Fig. 4

Inheritance of active transcriptional states propagating through cell division. a Representative images of dPspCas13b-EGFP tracking eppk1 re-activation with Cy3-geppk1s, recorded every 5 min from 4.5 to 8 hpf in EVL of cell cycle 14. See also Additional file 7: Movie 7. Green arrowheads indicate puncta 1, and magenta arrowheads indicate puncta 2. b Synchronized transcription of inter-allelic eppk1 in the cell cycle post the cell cycle having de novo transcription. Normalized signal intensity of transcriptional activity recorded over time in a in the EVL of cell cycle 14 in developing embryos. c Synchronized post-mitotic transcriptional re-activation of EVL cells. Statistics of the inherited active transcriptional sites of two genes eppk1 (left) and 100537515 (right) during re-activation in the next cell cycles (the EVL of cell cycles 14 and 15, respectively). Data were traced from Additional file 1: Fig. S9c, d for original data. See also Fig. 4d for detail. d Time difference in initial transcription between individual inter-alleles of eppk1(left) and 100537515 (right) during transcription re-activation in the next cell cycles (the EVL of cell cycles 14 and 15, respectively). Data were traced from Additional file 1: Fig. S9c, d. See also Additional file 1: Fig. S9f. e Inherited active transcriptional states propagating through cell division. Statistics of the time difference in initial transcription between inter-alleles, and among pairs of sister cells for eppk1 and 100537515 during de novo transcription and post-mitotic re-activation in different cell cycles. See also Figs. 3e, 4d and Additional file 1: Fig. S9f-h. f, g Rapid kinetics of post-mitotic transcriptional re-activation. Expression of both eppk1 (f) and 100537515 (g) is more rapidly re-activated and reached a transcriptional plateau in the post de novo transcription cell cycle of EVL cells. Normalized intensity in all cells (eppk1, n= 55 in the cell cycle 13, 0 time point indicating 4 hpf; n= 36 in the cell cycle 14, 0 time point indicating 4.5 hpf. 100537515 n= 24 in the cell cycle 14, 0 time point indicating 6 hpf; n = 36 in the cell cycle 15, 0 time point indicating 9.5 hpf) at each time point across de novo expression (cell cycle 13 for eppk1, and cell cycle 14 for 100537515) and post-mitotic transcriptional re-activation (cell cycle 14 for eppk1, and cell cycle 15 for 100537515) cell cycles. Sum of puncta 1 and puncta 2 for one cell transcriptional activity at each time point. Of note, the non-synchronized transcription of 100537515 inter-alleles in cell cycle 14 can be used as a control for the synchronized transcription of eppk1 inter-alleles in the same cell cycle, and vice versa. Data were traced from Additional file 1: Fig. S8f, g and S9c, d for original data. h–k Scatterplot of transcriptional output of pairs allelic eppk1 in the same cells (h, i) and random pairs allelic eppk1 from the different cells (j, k) during EVL cell cycles 13 (h, j) and 14 (i, k), respectively. Total RNA output was determined by summing the area under the time point traced at each allele (data from Additional file 1: Fig. S8f,9c). r: Pearson correlation coefficient, ρ: Spearman’s rank correlation coefficient, k: Slope. The pink line in each panel indicates slope. l, m Increased inter-allelic correlations of eppk1 (l) and 100537515 (m), respectively, during post-mitotic transcriptional re-activation, compared with those during de novo expression. Statistics of the Pearson correlation coefficient of all cells (eppk1, n = 55 in the cell cycle 13; n = 36 in the cell cycle 14. 100537515 n = 24 in the cell cycle 14; n = 36 in the cell cycle 15) at each time point. See also Additional file 1: Fig. S11e-h. Pearson correlation coefficient in real time. eppk1, p = 1.17e−06; 100537S515, p = 0.01. Data were traced from Additional file 1: Fig. S8f, g and S9c, d for original data. n The transcriptional activity has no difference in random pairs of eppk1 (left panel) and 100537515 (right panel) alleles from different cells between de novo expression and post-mitotic transcriptional re-activation, shown by Pearson correlation coefficient. eppk1, p = 0.85; 100537515, p = 0.91. In d–g, data are represented as mean ± SD. In d, e, data unpaired two-tail Student’s t test; ns, not significant; * p< 0.05, **** p< 0.0001. In l–n, data of the box, middle line is median; upper and lower horizontal lines are 25% and 75% quartiles respectively; the point is mean; unpaired two-tail Student’s t test