Hepatocyte Nuclear Factor 4 contributes to thyroid hormone homeostasis by cooperatively regulating the type1 Iodothyronine Deiodinase gene with GATA4 and Krüppel-like transcription factor 9

Laboratory of Systems Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo 153-8904, Japan; Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan; Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA; and Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92717-3900, USA

^* To whom correspondence should be addressed. Email: jmsakai-tky{at}umin.ac.jp.

	Abstract

Type1 iodothyronine deiodinase (Dio1), a selenoenzyme catalyzing the bioactivation of thyroid hormone, is highly expressed in the liver. Dio1 mRNA and enzyme activity are markedly reduced in the livers of hepatic hepatocyte nuclear factor 4 (HNF4)-null mice, thus accounting for its liver-specific expression. Consistent with this deficiency, serum T₄ and rT₃ concentrations are elevated in these mice compared with those of HNF4-floxed control littermates, however serum T₃ levels are unchanged. Promoter analysis of the mouse Dio1 gene demonstrated that HNF4 plays a key role in the trans-activation of the mouse Dio1 gene. Deletion and substitution mutation analyses demonstrated that a proximal HNF4 site (Direct Repeat-1: TGGACA A AGGTGC; HNF4-RE) is crucial for trans-activation of the mouse Dio1 gene by HNF4. Mouse Dio1 is also stimulated by thyroid hormone signaling but a direct role for thyroid hormone receptor action has not been reported. We also showed that the thyroid hormone inducible Krüppel-like factor 9 (KLF9) stimulates mouse Dio1 promoter very efficiently through two CACCC sequences that are located on either side of the HNF4-RE. Furthermore, KLF9 functions together with HNF4 and GATA4 to synergistically activate the mouse Dio1 promoter, suggesting that Dio1 regulation by thyroid hormone in the mouse is through an indirect mechanism requiring prior KLF9 induction. In addition, we showed that physical interactions between the C-terminal zinc finger domain (Cf) of GATA4 and activation function -2 of HNF4 and between the basic domain adjacent to Cf of GATA4 and a C-terminal domain of KLF9 are both required for this synergistic response. Taken together, these results suggest that HNF4 regulates thyroid hormone homeostasis through transcriptional regulation of the mouse Dio1 gene with GATA4 and KLF9.