Essential Role of GATA2 in the Negative Regulation of Thyrotropin β Gene by Thyroid Hormone and Its Receptors

0888-8809/07/$15.00/0 Printed in U.S.A. Molecular Endocrinology 21(4):865–884 Copyright © 2007 by The Endocrine Society doi: 10.1210/me.2006-0208 Essential Role of GATA2 in the Negative Regulation of Thyrotropin ␤ Gene by Thyroid Hormone and Its Receptors Akio Matsushita, Shigekazu Sasaki, Yumiko Kashiwabara, Koji Nagayama, Kenji Ohba, Hiroyuki Iwaki, Hiroko Misawa, Keiko Ishizuka, and Hirotoshi Nakamura Previously we reported that the negative regulation of the TSH␤ gene by T3 and its receptor [thyroid hormone receptor (TR)] is observed in CV1 cells when GATA2 and Pit1 are introduced. Using this system, we further studied the mechanism of TSH␤ inhibition. The negative regulatory element (NRE), which had been reported to mediate T3-bound TR (T3-TR)-dependent inhibition, is dispensable, because deletion or mutation of NRE did not impair suppression. The reporter construct, TSH␤-D4chloramphenicol acetyltransferase, which possesses only the binding sites for Pit1 and GATA2, was activated by GATA2 alone, and this transactivation was specifically inhibited by T3-TR. The Zn finger region of GATA2 interacts with the DNAbinding domain of TR in a T3-independent manner. The suppression by T3-TR was impaired by overexpression of a dominant-negative type TR-asso- ciated protein (TRAP) 220, an N- and C-terminal deletion construct, indicating the participation of TRAP220. Chromatin immunoprecipitation assays with a thyrotroph cell line, T␣T1, revealed that T3 treatment recruited histone deacetylase 3, reduced the acetylation of histone H4, and caused the dissociation of TRAP220 within 15–30 min. The reduction of histone H4 acetylation was transient, whereas the dissociation of TRAP220 persisted for a longer period. In the negative regulation of the TSH␤ gene by T3-TR we report that 1) GATA2 is the major transcriptional activator of the TSH␤ gene, 2) the putative NRE previously reported is not required, 3) TR-DNA-binding domain directly interacts with the Zn finger region of GATA2, and 4) histone deacetylation and TRAP220 dissociation are important. (Molecular Endocrinology 21: 865–884, 2007) SH IS A HETERODIMER consisting of ␣- and ␤-chains. The ␤-chain (TSH␤) is specific to TSH, whereas the ␣-chain (TSH␣) is common to all glycoprotein hormones. Transcription for both TSH␣ and ␤ genes is known to be repressed by thyroid hormone (T3) in thyrotrophs (1). The effect of T3 is mainly medi- ated through thyroid hormone receptors (TRs), which are encoded by TR␣ and -␤ genes. The TR␣ locus generates mainly TR␣l and -␣2 through alternative splicing, whereas different promoters in the TR␤ locus generate TR␤l and ␤2. In patients with resistance to thyroid hormone who exhibit the syndrome of the inappropriate secretion of TSH (2), abnormalities have been identified exclusively in the TR␤, not the TR␣, gene. In agreement, mice lacking the TR␤ gene display inappropriate secretion of TSH (3), whereas no apparent alteration of TSH␤ expression is detected in TR␣ null mice (4), and mice having neither TR␣ nor TR␤ show dramatic overexpression of the TSH␤ gene (5). We recently reported that TR␤2 is the major TR isoform expressed in T␣T1, a thyrotroph cell line (6, 7). This suggests that TR␤2 plays a central role in the T3-dependent negative regulation of TSH genes. Moreover, TR␤2 null mice were reported to exhibit central resistance to thyroid hormone not only in the pituitary but also in the hypothalamus (8, 9). For transcriptional activation by T3 in positive regulation, TR heterodimerizes with the retinoid X receptor (RXR) on the positive T3-responsive element (positive TRE) (10). Unliganded TR recruits corepressors including the nuclear receptor corepressor (NCoR) and silencing mediator of retinoic acid and thyroid hormone receptors (SMRT), resulting in the association of his- T First Published Online January 23, 2007 Abbreviations: Ad4BP, Adrenal 4-binding protein; AF-2, activation function-2; CBP, CREB-binding protein; ChIP, chromatin immunoprecipitation; CTD, carboxyl-terminal domain; DBD, DNA-binding domain; dnTRAP220, dominantnegative TRAP220; DOC, deoxycholate; E2, estrogen; ER, estrogen receptor; FOG, Friend of GATA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GATA-RE, GATA-responsive element; GATA2-Zf, Zn-finger domain of GATA2; GR, glucocorticoid receptor; GST, glutathione S-transferase; GST-Zf, Zn finger domain of GST; HAT, histone acetyltransferase; HDAC, histone deacetylase; NCoR, nuclear receptor corepressor; NF-␬B, nuclear factor-␬B; NRE, negative regulatory element; nTRE, negative TRE; RAR, retinoic acid receptor; SF-1, steroidogenic factor 1; SMCC, SRB/MED-containing cofactor; SMRT, silencing mediator of retinoic acid and thyroid hormone receptor; SRC-1, steroid receptor coactivator 1; TR, thyroid hormone receptor; TRAP, TR-associated protein; TRE, T3-responsive element; VDR, vitamin D receptor. Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community. 865 Second Division, Department of Internal Medicine, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka 431-3192, Japan 866 Mol Endocrinol, April 2007, 21(4):865–884 to the thyrotroph lineage. In our previous study, we reported that negative regulation of the TSH␤ promoter is easily detected even in CV1 cells when TR is coexpressed with Pit1 and GATA2 (6). This observation suggested that thyrotroph-specific factors other than Pit1 and GATA2 may not be essential to mediate T3-TR-dependent negative regulation of the TSH␤ gene. Receptor-ligand specificity was observed as in the positive regulation of T3-target genes (6). Deletion analyses of TR␤ in CV1 cells revealed that the DNAbinding domain (DBD) of TR is indispensable for T3dependent negative regulation, consistent with the findings of in vivo analysis (30) and also the study of the TSH␣ gene (31). On the other hand, mutant TR␤2, E457A, which has a glutamic acid to alanine substitution in AF-2, fails to interact with coactivators. Recently, Ortiga-Carvalho et al. (32) reported that TSH secretion was elevated in homozygotic mice with E457A mutation. Thus, T3-dependent inhibition may require an intact DBD and AF-2 domain. The reporter assay system using CV1 cells provides an ideal experimental platform with which to study the difference between positive and negative regulation by T3, because the CV1 cell line is one of the cultured cells most frequently used for the study of positive regulation. Using CV1 cells cotransfected with GATA2 and Pit1, we have reevaluated the function of putative NRE in the TSH␤ promoter and found that negative regulation by T3-TR was preserved after complete destruction of NRE. The TSH␤-D4-chloramphenicol acetyltransferase (CAT) construct containing only the binding sites for Pit1 and GATA2 was stimulated by GATA2 alone without Pit1, and this transactivation was specifically inhibited by T3-TR. We found that GATA2 asso (...truncated)