Fig. 6: DPPA3 binds nuclear UHRF1 with high affinity prompting its release from chromatin in naïve ESCs.

a Overview of RICS and ccRICS. Confocal image series are acquired on a laser scanning confocal microscope, containing spatiotemporal fluorescence information on the microsecond and millisecond timescales. A spatial autocorrelation function (SACF) is calculated from the fluorescence image and fit to a diffusive model. The cross-correlation of intensity between two channels is used to estimate the co-occurrence of two fluorescent molecules in live cells. The mean cross-correlation of the fluctuations is calculated and shown in the 3D plot color-coded according to the correlation value. b–e Representative plots of the spatial cross-correlation function (SCCF) between the depicted fluorescent molecules in cells from each cell line measured: (b) wild-type (U1WT:D3^WT) and (c) K85E/R85E/R87E DPPA3 mutant (U1WT:D3^KRR), and control ESCs expressing (d) free eGFP, free mScarlet (eGFP + mScarlet) and (e) an eGFP-mScarlet tandem fusion (eGFP-mScarlet). f, g Mobile fraction of (f) mScarlet and (g) eGFP species in the cell lines depicted in (b, c, and e) and in Uhrf1KO ESCs expressing free eGFP and wild-type DPPA3-mScarlet (U1KO:D3^WT). The mobile fraction was derived from a two-component model fit of the autocorrelation function. Data are pooled from three (U1WT:D3^WT, U1WT:D3^KRR) or two (U1KO:D3^WT, eGFP-mScar) independent experiments. h Mean cross-correlation values of mobile eGFP and mScarlet measured in the cell lines depicted in (b–e). The spatial lag in the x-dimension (sensitive to fast fluctuations) is indicated by ξ, and the spatial lag in the y-dimension (sensitive to slower fluctuations) is indicated by ψ. Data are pooled from two independent experiments. i Microscale thermophoresis measurements of UHRF1-eGFP binding to GST-DPPA3 WT (D3^WT) or GST-DPPA3 1–60 (D3^1–60). Error bars indicate the mean ± SEM of n = 2 technical replicates from n = 4 independent experiments. In (f–h), each data point represents the measured and fit values from a single cell where n = number of cells measured (indicated in the plots). In the boxplots, darker horizontal lines within boxes represent median values. The limits of the boxes indicate the upper and lower quartiles; the whiskers extend to the most extreme value within 1.5 x the interquartile range from each hinge. Source data are provided as a Source Data file.