MED220/thyroid receptor-associated protein 220 functions as a transcriptional coactivator with Pit-1 and GATA-2 on the thyrotropin-β promoter in thyrotropes
MED220/thyroid receptor-associated protein 220 functions as a transcriptional coactivator with Pit-1 and GATA-2 on the thyrotropin-β promoter in thyrotropes
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DOI：
10.1210/me.2005-0115
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发表时间：
2006-05-01
影响因子：
--
通讯作者：
Ridgway, EC
中科院分区：
文献类型：
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作者：
Gordon, DF;Tucker, EA;Ridgway, EC
介体（MED） 220/甲状腺受体相关蛋白（TRAP） 220是一种与配体甲状腺/类固醇激素受体相互作用的转录介体。MED220单倍体不足的杂合子表现出甲状腺功能减退和TSH β转录减少，表明TSH β转录具有特定功能。我们之前证明了Pit-1和GATA-2可以结合近端TSH β启动子内的复合元件并协同激活转录。我们通过Northern和Western blot分析检测了TtT-97甲状腺组织中MED220的表达。CV-1细胞的共转染显示，单独使用Pit-1、GATA-2或MED220不能显著刺激TSH β启动子。然而，Pit-1加GATA-2导致10倍激活，显示出协同合作。MED220的滴定导致进一步的剂量依赖性刺激高达25倍，这是启动子特异性的。谷胱甘肽- s -转移酶相互作用研究表明，MED220或GATA-2分别与Pit-1的同源结构域结合，而MED220与GATA-2的锌指相互作用独立，但不与任何一端相互作用。MED220在其N端广泛区域与GATA-2和Pit-1相互作用。这些相互作用的区域对最大功能也很重要。共免疫沉淀证实了这三个因子在促甲状腺素和染色质中相互作用，免疫沉淀在体内占据近端TSH β启动子。因此，TSH β基因被三种促甲状腺激素转录因子的组合最大限度地激活，这些转录因子通过蛋白质- dna和蛋白质-蛋白质相互作用起作用。
Mediator (MED) 220/thyroid receptor-associated protein ( TRAP) 220 is a transcriptional mediator that interacts with liganded thyroid/steroid hormone receptors. MED220 haploinsufficient heterozygotes exhibited hypothyroidism and reduced TSH beta transcripts, suggesting a specific function for TSH beta transcription. We previously demonstrated that Pit-1 and GATA-2 can bind to a composite element within the proximal TSH beta promoter and synergistically activate transcription. We detected MED220 expression in TtT-97 thyrotropes by Northern and Western blot analysis. Cotransfections in CV-1 cells showed that Pit-1, GATA-2, or MED220 alone did not markedly stimulate the TSH beta promoter. However, Pit-1 plus GATA-2 resulted in an 10-fold activation, demonstrating synergistic cooperativity. Titration of MED220 resulted in a further dose-dependent stimulation up to 25-fold that was promoter specific. Glutathione-S-transferase interaction studies showed that MED220 or GATA-2 each bound the homeodomain of Pit-1, whereas MED220 interacted independently with each zinc finger of GATA-2 but not with either terminus. MED220 interacted with GATA-2 and Pit-1 over a broad region of its N terminus. These regions of interaction were also important for maximal function. Coimmunoprecipitation confirmed that all three factors can interact in thyrotropes and chromatin immunoprecipitation demonstrated in vivo occupancy on the proximal TSH beta promoter. Thus, the TSH beta gene is maximally activated by a combination of three thyrotrope transcription factors that act via both protein-DNA and protein-protein interactions.