Fig 2: Expression patterns of Sbno1 during mouse preimplantation development and phenotypes of Sbno1 knockout embryos (Sbno1Δ/Δ).(a) Expression levels of Sbno1 transcripts during the preimplantation period were analyzed by semi-quantitative RT-PCR. β-Actin was used as an internal control. (b) Immunohistochemistry showed that Sbno1 (green) is clearly localized in the nuclei from the two-cell to blastocyst stages. β-Catenin staining (red) demarcates the cell membrane, and DAPI staining (blue) identifies the nuclei. (c,d) At E3.5, control Sbno1Δ/+ embryos develop to blastocysts with an inner cell mass (ICM), trophectoderm (TE) and blastocoel (BC), whereas development of Sbno1Δ/Δ embryos was halted with a morula-like morphology. (e,f) In Sbno1Δ/Δ embryos, Sbno1 protein (green) was absent from the two-cell stage. (g,h) When two-cell stage embryos were cultured in vitro, Sbno1Δ/Δ embryos developed normally to the compacted morula stage, but failed to form blastocysts and collapsed. (i–m) TUNEL (green in i,j) and phospho-histone H3 staining (pHH3, red in k,l) revealed increased cell death and decreased proliferation in Sbno1Δ/Δ embryos, respectively. Uncropped image of gel is shown in Supplementary Fig. 9.

Fig 3: Effects of human SBNO1 and SBNO1 mutants on transcription and preimplantation development.(a) DExH box, disorder region with two nuclear localization signals (NLS), coiled-coil, helicase-C domains and cysteine cluster in human SBNO1 are indicated. Seven conserved motifs (I to VI) were found in the DExH box and helicase-C domains. Deletion and E437Q mutants of hSBNO1 used in Gal4-luciferase assays are shown. (b) Transcriptional activation profiles of the full length, deletion and E437Q mutants were obtained by Gal4-luciferase assays in 293 T cells. Gal4-hSBNO1 acted as a robust transcriptional activator. The N-terminal region of hSBNO1 retained its activator function, whereas the middle and C-terminal regions did not convey activity and produced a repressive effect. Note that the E437Q mutant completely lost activity on transcription. All data are presented as means ± SD. (c-g) mRNA injection of GFP-hSBNO1-E437Q to zygotes resulted in developmental arrest and Cdx2 reduction after 3days culture, on the other hand, embryos showed little effect by GFP-hSBNO1-Wt mRNA injection.

Fig 4: Physical interaction between SBNO1 and Yap/Tead4 or Notch/Rbpj.(a) SBNO1-Myc, HA-Yap, and/or FLAG-Tead4 were co-expressed in 293 T cells, and immunoprecipitated with indicated antibodies (α-Myc, α-HA and α-FLAG, respectively). Interactions between HA-Yap and FLAG-Tead4, and SBNO1-Myc and FLAG-Tead4 were observed by co-precipitation. Expression of HA-Yap attenuated the interaction between SBNO1-Myc and FLAG-Tead4, as observed by faint bands of co-precipitation (red arrowheads). Myc-SBNO1 co-precipitated HA-Yap, whereas HA-Yap did not co-precipitate SBNO1-Myc. (b) SBNO1-V5, Myc-NICD1, and/or FLAG-Rbpj were co-expressed in 293 T cells, and immunoprecipitated with indicated antibodies (α-V5, α-Myc and α-FLAG, respectively). FLAG-Rbpj was co-immunoprecipitated with SBNO1-V5 (α-V5) and Myc-NICD1 (α-Myc), indicating their interaction. This was further confirmed by co-precipitation of SBNO1-V5 and Myc-NICD1 with FLAG-Rbpj (α-FLAG). The interaction between SBNO1 and Rbpj was weak in the presence of NICD1, as observed by faint bands (red arrowheads). (c–e) Interaction between Sbno1 or Yap and Tead4 in E3.5 embryos was analyzed by in situ PLA. Green signals indicate the ligated antibodies, which represent physical interaction of the antigens. Antibodies against Sbno1 or Yap with Tead4 gave clear signals in the nuclei of outer cells (d,e), while α-Tead4 antibody alone did not give any signal (c). Uncropped image of blots are shown in Supplementary Fig. 11.

Fig 5: Involvement of Sbno1 in transcriptional activation of the Cdx2 trophectoderm enhancer (Cdx2-TEE) in 293 T and E14Tg2a ES cells.(a) Location and core sequences of the Cdx2-TEE are shown. Tetramerized 47 bp Cdx2-TEE was ligated to δ-crystalline minimal promoter-luciferase (4xCdx2-TEE47bp). (b) Expression of Yap alone or NotchΔE alone activated the 4xCdx2-TEE47bp reporter (39- and 3.9-fold, respectively), yet when both Yap and NotchΔE were co-expressed, this reporter was synergistically and robustly activated (1287-fold). As expected, this activation was repressed by SBNO1 siRNA (664-fold). Synergism between Yap and Tead4 was observed (367-fold); however, expression of Tead4 did not affect the synergistic activation by Yap and NotchΔE (1287- versus 1404-fold activation). (c) Lack of transcriptional activation of the 4xCdx2-TEE47bp reporter was evident when the SBNO1-E437Q mutant was expressed. (d) Transcriptional activity of the 4xCdx2-TEE47bp reporter was synergistically upregulated by Yap and NotchΔE in E14Tg2a ES cells, and the SBNO1-E437Q mutant significantly decreased the activity (6.9- and 3.2 fold, respectively). In contrast, SBNO1-Wt increased the Yap and NotchΔE-induced transcriptional activity (16.8 fold). (e) Synergistic activation of the 4xCdx2-TEE47bp reporter by Yap and NotchΔE was observed at both high and low cell densities in the absence of mechanical stretch (713- and 651-fold, respectively). In contrast, when cells were stretched, this synergistic activation was super-enhanced to 1764-fold only in the high cell density culture. At low cell density, mechanical enhancement of transactivation was not observed, and was slightly repressed (490-fold). Pictures of cell cultures are shown. Note that cells make mutual contacts at high density, while at low density cells are isolated or clustered in small separated islands of cells. All data are presented as means ± SD. **p < 0.01 versus relevant control.