Aberrant Dynamics of Histone Deacetylation at the Thyrotropin-Releasing Hormone Gene in Resistance to Thyroid Hormone

0888-8809/04/$15.00/0 Printed in U.S.A. Molecular Endocrinology 18(7):1708–1720 Copyright © 2004 by The Endocrine Society doi: 10.1210/me.2004-0067 Aberrant Dynamics of Histone Deacetylation at the Thyrotropin-Releasing Hormone Gene in Resistance to Thyroid Hormone S. ISHII, M. YAMADA, T. SATOH, T. MONDEN, K. HASHIMOTO, N. SHIBUSAWA, K. ONIGATA, A. MORIKAWA, AND M. MORI Department of Medicine and Molecular Science (S.I., M.Y., T.S., T.M., K.H., N.S., M.M.) and Department of Pediatrics and Developmental Medicine (K.O., A.M.), Gunma University Graduate School of Medicine, Maebashi 371-8511, Japan Histone acetylation status influences transcriptional activity, and the mechanism of negative gene regulation by thyroid hormone remains unclear, although its impairment by a mutant thyroid hormone receptor (TR) is critical for resistance to thyroid hormone (RTH). We found a novel RTH mutant, F455S, that exhibited impaired repression of the TRH gene and had a strong dominant-negative effect on the gene. F455S strongly interacted with nuclear receptor corepressor (NCoR) and was hard to dissociate from it. To analyze the dynamics of histone acetylation status in vivo, we established cell lines stably expressing the TRH promoter and wild-type or F455S TR. Treatment with a histone deacetylase (HDAC) inhibitor completely abolished the repression of the gene by T3. The histones H3 and H4 at the TRH promoter were acetylated, and addition of T3 caused recruitment of HDACs 2 and 3 within 15 min, resulting in a transient deacetylation of the histone tails. TR and NCoR were located on the promoter, and T3 caused NCoR dissociation and steroid receptor coactivator-1 recruitment. In the presence of F455S, the histones were hyperacetylated, and HDAC recruitment and histone deacetylation were significantly impaired. This is the first report demonstrating the direct involvement of aberrant dynamics of chromatin modification in RTH. (Molecular Endocrinology 18: 1708–1720, 2004) R fore, impairment of the negative gene regulation by thyroid hormone in the hypothalamic-pituitary-thyroid axis plays a critical role in the pathophysiology of RTH. On the genes positively regulated by thyroid hormone, TR binds to target promoters as a homodimer or a heterodimer with the retinoid X receptor (RXR) and regulates promoter activity by recruiting specific coregulatory protein complexes (3, 4). In the unliganded state, TR assumes a conformation that stably interacts with corepressor molecules such as nuclear receptor corepressor (NCoR) and silencing mediator of retinoic and thyroid hormone receptors (SMRT). Numerous histone deacetylases (HDACs), including HDAC-1, -2, -3, -4, -5, -7, and -9, have been shown to interact with NCoR and SMRT in one context or another, and then repress basal transcriptional activity. Recent chromatin immunoprecipitation (ChIP) experiments have demonstrated that HDAC3 on NCoR, not on SMRT, is most important for the repression by unliganded TR (5). Stimulation with T3 leads to the dissociation of corepressors and recruitment of coactivators including members of the p160/steroid receptor coactivator (SRC) family and TR-associated protein/vitamin D receptor-interacting protein mediators. These proteins are thought to function in part by associating with potent histone acetylytransferases (HATs) such as p300/cAMP response element binding protein-binding protein and ultimately import the HAT activity to promoter-bound TR, resulting in the acetylation of nu- ESISTANCE TO THYROID hormone (RTH) is an autosomal dominant disorder caused mainly by mutations in the thyroid hormone receptor (TR) ␤ gene (1, 2). RTH is characterized by elevated serum thyroid hormone levels associated with a failure to suppress pituitary TSH secretion. The high levels of thyroid hormone result in various symptoms according to the degree of refractoriness to hormone in peripheral tissues. TRs are members of the nuclear receptor superfamily and function as ligand-regulated transcription factors that increase (positively regulate) or decrease (negatively regulate) the expression of target genes. The serum thyroid hormone levels in RTH patients depend on the resistance in the hypothalamicpituitary-thyroid hormone axis, in which all critical genes including TRH, TRH-receptor, and TSH genes are negatively regulated by thyroid hormone. ThereAbbreviations: ChIP, Chromatin immunoprecipitation; FBS, fetal bovine serum; GST, glutathione-S-transferase; HAT, histone acetylytransferase; HDAC, histone deacetylase; NCoR, nuclear receptor corepressor; PML, promyelocytic leukemia; RAR, retinoic acid receptor; RTH, resistance to thyroid hormone; RXR, retinoid X receptor; SDS, sodium dodecyl sulfate; SMRT, silencing mediator of retinoic and thyroid hormone receptor; SRC, steroid receptor coactivator; TR, thyroid hormone receptor; TRE, thyroid hormone receptor response element; TSA, trichostatin A. Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community. Ishii et al. • Aberrant Histone Deacetylation in RTH cleosome histones. Additionally, some p160/SRC family members have intrinsic HAT activity, further supporting a functional role for these factors in chromatin modification (6, 7). It was reported that the ordered recruitment and release of coactivators are important for transcriptional activation (8). In contrast to the mechanism of positive regulation, the mechanism of trans-repression of the hypothalamic TRH and pituitary TSH subunit genes remains poorly understood. It remains to be elucidated whether direct binding of TR to DNA is necessary for the negative regulation. A detailed analysis of TR knockout mice demonstrated that at least the ␤ isoform of the TR (TR␤) has a key role in the negative feedback regulation of the hypothalamic-pituitarythyroid axis (9). Although a number of distinct mechanisms for TR␤-mediated negative regulation by thyroid hormone have been proposed, several investigators reported that coregulators including NCoR, SMRT, and SRC-1 are involved in the negative regulation by thyroid hormone and the dominant-negative effect of the mutant TR observed in RTH patients. It is of interest how such cofactors affect histone acetylation status and chromatin structure in the negative gene regulation by thyroid hormone. In the present study, we first report a novel RTH mutant, F455S, characterized using conventional molecular methods including transient transfection analysis, glutathione-S-transferase (GST) pull-down assay, and EMSA. In addition, to investigate the chromatin structure, we established cell lines stably expressing the TRH gene, a typical gene negatively regulated by thyroid hormone, together with the wild-type or mutant TR, and then performed ChIP analysis. We found that transcriptional repression by thyroid hormone of the TRH gene is associated with rapid local histone deacetylation. T (...truncated)