FIGURE 5.

The mSin3A-HDAC complex interacts with Sox2 to cooperatively stimulate Nanog transcription. A, in vitro NusA-Sox2 pulldown from ES cell nuclear extract. mSin3A-HDAC complex subunits were detected by Western blot. B, immunoprecipitation of endogenous Sox2 from nuclear and cytoplasmic fractions of ES cells treated for 6 h with 5 μm RA or vehicle. mSin3A-HDAC complex subunits were detected by Western blot using the indicated antibodies. Western blots for H3 and tubulin indicate the purity of the fractions. C, fractionation of ES cell nuclear extract. Western blots of every fifth fraction indicate that mSin3A and Sox2 are present in majority of the same fractions. D, schematic diagram of the pGL3-TK-Luc and pGL3-O/S-TK-Luc constructs. pGL3-TK-Luc has luciferase driven by the TK promoter. pGL3-O/S-TK-Luc contains three tandem copies of the Oct4-Sox2 binding site from the Nanog promoter inserted upstream of the TK promoter. E, increase in luciferase activity by the addition of Oct4/Sox2, and mSin3A-HDAC complex members. The graph represents the -fold increase in luciferase activity between pGL3-O/S-TK-Luc and pGL3-TK-Luc after adding the indicated factors.