Figure 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.