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

据推测，在TSH β启动子中，一个直接下游到转录起始位点的DMA序列可能在介导甲状腺激素（T3）及其受体（TR）驱动的负调控中起关键作用（Chin, w.w. et al.(1993)）。Prog.Horm。Res. 48: 393-414)。我们将该区域指定为负调控元件（NRE），并报道了组蛋白去乙酰化酶（HDAC） 2可能以T3依赖的方式被TR募集(Sasaki S, et al. （1999) EMBO J. 18(19):5389-5398）。通过对NRE序列的检测，预测了高迁移率组（high mobility group， HMG）蛋白与NRE结合并调节T3结合的TR功能的可能性。通过凝胶移位实验，我们证实了HMG蛋白确实与新近建立的甲状腺营养细胞系T α T1细胞NRE结合。此外，我们使用退化引物RT-PCR鉴定了HMG蛋白Sox11。用特异性抗体凝胶筛选法证实Sox11的存在。此外，我们还建立了一个实验系统来检测猴肾细胞株CV 1中TSH β基因的负调控。该实验系统的建立使我们比以往更容易分析TSH β基因负调控的精确机制。通过该系统，我们发现Sox11的过表达消除了T3和TR对TSH β基因的负调控，然而令人惊讶的是，该系统显示即使在NRE被破坏后，这种负调控仍然保持。进一步的研究表明，转录因子Pit1和GATA2可能不仅是激活TSH β启动子的关键，也是配体TR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.