Figure 6.

TRα1 directly binds at the PLB core promoter in HL-1 cells, and addition of T₃ triggers changes in covalent histone modifications associated with transcriptional repression. A, Schematic representation of the mouse PLB promoter including the relative positions of the three PCR primer sets. B and C, TRα1 binds at the PLB core promoter region and its occupancy increases in the presence of T₃. Formaldehyde cross-linked chromatin was prepared from HL-1 cells cultured in normal serum (B) or additionally cultured in the presence of T₃ (1 μm) for 24 h (C). ChIP was then carried out using an antibody specific for TRα1 or a nonspecific control (Con.) rabbit IgG and then analyzed by semiquantitative PCR using all three PCR primer sets (B) or primer set B alone (C). D and E, T₃-dependent histone H3 deacetylation and demethylation at the PLB core promoter. ChIP was carried out on formaldehyde cross-linked chromatin prepared from HL-1 cells cultured with or without T₃ using antibodies specific for H3-Ac or H3K4–2me and then analyzed by PCR using primer set B. The ethidium bromide-stained PCR bands in C–E were quantitated by gel documentation and are presented as the fold enrichment over the PCR signal generated by the control IgG. Error bars represent the ±sd calculated from three separate experiments. Statistical significant difference: *, P < 0.001; **, P < 0.005; ***, P < 0.004.