FIG. 6.

GCNF recruits MBD2 and MBD3 to bind to the Oct4 promoter in ES cells. (A) Expression of GCNF, Oct4, MBD2, and MBD3 was detected in differentiated WT and GCNF^−/− ES cells by RT-PCR and Western blotting. (B) Binding of GCNF, MBD2, and MBD3 to the Oct4 promoter in WT and GCNF^−/− ES cells was detected by ChIP assay (normal PCR and ³²P-labeling PCR). (C) Quantitation of ³²P-labeled PCR signals. The strength of LRH-1-, GCNF-, MBD2-, and MBD3-bound signals at the undifferentiated time point was set as 1. The bound LRH-1 signal at 72 h of RA differentiation was set as 1. (D) Effect of MBD2 and MBD3 siRNA treatment on Oct4 repression in ES cells. The RNA levels of GCNF, Oct4, MBD2, and MBD3 were determined using RT-PCR. ES cells were treated with 80 nM siRNA duplexes for 24 and 40 h in the presence of LIF or 1 μM RA. (E) Quantitation of ³²P-labeled PCR signals. The signal strength at the undifferentiated time point without siRNA treatment was set as 1. The average ratio of Oct4 to actin at each point was determined from two experiments with siRNA treatment.