Nanog occupancy as detected by FitCUT&RUN during zebrafish ZGA. (A) The genome browser view shows a gradual increase in the Nanog FitCUT&RUN signal during the zebrafish ZGA process (256-cell, 1k-cell, and dome stage; three replicates of each). Multiple adjacent Nanog binding sites gradually accumulated as Nanog-binding cluster (NBC) regions; a representative example is indicated by the dashed box. (B) Venn diagram showing the overlap status of Nanog binding sites at different stages (256-cell, 1k-cell, and dome stage). (C) Dot plots showing the Gene Ontology (GO) enrichment analysis for Nanog target genes. Genes targeted by Nanog at pre-MBT had higher enrichment scores in the embryonic morphogenesis and regulation of cell fate commitment function categories, especially for NBC target genes. (D) Line plots showing the z-score-transformed expression level (Supplemental Table S3; White et al. 2017) of the first-wave genes (Lee et al. 2013) targeted by Nanog (pre-MBT target, MBT new target, and post-MBT new target) at different developmental stages. Pre-MBT target genes reached their expression peak (dome) earlier than the MBT new target genes (8 hpf) and post-MBT new target genes (50%-epiboly). (E) Bar plots showing the percentage of first-wave genes down-regulated at 4 hpf and 6 hpf upon nanog MO compared to that of the wild-type samples (Lee et al. 2013). The earlier the genes were targeted by Nanog, the larger the proportion with a down-regulation status, indicating that Nanog pre-MBT binding is critical for zebrafish ZGA. (F) Relative expression level of genes corresponding to the mutated Nanog-binding sites detected by RT-qPCR at the 1k-cell stage (dharma, fgfr4, and hist1h4l) or 4 hpf (fox3). Out of the four genes, foxa3 has two Nanog binding sites near its TSS; whereas injecting one gRNA did not change its expression, co-injecting two gRNA targeting two binding sites together displayed significant expression decrease. For the remaining three genes, two showed a significant expression decrease upon corresponding gRNA injection. (G) Bar plots showing the Nanog motif frequency in three groups of peaks within NBCs, that is, emerged from the 256-cell stage, 1k-cell stage, and dome stage. Peaks that emerged earlier show higher Nanog motif frequency.
