Chromatin receptors for thyroid hormones. Interactions of the solubilized proteins with DNA

Thyroid hormone-responsive tissues contain chromatin "receptor" proteins that are concentrated in chromatin subfractions enriched in DNA. These receptors appear to be DNA-binding proteins. In the present study, we utilized a DNA-cellulose binding assay to further examine the interactions of solubilized receptors with DNA. [125I]Triiodothyronine associates with receptors bound to DNA-cellulose, whereas free [I]triiodothyronine and [125I]triiodothyronine associated with other proteins does not. The DNA-receptor interactions appear to be strong enough to exist at physiological ionic strength since binding is 50% maximal ag 0.175 M NaCl and is only partly inhibited by Ca2+ and Mg2+ in the 1 to 5 mM range. Most, if not all, of the receptors are capable of DNA binding, and there are at least 80,000 receptor binding sites/diploid DNA (assuming one triiodothyronine binding site/receptor). Binding of the receptor-[125I]triiodothyronine complexes to other DNAs and analogs was examined using a competition assay. There is similar binding by native and denatured DNA, gy eukaryotic DNA from different species and by prokaryotic DNA (Bacillus subtilis). Binding by natural DNAs is more avid than by cytoplasmic RNA, nuclear RNA, poly(dA-dT)-poly(dA-dT), or poly(dG-dC)-poly(dG-dC). Under these conditions, binding by tRNA and poly(dA) is insignificant, and the nucleotide monomers ATP and GTP have no detectable binding. These studies support the idea that the thyroid hormone receptor is a DNA-binding protein and that the interaction is a major determinant for receptor localization in chromatin. The competition studies suggest that the polynucleotide composition and/or conformation can have marked influences on the binding, and that multiple orders of binding affinity can exist. The presence of specific sequences cannot be excluded. However, the finding that receptors bind extensively and tightly to DNA suggests that receptors in chromatin may randomly bind to any available DNA, resulting in some of the receptors being at physiologically unimportant sites. If so, the several thousand hormone receptors present in each target cell may be required to enhance the possibility that some of the receptors are present at the actual sites of action.