The study of the negative regulation of thyrotropin b by thyroid hormone and its receptor

有人推测，在TSHβ启动子中，直接下游到转录起始点的DMA序列可能在介导甲状腺激素(T3)及其受体(TR)驱动的负调控中发挥关键作用(Chin，W.W.等人(1993))。进展霍姆。结果48：393-414)。我们将该区域命名为负性调控元件(NRE)，并报道了组蛋白脱乙酰基酶(HDAC2)可能以一种依赖于T3的方式被TR招募(Sasaki S等人)。(1999)EMBO J.18(19)：5389-5398)。对NRE序列的检测预测了高迁移率族(HMG)蛋白与NRE结合的可能性，并通过凝胶漂移实验证实了HMG蛋白确实与NRE结合在一起。此外，我们还用简并的RT-PCR方法鉴定了一个HMG蛋白sox11。用特异性抗体进行凝胶筛选实验，证实了Sox11的存在。此外，我们还建立了一套检测猴肾细胞系CV1中TSHβ基因负调控的实验系统。该实验系统的建立使我们能够更容易地分析TSHβ基因负调控的精确机制。利用该系统，我们发现Sox11的过表达消除了T3和TR对TSHβ基因的负调控。然而，令人惊讶的是，这一系统显示，即使在NRE被摧毁后，负调控仍保持不变。进一步的研究表明，转录因子PIT1和GATA2可能不仅是激活TSHβ启动子的关键因素，也可能是配体受体负调控的主要靶点。

It has been postulated that, in the TSH beta promoter, a DMA sequence immediately down stream to the transcription start site may have critical role to mediate the negative regulation driven by thyroid hormone (T3) and its receptor (TR) (Chin, W. W. et al.(1993) Recent. Prog.Horm. Res. 48 : 393-414). We designated this region as Negative Regulatory Element (NRE) and reported that histone deacetylase (HDAC) 2 may be recruited by TR in a T3 dependent manner (Sasaki S, et al. (1999) EMBO J. 18(19):5389-5398). The inspection of NRE sequence predicted the possibility that high mobility group (HMG) proteins may bind NRE and modulate the function of T3 bound TR Using gel shift assay, we confirmed that HMG protein really bind to NRE in T alpha T1 cell which has been established recently as thyrotroph cell line. In addition, we identified a HMG protein, Sox11, using RT-PCR with degenerated primers. The existence of Sox11 was confirmed by the gel sift assay with specific antibody. Furthermore, we established an experimental system in which we can detect the negative regulation of TSH beta gene in monkey kidney cell line, CV 1. The establishment of this experimental system enabled us to analyze the precise mechanisms of the negative regulation of TSH beta gene much easier than before. Using this system, we found that the overexpression of Sox11 eliminated the negative regulation of TSH beta gene by T3 and TR. Surprisingly, however, this system revealed that the negative regulation was maintained even after the destruction of NRE. Further studies suggested the possibility that the transcription factors, Pit1 and GATA2, may not only be critical to activate TSH beta promoter but also the main target of negative regulation by liganded TR.