Negative Regulation of TSHα Target Gene by Thyroid Hormone Involves Histone Acetylation and Corepressor Complex Dissociation

ORIGINAL RESEARCH Negative Regulation of TSH␣ Target Gene by Thyroid Hormone Involves Histone Acetylation and Corepressor Complex Dissociation Dongqing Wang,* Xianmin Xia,* Ying Liu, Alexis Oetting, Robert L. Walker, Yuelin Zhu, Paul Meltzer, Philip A. Cole, Yun-Bo Shi, and Paul M. Yen Endocrinology Division (D.W., X.X., P.M.Y.), Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224; Department of Pharmacology (P.A.C.), Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; Developmental Endocrinology Branch (Y.L.), and Laboratory of Gene Regulation and Development (A.O., Y.-B.S.), National Institutes of Child Health and Human Development, National Institutes of Health and Genetics Branch (R.L.W., Y.Z., P.M.), National Cancer Institute, Bethesda, Maryland 20892 Currently, little is known about histone modifications and molecular mechanisms of negatively regulated transcription. In pituitary cells, thyroid hormone (T3) decreased transcription, and surprisingly increased histone acetylation, of TSH␣ promoter. This increase was mediated directly by thyroid hormone receptor. Histone acetylation of H3K9 and H3K18 sites, two modifications usually associated with transcriptional activation, occur in negative regulation of TSH␣ promoter. T3 also caused release of a corepressor complex composed of histone deacetylase 3 (HDAC3), transducin ␤-like protein 1, and nuclear receptor coprepressor (NCoR)/ silencing mediator for retinoic and thyroid hormone receptor from TSH␣ promoter in chromatin immunoprecipitation assays. NCoR and HDAC3 overexpression selectively increased ligand-independent basal transcription. Two histone acetyltransferase inhibitors increased overall transcription but did not abrogate negative regulation or NCoR/HDAC3 complex release by T3. Chromatin immunoprecipitation analyses of an endogenous positively regulated target gene showed increased histone acetylation and corepressor complex release with T3 treatment. Finally, microarray analyses suggested there is a subset of negatively regulated genes with increased histone acetylation. These findings demonstrate the critical role of NCoR/HDAC3 complex in negative regulation of TSH␣ gene expression and show that similar complexes and overlapping epigenetic modifications can participate in both negative and positive transcriptional regulation. (Molecular Endocrinology 23: 600 – 609, 2009) T hyroid hormone receptors (TRs) belong to a superfamily of nuclear hormone receptors that act as ligand-regulatable transcription factors (1, 2). There are two major TR isoforms, TR␣ and TR␤, encoded on separate genes. TRs bind to thyroid hormone response elements in the promoters of target genes to regulate their transcription. In positively regulated target genes, unliganded TRs bind to corepressors such as nuclear receptor corepressor (NCoR) or silencing mediator for retinoic and thyroid hormone receptors (SMRT) that form corepressor complexes containing transducin ␤-like protein 1 (TBL1) and histone deacetylase 3 (HDAC3), and mediate basal transcriptional repression by unliganded thyroid hormone receptor in positively regulated target genes (3–5). In the presence of T3, corepressor complexes are released from liganded TRs that, in turn, associate with coactivator complexes that contain steroid receptor coactivator (SRCs), cAMP response element-binding protein (CREB)-binding protein (CBP), and P/CAF. These complexes cause increased histone acetylation near the TRE of the promoter (1, 2, 6). ATP-dependent chromatin remodeling complexes similar to the SWI/SNF family in yeast that contains the adenosine triphosphatase subunit, Brahma-related gene 1, also are recruited to the promoter (7, 8) and critical for transcrip- ISSN Print 0888-8809 ISSN Online 1944-9917 Printed in U.S.A. Copyright © 2009 by The Endocrine Society doi: 10.1210/me.2008-0389 Received October 14, 2008. Accepted January 27, 2009. First Published Online February 5, 2009 * D.W. and X.X. contributed equally to this work. Abbreviations: CBP, CREB-binding protein; ChIP, chromatin immunoprecipitation; CoA, coenzyme A; CREB, cAMP response element binding protein; FBS, fetal bovine serum; HAT, histone acetyl transferase; HDAC3, histone deacetylase 3; NCoR, nuclear receptor corepressor; PEPCK, phosphoenolpyruvate carboxykinase; RNA Pol II, RNA polymerase II; SMRT, silencing mediator for retinoic and thyroid hormone receptor; SRC, steroid receptor coactivator; TBL1, transducin ␤-like protein 1; TR, thyroid hormone receptor; TSA, trichostatin A. 600 mend.endojournals.org Mol Endocrinol, May 2009, 23(5):600 – 609 Mol Endocrinol, May 2009, 23(5):600 – 609 Results Establishment of a stable rat pituitary GH3 cell line We generated a stable rat pituitary GH3 cell line, ␣-23, containing the human TSH␣ promoter (⫺840 to ⫹1) in tandem with a luciferase reporter cDNA to study negative regulation by T3 in a native chromatin context. This construct was shown 601 A Relative luciferase activity % of control previously to be sufficient for negatively regulated transcription by T3 in transient transfection studies (24). As expected, ␣-23 cells exhibited a dose-dependent decrease in luciferase activity by T3 (Fig. 1A). A decrease was observed at 1 nM T3 and a maximal decrease (⬃90%) was seen at 0.1 ␮M T3 (Fig. 1A). These results demonstrate that the stably integrated TSH␣ promoter is negatively regulated by T3 in the GH3 cells, and mimics the ligand-dependent negative-feedback regulation of the TSH␣ gene by the pituitary in vivo. We found minimal expression of endogenous TSH␣ gene in the absence or presence of T3 by microarray analyses and quantitative RT-PCR so it apparently is silenced in parent GH3 cells. 120 100 80 * 60 40 * * * 10 100 1000 20 0 0 1 0.1 120 100 80 * 60 * 40 20 l 1 3 ( co nt ro T C * * TS A uM ) 0 * ** * * * * * * * 50 ng TS /m A1 l 00 n g/ TS m A1 l 50 T ng 3+ 50 /m ng l /m T 3+ lT 10 S 0n A g/ T m 3+ lT 15 S 0n A g/ m lT SA B Relative luciferase activity % of control T3 (nM) Luciferase mRNA level normalized to beta-actin tional activation. Another major complex, Mediator complex, also can interact with the promoter and serves to recruit RNA polymerase II (RNA pol II) (9 –11). Recently, chromatin immunoprecipitation (ChIP) studies have suggested that liganded TRs and nuclear hormone receptors recruit coactivators in a cyclical pattern on the promoters of some target genes (12–16). In contrast to positively regulated target genes, negatively regulated genes can be stimulated in the absence of T3 and decreased by its presence. The mechanism(s) for negative transcriptional regulation by T3 is not well understood; however, corepressors and coactivators may be involved. NCoR and SMRT can increase basal transcription of some target genes in the absence of T3 (17–20). Coactivators also can play an apparently paradoxical role in T3-dependent negative regulation of s (...truncated)