grayscale indicates CV calculated from representative images (n = 10; Fig. S8). 1,6-HD, 1,6-hexanediol; TDG, thymine DNA glycosylase.

Fig 4: TDG-mediated chromatin phase separation is regulated by its IDRs.A, TDG domains and truncated variants discussed in this work. B, phase diagrams of TFF1e-Cy5 chromatin and TDG truncations under varying conditions. Red circles indicate phase separation. The grayscale indicates CV as described for Figure 3E (Fig. S9). IDR, intrinsically disordered region; TDG, thymine DNA glycosylase.

Fig 5: TDG’s IDRs induce phase separation of chromatin.A, diagram of the structural domains of TDG and their predicted disorder probability (PrDOS) (68). B, representative confocal fluorescent microscopy images of 12-NCP-Cy5 chromatin (25 nM) in the presence of the indicated [IDR]. Droplets were formed in the presence of LLPS buffer (10 mM Hepes [pH 7.2], 100 mM KCl, and 1 mM MgCl2) containing 5% PEG. C and D, circularity and diameter of individual chromatin droplets formed by TDG’s IDRs. Data are mean ± SD (n > 600 droplets). **p < 0.01; ****p < 0.0001. E, time-lapse images of an IDRN-chromatin droplet fusion event. Droplets were formed by combining 25 nM 12-NCP-Cy5 with 5 µM IDRN. F and G, representative confocal fluorescent microscopy images demonstrating that 12-NCP-Cy3 chromatin (50 nM) penetrates into preformed IDRN-12-NCP-Cy5 (F) and IDRC-12-NCP-Cy5 (G) droplets generated by mixing 5 µM of the IDR with 12.5 nM chromatin. Scale bars for zoom insets in B represents 2 µm. All other scale bars represent 5 µm. IDR, intrinsically disordered region; LLPS, liquid–liquid phase separation; TDG, thymine DNA glycosylase.