Triiodothyronine receptor from rat liver nuclei Interaction, after partial purification, with DNA and chromatin

Abstract

Nuclear triiodothyronine-binding proteins (NTBP) which are at present considered as a nuclear receptor for triiodothyronine (T 3) have been partially purified (about 100-fold) from rat liver nuclei by Sephadex G-100 gel filtration and DEAE-Sephadex chromatography and incubated with [ 125I]-T 3([ 125I]T 3-NTBP complexes). Their ability to bind to DNA and chromatin was analyzed by using in the first case rat liver or calf thymus DNA either soluble or coupled to Sepharose 4B and in the second case expanded chromatin preparations from residual chromatin after NTBP extraction (extracted chromatin) or from whole nuclei (total chromatin). [ 125I]T 3-NTBP complexes could be rapidly and totally bound to DNA; chromatin binding was slower and less efficient on a DNA weight basis, particularly with total chromatin. In both cases, binding presented similar characteristics: it was highly sensitive to concentration of KCl and divalent cations; it depended upon the presence of reducing agents and probably the maintenance of a proper conformation of the receptor molecule; it did not need the presence of bound hormone; it was inhibited by ethidium bromide, actinomycin D and heparin. DNA binding of [ 125I]T 3-NTBP complexes was similar with eukaryotic DNAs (rat, calf), reduced with Escherichia coli DNA, synthetic poly[d(A-T)] and heat-denatured DNA, and almost non-existent with poly(dA) or yeast RNA. No saturation of rat liver DNA could be detected, suggesting a capacity higher than 130 pmol NTBP/mg DNA. Saturation curves were observed in only three experiments out of six using total chromatin preparations and suggested capacities about 50-fold higher than the physiological concentrations of NTBP in nuclei (0.5 pmol/mg DNA). Furthermore, NTBP binding was significantly lower with spleen chromatin preparations as compared to liver ones. Thus, the T 3 receptor appears as a DNA binding protein; it is suggested from our in vitro chromatin binding studies that DNA could be the major locus of NTBP binding in chromatin. The extent of DNA binding in chromatin is limited by other chromatin components in a more pronounced manner with the least modified chromatin preparations and with chromatin preparations from spleen, a tissue poorly responsive to thyroid hormones. Nevertheless, another chromatin localization of the T 3 receptor cannot be excluded.