Fig. 4: KAP1Hbox enhances the phase separation and DNA binding abilities of HP1α.
a EMSA of 147 bp Widom 601 DNA (DNA147) in the presence of increasing amounts of HP1α with (top) and without (bottom) tenfold molar excess of KAP1Hbox. DNA:protein ratio is shown below the gel images. b EMSA of DNA147 in the presence of increasing amounts of HP1α with tenfold molar excess of indicated modified KAP1Hbox. c Immunofluorescence images show the cellular distribution of GFP-KAP1 and endogenous HP1α in KAP1−/− ESCs stably expressing GFP-KAP1RF or GFP-KAP1FR-PVLmut stained with an anti-HP1α antibody (red). DNA was counterstained using DAPI (blue). Scale bar: 10 µm. d Half-FRAP recovery curves for HP1α in GFP+ (green) or GFP-KAP1FR+ (purple) ESCs for 8–12 heterochromatin compartments. The bleach half recovery is indicated by thin lines, and a Savitzky-Golay fit was performed to show the recovery of the non-bleached half and calculate the dip (thick lines)36. The non-bleached data points represent average ±SEM between 8 and 12 measurements. n > 8 The gray area indicates the range of dip depths in the non-bleached half that correspond to LLPS36. e Representative confocal images of DNA-induced HP1α phase separated droplets in the presence of GFP-KAP1FR wild type (green) or GFP-KAP1FR-PVLmut (green) and 647 N labeled HP1α (red). DIC, Differential interference contrast. Scale bar: 5 µm. f The box plot displays the fluorescence of 647 N labeled HP1α within phase-separated droplets. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. n = 169 (wild type GFP-KAP1FR), 175 (GFP-KAP1FR-PVLmut). Statistical analysis was performed by a two-sided Student’s t test, p-value is indicated. Source data are provided as a Source Data file.
