A distinct isoform of ZNF207 controls self-renewal and pluripotency of human embryonic stem cells

Abstract

Self-renewal and pluripotency in human embryonic stem cells (hESCs) depends upon the function of a remarkably small number of master transcription factors (TFs) that include OCT4, SOX2, and NANOG. Endogenous factors that regulate and maintain the expression of master TFs in hESCs remain largely unknown and/or uncharacterized. Here, we use a genome-wide, proteomics approach to identify proteins associated with the OCT4 enhancer. We identify known OCT4 regulators, plus a subset of potential regulators including a zinc finger protein, ZNF207, that plays diverse roles during development. In hESCs, ZNF207 partners with master pluripotency TFs to govern self-renewal and pluripotency while simultaneously controlling commitment of cells towards ectoderm through direct regulation of neuronal TFs, including OTX2. The distinct roles of ZNF207 during differentiation occur via isoform switching. Thus, a distinct isoform of ZNF207 functions in hESCs at the nexus that balances pluripotency and differentiation to ectoderm.

Fig. 1

Locus-specific proteomics identified proteins located at the proximal enhancer of OCT4 gene in hESCs. a Schematic overview of locus-specific proteomics in hESCs. A representation of OCT4 locus is shown on the top. Dark boxes represent exons, and the white box represents the proximal enhancer that is bound by the transcription factory. TALEN protein with a 3xFLAG tag was designed to bind to the proximal enhancer. The colored ovals represent the “repeat-variable di-residues” (RVD) of TALEN protein that determines binding specificity to DNA bases. They follow the code that NG, NI, HD, and NN respectively recognizes thymine, adenine, cytosine, and guanine. Chromatin is crosslinked by formaldehyde and fragmented by sonication. Then, FLAG antibody was used for immunoprecipitation of proteins bound to the proximal enhancer. The pull-down complex was de-crosslinked of the identity of isolated proteins was discovered by mass spectrometry. b Validation of the binding of dTALE protein by ChIP. A ChIP assay was performed using anti-flag antibody to detect enriched fragments in hESCs. Fold enrichment is the relative abundance of DNA fragments at the amplified region over a control amplified region. IgG ChIP is served as control. The locations of the amplified products are indicated by arrows along the proximal promoter of OCT4. Data are presented as the mean ± SEM and are derived from three independent experiments. c Gene ontology analysis of the proteins identified by locus-specific proteomics. Blue bars represent the number of genes and red bars represent p-value. p-value is calculated using hypergeometric distribution

Fig. 2

ZNF207 is required for hESCs maintenance and enhances reprogramming efficiency through direct regulation of OCT4. a Bright field view of cell morphology after knockdown. Scale bars represent 50 µm. b Alkaline phosphatase staining of cells after knockdown. Scale bars represent 50 µm. c Validation of the binding of ZNF207 by ChIP. A ChIP assay was performed using anti-ZNF207 antibody to detect enriched fragments in hESCs. IgG ChIP is served as control. The locations of the amplified products are labeled as Fig. 1b. d Immunofluorescence staining of control and ZNF207 KD hESCs. Scale bars represent 50 µm. e Overexpression of ZNF207 (ZNF207 OE) enhanced reprogramming efficiency, while knockdown of ZNF207 (ZNF207 KD) reduced the efficiency. Top: Colony formation shown by AP staining. Bottom: Counts of colonies. Scale bars represent 5 cm. f Deletion of ZNF207 DNA-binding domain abolished its effect on reprogramming. Top: Colony formation shown by AP staining. Bottom: Counts of colonies. Scale bars represent 11 mm. ZNF207 WT: wild-type ZNF207 protein; ZNF207 MUT: ZNF207 protein without DNA binding domain. Data are presented as the mean ± SEM and are derived from three independent experiments

Fig. 3

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. 4

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

Fig. 5

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. 6

ZNF207 works upstream of OTX2 in hESCs to govern specification to ectoderm. a Immunofluorescence staining of control and ZNF207 KD hESCs for lineage markers. Scale bars represent 50 µm. b Counts and percentage of cells that were stained positive for each lineage. c KD of ZNF207 disrupted pluripotency of hESCs. HESCs have equal potential to differentiate into any of the three lineages, while KD of ZNF207 tilts the potential to endoderm and mesoderm. The potential to differentiate into ectoderm is significantly impaired by KD of ZNF207. d Immunofluorescence staining of control, ZNF207 KD and ZNF207 KD, OTX2 OE hESCs for ectodermal proteins. Top: Scale bars represent 100 µm; Bottom: Scale bars represent 25 µm. e Counts of cells that were stained positive for ectodermal proteins. f ZNF207-centered transcriptional network to regulate self-renewal and pluripotency in hESCs. ZNF207 works with master transcription factors and co-factors, such as OCT4, SOX2, and P300, to form the core transcriptional network in hESCs. They bind together at pluripotency, development, and cell cycle-related genes to govern self-renewal and pluripotency. OTX2 is targeted and regulated by ZNF207 to ensure the developmental potential towards ectoderm. Once ZNF207 is knocked down, the expression of OCT4, SOX2, and P300 is reduced. The level of cell cycle-related genes has been changed, which leads to reduced ability of self-renewal. The expression of OTX2, which is downstream of ZNF207, is also downregulated, which impedes the differentiation potential towards ectoderm. In the meantime, the potential to endoderm and mesoderm is enhanced. Data are presented as the mean ± SEM and are derived from three independent experiments

Fig. 7

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