Extended Data Fig. 8: identification and in vitro functional validation of totipotency-related TFs in totipotency induction.

(a) Clustering synthetic cells by posterior probability of different chromatin states in transcription start site (TSS) regions. (b) Progressive changes in expression of totipotency related genes (left) or progressive establishment of totipotency-related chromatin states (right) from zygote to 8-cell developmental stages. The median expression of totipotency related genes is calculated for each synthetic cell (black line, RNA). (c) Bar charts showing enrichment of 2,583 classifier bins in promoter, gene body, distal, and repeat regions. Promoter is defined as the ± 1 kb genomic region around TSS. (d) Overlap of the active cluster of totipotency-related classifier bins (see Fig. 4e) with totipotency feature genes. The totipotency gene list was downloaded from Hui Shen et al. 2021. (e) Bar plots showing GO terms enriched in 120 totipotency-related TFs (listed in Supplementary Table 8). P value, one-sided binomial test. (f) Venn diagram showing overlap of TF motifs enriched on totipotency-related classifier bins generated from the two machine learning models. Both known totipotency-related TF motifs (ZSCAN4 and NR5A2) and newly identified totipotency-potential TF motifs (MEF2D, LBX1, ESR1, ETS1, CEBPG) are shown. P value, one-sided hypergeometric test. (g) Schematic for CRISPRa experiment in mESCs. (h) Scatter plot showing the sgRNA UMI fraction within a given cell (x axis) as a function of the log2 (UMI per sgRNA) received in that cell (y axis). Each dot represents an individual sgRNA species in a specific cell. sgRNAs with less than 16 UMI were not considered for further analyses. (i) Data quality control for detected sgRNA species per cell. (j) UMAP showing the individual cells in this study (n = 10,178) and public scRNA-seq datasets. Public scRNA-seq data were downloaded from GSM5195024.TBLCs, totipotent blastomere-like cells. PSCs, pluripotent stem cells. (k) Pseudotime analysis for individual cells shown in (c). (l) Expression level of totipotency gene signature along the pseudotime of totipotency activation. (m) Cell distribution along the pseudotime of with the increasing of cell totipotency. (n) The proportion of cells receiving different kinds of sgRNAs for pluripotent, intermediate, and TBLCs. NTC, non-targeting control. PC, positive control with DUX and ZSCAN4. (o) The overall perturbation effect ranking lists identified by MUSIC (Duan et al.⁷⁰, PMID: 31110232) using the cells taking only one sgRNA in the CRISPRa experiment. (p) Heatmap showing the gene–gene perturbation relationships for candidate TFs and positive control TFs (ZSCAN4 and DUX). (q) Violin plot showing the scaled totipotency score for cells with different combination of gene perturbarions. Box plots center lines indicate the median, box limits indicate the first and third quartiles, and whiskers indicate 1.5× IQR. The cell number for each of the eight perturbation combinations is as follows: 1,946, 3,734, 746, 1,068, 651, 1,119, 800, and 1,588. (r) Projection of cells with different combination of gene perturbarions shown in (j) along pseudotime. The cell with median pseudotime distance was labeled by red for each group.

Source data