Fig. 4: The zinc finger domain of Tet1 impedes S-phase dependent heterochromatin association and prevents aberrant 5mC oxidation.

A Domain organization of Tet1 with locations of amino acids corresponding to N-terminal deletion mutants. Representative images of C2C12 cells in late S-phase expressing EGFP-tagged Tet1/Tet1∆1-389/Tet1∆566−833/Tet1-ZF domain and mRFP-PCNA. Boxplot shows quantification of relative accumulation. B C2C12 cells were transfected with mcherry-Tet1CD/Tet1-CD-ZF and EGFP-PCNA. Representative images of cells in late S-phase are shown. In situ 5hmC levels were analyzed 24 h after transfection by wide-field high-content microscopy. Boxplots show sum nuclear 5hmC levels normalized by averaged levels in non-transfected cells and sum nuclear DAPI. Cells were grouped by their mean mcherry fluorescence intensities into low, mid, and high expressing (see Supplementary Fig. 2B, C). C Representative images of C2C12 from 4B. Selected ROIs in pericentric heterochromatic regions were magnified. Fiji-based in situ 5hmC quantification was performed and results are shown in boxplot (5hmC levels in pericentric heterochromatin in dark-gray). D FRAP experiments scheme in cells expressing mcherry-tagged Tet1-CD/Tet1-CD-ZF. The mcherry signal was photobleached with a 561 nm laser and recovery of fluorescence was followed by time lapse microscopy. E Fluorescence recovery curves and T-half times were calculated using easyFRAP. Line plots show normalized averaged fluorescence recovery values, and error bands show the respective standard deviation. 95% confidence intervals are indicated in the plot. Representative images are shown in Supplementary Fig. 3C. For all boxplots, the box represents 50% of the data, starting in the first quartile (25%) and ending in the third (75%). The line inside represents the median. The whiskers represent the upper and lower quartile. Statistical significance was tested with a paired two-samples Wilcoxon test (n.s. not significant, is given for p-values ≥ 0.05; one star (*) for p-values < 0.05 and ≥ 0.005; two stars (**) is given for values < 0.005 and ≥ 0.0005; three stars (***) is given for values < 0.0005). N-numbers and p-values are shown on Supplementary Data 1. Source data are provided as a Source Data file. Scale bar = 5 μm. White scale bar = 2.5 μm.