Figure 5: RAD21- and CTCF-mediated OCT4-induced changes in the 3D configuration of Sox-17 locus.

(a) NIBPL was downregulated by a siRNA. Anti-NIPBL immunostaining of ZHBTc6 cells before differentiation within embryoid bodies of OCT4OE induced mesendodermal cells transfected with a scrambled siRNA or the NIPBL siRNA. Inset: OCT4 and NANOG expressions were found not affected in NIPBL-downregulated cells. Scale bar, 50 μm. (b) Anti-OCT4 ChIP was performed in control ESCs (CTRL) and OCT4-overexpressing mesendodermal cells (OCT4OE) in the presence or absence of NIPBL. Both the Sox-2C and the Sox-17 enhancers were interrogated. (c) Anti-CTCF and anti-RAD21 ChIP–PCR were performed from chromatin of control ZHBTc6 (+tetracyclin) ESCs (CTRL) or OCT4-overexpressing (ZHBTc6–tetracyclin) mesendodermal cells (Oct4OE). (d) Sequential ChIP–PCR anti-OCT4 and then anti-RAD21 were performed from chromatin of control ZHBTc6 ESCs (CTRL) or OCT4-overexpressing mesendodermal cells (OCT4OE). Both Sox-2C (blue bars; c,d) and Sox-17 (green bars; c,d) enhancers were interrogated in real-time PCR. (e) Anti-OCT4 immunoprecipitation of whole cell extract from control (Ctrl) or OCT4-overexpressing mesendodermal cells (Oct4OE). The blot was incubated with both anti-OCT4 and anti-RAD21 antibodies. (f) Anti-OCT4 ChIP–PCR was performed from chromatin of control ZHBTc6 ESCs (Ctrl) or OCT4-overexpressing mesendodermal cells (Oct4OE). Sox-17 promoter was interrogated. Data are representative of three to four experiments (mean±s.e.m.). *Student’s t-test, P≤0.01.