Functions of PIT1 in GATA2-dependent transactivation of the thyrotropin β promoter

Abstract

Thyrotropin (TSH) is a heterodimer consisting of a and b chains, and the b chain (TSHb) is specific to TSH. The coexistence of two transcription factors, PIT1 and GATA2, is known to be essential for TSHb expression. Using kidneyderived CV1 cells, we investigated the role of PIT1 in the expression of Tshb gene. GATA2 Zn finger domain, which is known to recognize GATA-responsive elements (GATA-REs), is essential for cooperation by PIT1. Transactivation of TSHb promoter requires PIT1-binding site upstream to GATA-REs (PIT1-US), and the spacing between PIT1-US and GATA-REs strictly determines the cooperation between PIT1 and GATA2. Moreover, truncation of the sequence downstream to GATA-REs enabled GATA2 to transactivate the TSHb promoter without PIT1. The deleted region (nt K82/K52) designated as a suppressor region (SR) was considered to inhibit transactivation by GATA2. The cooperation of PIT1 with GATA2 was not conventional synergism but rather counteracted SR-induced suppression (derepression). The minimal sequence for SR was mapped to the 9 bp sequence downstream to GATA-REs. Electrophoretic mobility shift assay suggested that some nuclear factor exists in CV1 cells, which binds with SR and this interaction was blocked by recombinant PIT1. Our study indicates that major activator for the TSHb promoter is GATA2 and that PIT1 protects the function of GATA2 from the inhibition by SR-binding protein.

Negative Regulation of the Thyrotropin β Gene by Thyroid Hormone

“…First, they showed that as long as T3 is at low concentrations or absent, expression of the TSH gene is maintained by Pit1 and GATA2 without stimulation by TRH signaling (Fig. 7(a)) (Gordon et al, 1997;Kashiwabara et al, 2009;Ohba et al, 2011). In agreement with this, the signaling and the number of TSH -positive cells in the pituitary of TRH-deficient mice were comparable with those of wild-type mice (Shibusawa et al, 2000).…”

“…Thus, cooperation of Pit1 with GATA2 is not synergistic, but Pit1 protects GATA2 from inhibition by SR (de-repression). These findings not only provide an insight as to why TSH expression is restricted in thyrotrophs where Pit1 and GATA2 coexist, but also imply that the true activator that drives the TSH promoter activity is GATA2 but not Pit1 (Kashiwabara et al, 2009). …”

“…These findings suggest that direct DNA binding of TR is unnecessary and that the mechanism for T3-dependent inhibition may be mediated by the crosstalk of T3/TR with Pit1 or GATA2 (Matsushita et al, 2007). As mentioned above, the true activator that drives the TSH promoter is GATA2 but not Pit1 (Kashiwabara et al, 2009) and the deletion of SR enables GATA2 to transactivate the TSH promoter without Pit1. Using the reporter gene lacking for SR, Matsushita et al (Matsushita et al, 2007) found that T3/TR 2 inhibits the transactivation by GATA2 alone.…”

“…Subsequent analysis with various transgenic mice revealed that co-expression of Pit1 and GATA2 is crucial for the differentiation of thyrotrophs (Dasen et al, 1999). Kashiwabara et al (Kashiwabara et al, 2009) reported that the co-operation of Pit1 with GATA2 is strictly determined by the number of nucleotides between the Pit1-US and GATA-REs (Fig 4), and suggested the possibility that a configuration of the Pit1-US and GATA-REs may be critical for the recruitment of CBP/p300 (Fig 6). Fig.…”

“…As shown in Fig. 4, a functional Pit1-binding site is included in this region (Haugen et al, 1993) and was designated as Pit1-US (Kashiwabara et al, 2009). Interestingly, comparison of the pattern of DNA foot printing using nuclear extracts from TtT97 cells and that from GH3 cells revealed that this promoter region also has binding sites for the transcription factor, GATA2 (Fig 4 and 5B) (Gordon et al, 1997), which was originally identified to be involved in the gene regulation of a hematopoietic cell lineage .…”