Fig. 3: LOC644656 interacts with the POU5F1 complex to attenuate pluripotency in hESCs. | Nature Communications

Fig. 3: LOC644656 interacts with the POU5F1 complex to attenuate pluripotency in hESCs.

From: p53-inducible lncRNA LOC644656 causes genotoxic stress-induced stem cell maldifferentiation and cancer chemoresistance

Fig. 3

a Schematic of the RNA pulldown assay. b ESCAPE database analysis of the 1,839 LOC644656-interacting proteins (±5-FU). Key pluripotency transcription factors are highlighted. c Overlap between LOC644656 interactors and the POU5F1/NANOG/SOX2 complex. d Transcription factor protein–protein interaction network of the 315 overlapping proteins. e Functional categories and GO terms enriched among the identified interactors. f Experimental workflow for hESC::TetLOC644656 induction. g LOC644656 expression ± doxycycline (Dox). Data are mean ± SEM (n = 3), **p < 0.01, ***p < 0.001, ****p < 0.0001 by two-sided one-way ANOVA. h RNA-FISH of LOC644656 (green) and FITC–rBC2LCN (red) ± Dox for four days. Images are representative of n = 3 independent experiments with similar results. Scale bars: 100 μm (main images), 20 μm (insets). i, j RT–PCR analysis of POU5F1(i) and NANOG (j) ± Dox. Data means SEM (n = 3), **p < 0.01 by two-sided one-way ANOVA. k Immunofluorescence for POU5F1 ± Dox for three days. Representative of n = 3 independent experiments. Scale bar, 50 μm. l RNA pulldown assay using biotinylated sense or antisense LOC644656. Representative of n = 3 independent experiments. Uncropped blots are provided in Source Data. The samples derive from the same experiment but different gels for POU5F1, NANOG, another for LSD1 and another for HDAC1 were processed in parallel. m RIP assay with an anti-POU5F1 antibody in hESC::TetLOC644656 ± Dox. Data means SEM (n = 3), *p < 0.05 by two-sided paired t-test. n ChIP assay of POU5F1 binding at target loci ± Dox. Data means SEM (n = 3), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by two-sided one-way ANOVA. o AlphaFold3 structural modeling of POU5F1 bound to DNA (left) or LOC644656 RNA (right). Exact p-values and 95% confidence intervals: Panel g: p = 0.001, 95% CI [−1.282, −0.3534] (WT Dox - vs WT::TetLOC644656 Dox +); p = 0.0003, 95% CI[−1.423, −0.4947] (WT Dox + vs WT::TetLOC644656 Dox +); p < 0.0001, 95% CI [−1.517, −0.7135] (WT::TetLOC644656 Dox - vs WT::TetLOC644656 Dox +). Panel i: p = 0.0011, 95% CI [0.4657, 1.430] (WT Dox - vs WT::TetLOC644656 Dox +); p = 0.0082, 95% CI[0.2014, 1.166] (WT Dox + vs WT::TetLOC644656 Dox +); p = 0.0015, 95% CI [0.4160, 1.381] (WT::TetLOC644656 Dox - vs WT::TetLOC644656 Dox +). Panel j: p < 0.0001, 95% CI [0.7270, 1.213] (WT Dox - vs WT::TetLOC644656 Dox +); p < 0.0001, 95% CI[0.5330, 1.052] (WT Dox + vs WT::TetLOC644656 Dox +); p < 0.0001, 95% CI [0.5568, 1.138] (WT::TetLOC644656 Dox - vs WT::TetLOC644656 Dox +). Panel m: p = 0.0368, 95% CI [0.7986, 15.18] (Day 0 vs Day 1, Dox +). Panel n: POU5F1 locus: p = 0.0002, 95% CI [0.06560, 0.1564] (Day 0 vs Day 1, Dox +); p < 0.0001, 95% CI[0.08524, 0.1868] (Day 0 vs Day 3, Dox +); NANOG locus: p = 0.0057, 95% CI [0.02939, 0.1483] (Day 0 vs Day 3, Dox +).

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