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

Abstract

Previously we reported that the negative regulation of the TSHbeta gene by T(3) 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 TSHbeta inhibition. The negative regulatory element (NRE), which had been reported to mediate T(3)-bound TR (T(3)-TR)-dependent inhibition, is dispensable, because deletion or mutation of NRE did not impair suppression. The reporter construct, TSHbeta-D4-chloramphenicol acetyltransferase, which possesses only the binding sites for Pit1 and GATA2, was activated by GATA2 alone, and this transactivation was specifically inhibited by T(3)-TR. The Zn finger region of GATA2 interacts with the DNA-binding domain of TR in a T(3)-independent manner. The suppression by T(3)-TR was impaired by overexpression of a dominant-negative type TR-associated protein (TRAP) 220, an N- and C-terminal deletion construct, indicating the participation of TRAP220. Chromatin immunoprecipitation assays with a thyrotroph cell line, TalphaT1, revealed that T(3) 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 TSHbeta gene by T(3)-TR we report that 1) GATA2 is the major transcriptional activator of the TSHbeta 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.

The role of thyroid hormone receptor DNA binding in negative thyroid hormone-mediated gene transcription

“…The regulation of TSH in the pituitary might be additionally regulated by interferance of TR with limiting amounts of pituitary-specific transcription factors, such as Pit1 and GATA2 (Sanchez-Pacheco et al 1995, Nakano et al 2004. A direct DNA binding of TR has been shown to be dispensable for the negative regulation of the TSHb gene whereas a critical interaction between the DBD of TR with GATA2 has been reported recently (Matsushita et al 2007). Positive regulation of mGPDH is accomplished by a welldescribed TRE that is located close to the transcriptional start site (Weitzel et al 2001b).…”

“…TR has been shown to bind other transcription factors and cofactors via its DBD (Poirier et al 2005, Matsushita et al 2007). An interesting interaction is the reported interaction of TR with cAMP response element binding protein (CREB), which is ensured via the DBD of TR (Mendez-Pertuz et al 2003, Furumoto et al 2005.…”

Novel aspects of T3 actions on GH and TSH synthesis and secretion: physiological implications

“…However, recent studies have shown that the negative regulation of human TSHB gene exerted by T 3 is independent of nTRE (Matsushita et al 2007, Matsunaga et al 2015.…”

Negative Regulation of the Thyrotropin β Gene by Thyroid Hormone

“…However, the function of the DBD of NHR is not limited to DNA recognition. It is known that the DBD also interacts with other DNA-binding transcription factors including NFkB (De Bosscher et al, 2003;Kalaitzidis and Gilmore, 2005;Kalkhoven et al, 1996;Ray and Prefontaine, 1994;Scheinman et al, 1995;Stein and Yang, 1995;Tao et al, 2001;Wissink et al, 1997), AP-1 (typically Jun/Fos heterodimers) (De Bosscher et al, 2001;Heck et al, 1994;Lopez et al, 1993;Schule et al, 1990;Webb et al, 1995), Nur77 (Martens et al, 2005) and GATA family transcription factors (Clabby et al, 2003;Matsushita et al, 2007), resulting in their inhibition by NHRs in a ligand-dependent fashion.…”

“…Thus, while mutation of the TR DBD abrogates the negative regulation of the TSH gene by T3, it does not always imply a direct interaction of the TR with DNA. Of note, there are ligand/receptor specificities in the repression by liganded NHRs via the tethering mechanism (Caldenhoven et al, 1995;Liden et al, 1997;Matsushita et al, 2007). Moreover, dimer formation is not always required for ligand-dependent inhibition via the tethering mechanism.…”