Fig. 5: DPPA3-mediated demethylation is achieved via inhibition of UHRF1 chromatin binding and attenuated by nuclear export.

a Schematic illustration of murine DPPA3 with the nuclear localization signals (NLS), nuclear export signal (NES), and predicted domains (SAP-like and splicing factor-like²¹⁰) annotated. For the DPPA3 mutant forms used in this study, point mutations are indicated with arrows (∆NES, KRR, R107E) and the two truncations are denoted by the middle break (1–60, left half; 61–150, right half). b, c Nuclear export and the N-terminus of DPPA3 are dispensable for disrupting focal UHRF1 patterning and chromatin binding in ESCs. b Representative confocal images illustrating the localization of endogenous UHRF1-GFP and the indicated mDPPA3-mScarlet fusions in live U1G/D3KO + pSB-D3-mSC ESCs after doxycycline induction. DNA counterstain: SiR-Hoechst. Scale bar: 5 μm. c FRAP analysis of endogenous UHRF1-GFP in U1G/D3KO ESCs expressing the indicated mutant forms of DPPA3. FRAP Curves (solid lines) indicate double-exponential functions fitted to the FRAP data acquired from n cells (shown in the plots). For single-cell FRAP data and additional quantification, see Supplementary Fig. 6d–k. d DPPA3-mediated inhibition of UHRF1 chromatin binding is necessary and sufficient to promote DNA demethylation. Percentage of DNA methylation change at LINE-1 elements (%) in D3KO ESCs after induction of the indicated mutant forms of Dppa3 as measured by bisulfite sequencing of n = 4 biological replicates. In the boxplot in (d), horizontal lines within boxes represent median values, boxes indicate the upper and lower quartiles, whiskers extend to the most extreme value within 1.5 x the interquartile range from each hinge, and dots indicate outliers. Source data are provided as a Source Data file.