Fig. 4. TERRA drives LSD1 phase separation.

a Representative images of U2OS cells expressing mCh-eDHFR-LSD1 and Halo-GFP-TRF1 after adding TFH to induce dimerization at indicated time points. Montage on the right shows the fusion event of LSD1 foci over time, every 5 min between images. b Mean telomere number per cell after dimerizing mCh-eDHFR-LSD1 (WT and 3KE mutant) to Halo-GFP-TRF1 stably expressed in U2OS cells (n = 20 cells for LSD1^ΔIDR, n = 18 cells for LSD1^{ΔIDR, 3KE} over three independent experiments, two-tailed unpaired t test). c Representative DIC images of condensates formed by a mixture of purified LSD1 at 25 μM and different UUAGGG repeats as mimics of TERRA at 2.5 μM (n = three independent experiments). d-f Phase diagram of full-length LSD1 with the addition of 8× (d), 12× (e), 20× (f), TERRA. g Representative DIC images and h phase diagram of LSD1^ΔIDR with 8× TERRA. i Representative EMSA showing (UUAGGG)₈U RNA binding of truncated LSD1 CoREST (top) and full-length LSD1-CoREST (bottom) (n= two independent experiments). j Fusion of condensates containing 25 μM LSD1 and 25 μM 8× TERRA. k Fluorescent images of condensates containing Cy3-labeled 10 μM 12× TERRA and FITC-labeled 50 μM LSD1 (n = three independent experiments). l FRAP of condensates containing 10 μM 12× Cy3-TERRA and 50 μM LSD1 condensates. m A representative FRAP curve for TERRA is shown. The fit to an exponential curve generates mobile fraction and recovery time. Mean values of 19 condensates from three independent experiments are shown in Supplementary Table 1. n Representative DIC images of LSD1^ΔIDR, LSD1^{ΔIDR, 3KE}, and LSD1^{ΔIDR, K661A} with the addition of TERRA (n = three independent experiments). o In vitro pull-down assay was used to evaluate the binding of biotinylated (UUAGGG)₈ oligonucleotide to recombinant LSD1^ΔIDR, LSD1^{ΔIDR, 3KE}, and LSD1^{ΔIDR, K661A} from E. coli. (n = three independent experiments). Source data are provided as a Source Data file.