Extended Data Fig. 2: SHARP forms multivalent, concentration-dependent assemblies in the nucleus. | Nature Structural & Molecular Biology

Extended Data Fig. 2: SHARP forms multivalent, concentration-dependent assemblies in the nucleus.

From: Xist spatially amplifies SHARP/SPEN recruitment to balance chromosome-wide silencing and specificity to the X chromosome

Extended Data Fig. 2

a) Schematic of the domains included in the eGFP-tagged FL-SHARP and ΔIDR-SHARP, and the mCherry-tagged FUS-ΔIDR-SHARP rescue constructs used in Fig. 2 and Extended Data Fig. 2. b) FRAP recovery curve of eGFP-tagged FL-SHARP (red), positive control PTBP1 (forms assemblies; light blue), and negative control EED (does not form assemblies; dark blue). Points represent mean value; error bars represent standard deviation of at least five replicates. c) Schematic depicting physical characteristics of concentration-dependent assemblies, including foci formation, fission and fusion, and rapid diffusion of proteins within an assembly (inset). d) Images across nine time-points from a live-cell movie of eGFP-tagged FL-SHARP in transiently transfected HEK293T cells (Supplementary Video 1,2) showing non-diffused, focal organization of SHARP molecules. Top panel: 3D reconstructions of the fluorescent intensity signal; middle panel: 3D volume reconstructions color-coded based on the volume of the focus; bottom panel: zoom-in representing one region of the nucleus that changes volume; Fluorescent Intensity (FI) e) Comparison of diffused or non-diffused localization patterns of FL-SHARP at different dox concentrations. Left: images representing FL-SHARP expressed with either 0.1x dox (diffused) or 1x dox (non-diffused) in transiently transfected HEK293T cells; images shown as max projections; scale bars 10 μm. Right: histograms representing fluorescent intensities for two cells showing diffused and non-diffused localization patterns. The intensity at the 99th percentile of each distribution is shown with the dashed lines. f) Images representing nuclear localization pattern of eGFP-tagged proteins in transiently transfected HEK293T cells. Left: proteins that have not been reported to form assemblies (HALO and EED); Right: an eGFP tagged protein that has been reported to form assemblies (Ptbp1) and ΔSPOC-SHARP that also forms assemblies. Images shown as max projections; scale bars 10 μm.

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