Fig 2: Identification of the Elf5 protein interactome in TSCs. (A) Summary of high-confidence Elf5 interactors as identified in three independent immunoprecipitation (IP) and mass spectrometry experiments. Note the interaction of Elf5 with the two other TSC TFs: Eomes and Tfap2c. The full list of interactors is provided in Supplemental Table S1. (B) Independent validation of the interaction between Elf5 and Eomes by anti-Flag immunoprecipitation of the 3xFlag-tagged (3xF) Elf5 protein followed by Western blotting. (C) Independent validation of the interaction between Elf5 and Tfap2c by immunoprecipitation followed by Western blotting. The asterisk highlights the Elf5-3xFlag band in the input sample. Additional validation of the Elf5–Tfap2c interaction is provided in Figure 3F and Supplemental Figure S2, D and E. (D) Eomes interacts with Tfap2c and Elf5. Eomes-3xFlag immunoprecipitates analyzed by Western blot using anti-Elf5 and anti-Tfap2c antibodies. The asterisk demarcates the presence of Eomes in the input samples that can be appreciated upon longer exposure, shown in the panel below. (E) Tfap2c interacts with Eomes. Tfap2c-3xFlag immunoprecipitates analyzed by Western blot using anti-Eomes antibody.

Fig 3: Differential adhesion force in ETX embryos.(a) Representative images of cell doublets for homotypic and heterotypic cell pairs (green, Gata4; cyan, Tfap2c; gray, Oct4; red, F-actin). Experiments were repeated 3 times. Scale bar is 10 µm. (b) Use of ImSAnE ‘Unrolling’ algorithm to project 3D E-cadherin staining stacks onto a 2D plane. Cell contact angles were quantified using built-in correction methods. Geometric observables as well as generally distortions in projections can be correctly quantified. Scale bar is 100 µm. (c) Cell-cell contact angle measurements based on ImSAnE method in day 4 ETX and E5.5 natural embryos. Total measured cell pairs in ETX embryos: ES-ES: n = 24; TS-TS: n = 15; XEN-XEN: n = 16; ES-TS: n = 24; XEN-TS: n = 19; XEN-ES: n = 16; XENi-XENi: n = 16. Total measured cell pairs in natural embryos: EPI-EPI: n = 20; TE-TE: n = 17; VE-VE: n = 22; EPI-TE: n = 24; EPI-VE: n = 16; TE-VE: n = 19; VEi-VEi: n = 24. Data are presented as box-whisker plots, black line inside the box indicates the median value and the error bar shows min to max value. Statistics calculated by one-way ANOVA with a multiple comparison test. (d) Enlargement of the boundary area in a day4 ETX embryo stained with E-cadherin, with homotypic contacts highlighted in blue (TS-TS), red (ES-ES) and purple (XEN-XEN), and the heterophilic boundary interface in yellow. Angles formed at tricellular junctions between different types are indicated: EX, TX and ET, angles between heterotypic contacts (ES-XEN, TS-XEN and ES-TS); EE, TT and XX, angles between homotypic contacts (ES-ES, TS-TS and XEN-XEN). XXi indicates contact angles of XEN cells at cell-medium interface. Experiments were repeated 6 times. Scale bar represents 20 µm. (e) E-cadherin and P-cadherin mRNA expression in cells after downregulation of E- or P-cadherin by RNAi, scrambled siRNA was used as a control. P-cadherin mRNA expression in ES cells after overexpression of P-cadherin. N = 4 for all conditions. Data are presented as mean ± SD. Statistics calculated by unpaired two-tailed Student’s t test. Source numerical data are available in source data. Source data

Fig 4: Stoichiometry-dependent effects of Eomes, Elf5, and Tfap2c. (A) Schematic diagram of expression constructs used to generate stable TSC lines. (B) Characteristics of Tfap2c_Elf5-overexpressing cells after short-term selection (7 d after transfection and 5 d after selection): Cells lose the tight epithelial morphology characteristic for TSCs in the stem cell state and up-regulate a series of differentiation markers, most notably those indicative of giant cell differentiation (Prl8a9, Prl3d1 = Pl1; Prl3b1 = Pl2; and Prl2c2 = Plf). Additional data on Tfap2c_Elf5-overexpressing cells are provided in Supplemental Figure S3. (C) Phase contrast images of representative colonies of the various stable TSC lines after ∼6 wk in culture. Note the tight epithelial colony shape in Eomes-overexpressing TSCs (Eomes, also Eomes_Elf5) and the high frequency of trophoblast giant cells (characterized by large cell and nuclear size) in Elf5-, Elf5_Elf5-, and Tfap2c-overexpressing cell lines. Examples of giant cells are highlighted by arrows, and cell dimensions are outlined by the thin dotted line. Larger areas of differentiating cells are highlighted by the thick yellow dashed line. The Tfap2c_Elf5 line is not shown, as it had largely lost transgene expression by this stage (see Supplemental Fig. S3D). (D) RT-qPCR analysis of various TSC-specific and differentiation stage-specific marker genes in the stable cell lines shown in C. Note that the Tfap2c_Elf5 line marked with an asterisk had largely lost transgene expression, explaining the less pronounced up-regulation of trophoblast giant cell markers. (E) (Co-)transfection experiments of the indicated expression constructs with or without Eomes. Immunofluorescence staining for Flag identifies transfected cells. Numbers of transfected cells per colony were counted 3 d after transfection for 40–60 separate colonies. Only cells with very low-level expression of Elf5 and/or Tfap2c form larger colonies. The presence of Eomes rescues the differentiation-promoting, anti-proliferative phenotype of Elf5 and/or Tfap2c overexpression. (F) Transfection of Eomes into established Elf5 and Tfap2c_Elf5 cell lines reduces the enhanced expression of differentiation markers. (G) Seesaw model of the function of Elf5 at the fulcrum between TSC self-renewal and differentiation. The stem cell state is characterized by preferential binding of Elf5 to Eomes in the presence of comparatively low Tfap2c amounts. Differentiation is triggered by an increase in Elf5 and Tfap2c levels and their predominant interaction.

Fig 5: Elf5, Eomes, and Tfap2c triply bound regions are associated with active TSC genes. (A) Venn diagram of the combinatorial ChIP-seq binding peaks between Elf5, Eomes, and Tfap2c in TSCs. (B) Bioinformatic analysis assessing the statistical significance of the occurrence of cobound elements over randomly expected. (C) Venn and chord diagrams depicting the genome-wide binding overlap of Tfap2c, Sox2, Eomes, and Elf5. Note that the Elf5–Tfap2c network is largely distinct from that established by the interaction between Tfap2c and Sox2. (D) Genome browser views of the ChIP-seq peak distribution at the Eomes, Elf5, and Tfap2c loci. Triply bound elements are highlighted in gray. (E) Functional annotation chart of the genes associated with single, double, and triple peaks. Only significant hits are shown; the TF combinations not shown did not exhibit significantly enriched functional annotations. The thickness of the connecting bars is indicative of the proportion of genes near bound elements that are annotated to the enriched term. Genes associated with triply bound regions are most strongly associated with early trophoblast development at the blastocyst stage, whereas Elf5–Tfap2c-associated genes are more strongly associated with later stages of trophoblast differentiation. (F) Integration of RNA-seq data from TSCs grown in stem cell conditions and upon 1 d of differentiation with ChIP-seq peaks. The graph shows that genes more highly expressed in stem cell conditions are enriched for Elf5–Eomes–Tfap2c triply bound regions (red line) compared with all ChIP-seq loci.