Limited capacity binding sites for L-triiodothyronine in rat liver nuclei. Localization to the chromatin and partial characterization of the L-triiodothyronine-chromatin complex.

Abstract Using in vivo saturation techniques we have previously demonstrated limited capacity nuclear binding sites which exhibit a high degree of specificity for l-triiodothyronine and only minimal cross-reaction with l-thyroxine. The current studies were designed to determine the intranuclear localization of the specifically bound triiodothyronine in liver nuclei. Rat liver nuclei were isolated by sucrose density gradient centrifugation 30 min after injection of a small dose (less than 130 ng/100 g body weight) of [125I]triiodothyronine or [131I]thyroxine. These nuclei were treated sequentially with 0.2% Triton X-100 to remove the outer membrane, 0.15 n NaCl and 0.1 m Tris, to extract nucleoplasmic and ribonucleoproteins. After a small dose of triiodothyronine had been injected, 50 to 70% of the nuclear [125I]triiodothyronine resisted extraction by these agents and remained with the residual chromatin pellet. The chromatin localization of triiodothyronine was not a result of in vitro distribution of tracer since more than 50% of nuclear [125I]triiodothyronine was recovered with purified chromatin which was isolated by discontinuous sucrose density gradient centrifugation from nuclei disrupted by hypotonic shock. Moreover, when a large dose of triiodothyronine was injected (more than 8000 ng/100 g body weight), 70 to 90% of the nuclear radioactivity was removed by Triton X-100. [131I]Triiodothyronine which was added to the nuclei in vitro was similarly removed by Triton X-100. Finally, when a small dose of [131I]thyroxine was injected, most of this iodothyronine was also recovered with the nuclear outer membrane. These data thus suggest that specifically bound triiodothyronine is associated with the chromatin whereas nonspecific binding of either triiodothyronine or thyroxine occurs at the nuclear outer membrane. When nuclei previously labeled in vivo with a small dose of [125I]triiodothyronine were extracted with 0.4 m KCl, 60 to 80% of the nuclear [125I]triiodothyronine was removed. The [125I]triiodothyronine extracted by KCl appeared bound to a macromolecule since more than 80% of the radioactivity in these extracts was excluded from small Sephadex G-50 columns. The nuclear triiodothyronine-macromolecular complex was unstable at 25 to 37°. The instability appeared related to breakdown of the nuclear protein since the loss of [125I]triiodothyronine-macromolecular complexes was paralleled by a loss of protein from the excluded volume of these columns. Attempts to reduce the instability of these complexes with iodoacetate, mercaptoethanol, dithiothreitol, rat serum, or glycerol were unsuccessful. The binding of [125I]triiodothyronine was disrupted after treatment with proteolytic enzymes and was unaffected by DNase or RNase. Moreover, the efficiency of the KCl extraction was substantially enhanced in mild alkaline conditions. Thus the specific nuclear triiodothyronine binding site appears to be a chromatin non-histone protein. The apparent molecular weight of the nuclear [125I]triiodothyronine-protein complex appears to be 60,000 to 70,000 as determined by gel filtration. The specific association of triiodothyronine with chromatin non-histone proteins localizes triiodothyronine to the genome and raises the possibility that this association is related to the increase in DNA transcription which occurs after triiodothyronine administration.