Fig 2: ZNF207 co-localizes with enhancer markers to promote cell cycle of hESCs. a Top: Composite plot shows ZNF207, p300 (GSM1003513) and H3K27ac (GSM733718) ChIP-Seq signals are enriched and overlapped at TSSs. Bottom: Heatmap illustrates genome-wide association of ZNF207 with p300 and H3K27ac-binding sites. b Gene ontology analysis of genes that are co-bound by ZNF207, p300 and H3K27ac. Blue bars represent fold enrichment and orange bars represent p-value. p-value is calculated using hypergeometric distribution. c ChIP-Seq tracks show co-localization of ZNF207, P300, and H3K27ac at the regulatory sequences of cell cycle genes. The scale bars indicate the size of the chromosome. The light pink boxes highlight the co-bound regions. d Gene ontology analysis of cell cycle genes that are differentially expressed in ZNF207 KD cells. Blue bars represent p-values and orange bars represent FDR (false discovery rate). p-value is calculated using hypergeometric distribution. e Cell cycle progressions determined by flow cytometry. Histogram plot of flow cytometry analysis of control cells (red) and ZNF207 KD cells (blue). The percentage of cells that are in each phase of mitosis is shown. f ChIP-Seq tracks show binding of ZNF207 at promoter and enhancer regions of FGF2 gene. g The expression of FGF2 in control and ZNF207 KD cells. h Western blot to detect the protein level of FGF2 in KD, control, and OE cells. GAPDH is shown as the loading control. i Clonogenic assay to show the self-renewal ability of control and ZNF207 OE cells in E7 media with 20 ng/ml bFGF. Scale bars represent 17.5 mm. The counts of colonies are shown on the right. Data are presented as the mean ± SEM and are derived from three independent experiments

Fig 3: ZNF207 switches isoforms during cell differentiation. a Confocal images of mitotic cells with aligned chromosomes. ~100 mitotic cells were measured for each experiment and condition. Centromeres were detected by the CREST serum. Scale bar, 5 µm. b Counts of mitotic cells with misaligned chromosomes in control and ZNF207 KD cells. c Schematic representation of the three alternative splice isoforms of ZNF207. d RT-qPCR analysis to test the expression level of different isoforms during induced neuronal differentiation from hESCs. e Alkaline phosphatase staining of cells in different conditions. Scale bars represent 50 µm. f RT-qPCR analysis to test the expression level of SALL4 and OTX2. Data are presented as the mean ± SEM and are derived from three independent experiments. g The model of ZNF207 isoform change in hESCs and differentiated/cancer cells. Data are presented as the mean ± SEM and are derived from three independent experiments

Fig 4: High-throughput analysis in hESCs to identify direct targets of ZNF207. a Analysis of differential expression on the RNA-Seq data of ZNF207 KD and control hESCs. Genes significantly changed (>twofold change, FDR = 1%) are colored in yellow and blue for upregulated and downregulated, respectively. b ZNF207 ChIP-Seq analysis in hESCs. Left: Heatmap depicts ZNF207 ChIP-Seq signals at TSSs. Right: Composite plot shows ZNF207 ChIP-Seq signal is enriched at transcription start sites (TSSs). The gradient blue-to-red color indicates high-to-low counts in the corresponding region. c Enriched motif from de novo motif search of sequences under ZNF207 peaks. Wild-type (WT) sequence and mutant (MUT) sequence are shown in the bottom. Mutated nucleic acids are labeled by red fond color. d EMSA were performed to detect interaction of ZNF207 protein with DNA sequences. e ChIP-Seq tracks show co-localization of ZNF207, SOX2, NANOG, and OCT4 at the proximal enhancer of OCT4 gene. OCT4 gene, Proximal enhancer (PE) and distal enhancer (DE) are indicated by the boxes in the bottom. The scale bar indicates the size of the chromosome. The light pink boxes highlight the co-bound regions. f Venn diagrams show overlaps of ChIP-Seq bound genes with differentially expressed genes identified from RNA-Seq. g Gene ontology analysis of genes that are directed regulated by ZNF207. Blue bars represent fold enrichment and orange bars represent p-value. p-value is calculated using hypergeometric distribution

Fig 5: ZNF207 co-localizes with master transcription factors in hESCs to regulate pluripotency and neuronal gene expression. a Enriched motifs from de novo motif search of sequences under ZNF207 peaks. Note the identification of consensus OCT4/SOX2 binding but also other known transcription factor binding motifs. Statistical significance (E-values) is indicated below the motif logo. b Heatmap depicts ZNF207, OCT4 (GSM1124067), and SOX2 (GSM1701825) ChIP-Seq signals at TSSs. c Protein interactions between ZNF207 with OCT4 and SOX2. Co-IP using cell extracts from hESCs was performed using anti-OCT4 and anti-SOX2 antibody. Western blotting was carried out with anti-ZNF207 antibody. Control IP was performed using anti-IgG antibody. Reverse co-IP was performed using anti-ZNF207 antibody, and western blotting was then performed with anti-OCT4 or anti-SOX2 antibody. Input was shown as loading control. d Venn diagrams show overlaps of genes bound by OCT4, SOX2, and ZNF207 in hESCs. e Gene ontology analysis of genes that are directed regulated by ZNF207. Blue bars represent the number of genes and orange bars represent p-value. p-value is calculated using hypergeometric distribution. f ChIP-Seq tracks show co-localization of ZNF207, SOX2, and OCT4 at the regulatory sequences of pluripotency genes. The scale bars indicate the size of the chromosome. The light pink boxes highlight the co-bound regions. g ChIP-Seq tracks show co-localization of ZNF207, SOX2, and OCT4 at the regulatory sequences of neuronal genes. The scale bars indicate the size of the chromosome. The light pink boxes highlight the co-bound regions