Fig. 6: Lysine residue K852 in the CRD domain of Tet1 is crucial for Tet1s S-phase localization and targeted catalytic activity.

A Localization of the conserved lysine residue K852 in Tet1s and sequence alignment between human and mouse Tet1. B Model of mouse Tet1s generated by homology modeling on the refined human Tet2 crystal structure (4NM6). The CRD is shown in red (conserved lysine 852 in pink), and the DSBH in yellow. The bound DNA helix appears in blue with 5mC flipped out of the double helix in green. C Representative images of C2C12 cells expressing mRFP-PCNA and EGFP-Tet1s/Tet1s-K852R/Tet1s-K852E. Boxplots showing heterochromatin accumulation of Tet1-X. D C2C12 cells from C were immunostained against 5hmC 24 h after transfection. Nuclear 5hmC levels were measured by high-content microscopy. Boxplot shows sum nuclear 5hmC levels normalized to the averaged 5hmC levels of background cells and against sum nuclear DAPI intensity. Cells were grouped according to their mean EGFP fluorescence intensities (see Supplementary Fig. 2B, C). E, F Fiji-based in situ 5hmC analysis: Binary nucleoplasm and heterochromatin masks were generated and mean fluorescence intensities in the respective areas were measured. Boxplots in F show 5hmC levels in pericentric heterochromatin (dark-gray) and the surrounding nucleoplasm (light-gray). Representative images and constitutive heterochromatin relative areas are shown in Supplementary Fig. 5E, F. G Chromatin immunoprecipitation experiments followed by qPCR for MajSat sequences. C2C12 cells were transfected with Tet1s-K852R/Tet1s-K852E and Tet1-CD (positive control) and synchronized in G1/late S phase. Barplots show the average value of amplification levels in input and chromatin binding fractions normalized with GFP-input (red line). The error bars represent the standard deviation with a 95% confidence interval. H FRAP experiments in transfected C2C12 cells (EGFP-tagged Tet1s/Tet1s-K852R/Tet1s-K852E). The EGFP signal was photobleached with a 488 nm laser. I For analysis, 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. 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 in Supplementary Data 1. Source data are provided as a Source Data file. Scale bar = 5 µm.