FIG. 9.

H7 inhibits both T3-dependent DioI expression and Pol II phosphorylation at the DioI promoter. (A) α-2 cells were cultured with or without T3 (10⁻⁷ M) in the presence or absence of H7 (10 or 25 μM, as indicated) for 1 h and then harvested. Total RNA was processed by quantitative RT-PCR in real time using primers specific for DioI. (B) H7 inhibits Pol II CTD phosphorylation at the DioI promoter in vitro. PICs were assembled on biotinylated DioI promoter templates in the presence of RXR/TR/T3 and HeLa nuclear extract essentially as described for Fig. 3 except that H7 (10 μM) was added to the reaction mixtures as indicated (lanes 4 and 6). The PICs were then isolated by using streptavidin beads and washed, and in selected reactions, transcription was initiated upon the addition of NTPs (100 μM) for 2 min (lanes 5 and 6). The streptavidin conjugates for all reactions were then precipitated, fractionated by SDS-PAGE, and then probed by immunoblotting using antibodies specific for CDK8, Pol II, and phosphorylated Ser-5 Pol II CTD. In reaction 5 (lane 5), the transcription initiation reaction eluate was trichloroacetic acid precipitated and processed for immunoblotting as described above. (C) H7 inhibits Pol II CTD phosphorylation at the DioI promoter in vivo. α-2 cells were cultured with or without T3 in the presence or absence of H7 (10 μM) and then processed for ChIP analyses exactly as described in the Fig. 4 legend.