Pit-1 and GATA-2 interact and functionally cooperate to activate the thyrotropin beta-subunit promoter
Pit-1 and GATA-2 interact and functionally cooperate to activate the thyrotropin beta-subunit promoter
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DOI：
10.1074/jbc.272.39.24339
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发表时间：
1997-09-26
影响因子：
4.8
通讯作者：
Ridgway, EC
中科院分区：
文献类型：
--
作者：
Gordon, DF;Lewis, SR;Ridgway, EC
促甲状腺激素(TSH)β亚单位基因在垂体中的细胞特异性表达的分子决定因素尚不清楚，小鼠TSHβ启动子(-133至-88)的P1区与Pit-1和一个额外的50 kDa因子在邻近位置相互作用，类似于共识的GATA结合位点。Northern和Western印迹分析表明，在TTT-97甲状腺肿瘤中存在GATA-2转录本和蛋白。在凝胶迁移率改变分析中，观察到TTT-97核抽提物和GATA-2在COS细胞中表达的迁徙复合体，该复合体与P1区DNA探针具有结合特异性，并可被GATA-2抗体破坏，当Pit-1和GATA-2结合时，观察到较慢的迁移复合体，表明蛋白质-DNA相互作用。将Pit-1和GATA-2共转染到CV-1细胞中，协同刺激小鼠TSHβ启动子活性8.5倍，而单独使用这两个因子的影响很小，取消这一功能刺激作用的突变被映射到P1区域。最后，我们发现GATA-2在溶液中直接与Pit-1相互作用，综上所述，这些数据证明了同源异型盒和锌指因子之间的功能协同和物理相互作用，并为甲状腺激素特异性基因表达的转录机制提供了深入的见解。
The molecular determinants governing cell-specific expression of the thyrotropin (TSH) beta-subunit gene in pituitary thyrotropes are not well understood, The P1 region of the mouse TSH beta promoter (-133 to -88) region interacts with Pit-1 and an additional 50-kDa factor at an adjacent site that resembles a consensus GATA binding site. Northern and Western blot assays demonstrated the presence of GATA-2 transcripts and protein in TtT-97 thyrotropic tumors, In electrophoretic mobility shift assays, a comigrating complex was observed with both TtT-97 nuclear extracts and GATA-2 expressed in COS cells, The complex demonstrated binding specificity to the P1 region DNA probe and could be disrupted by a GATA-2 antibody, When both Pit-1 and GATA-2 were combined, a slower migrating complex, indicative of a ternary protein-DNA interaction was observed. Cotransfection of both Pit-1 and GATA-2 into CV-1 cells synergistically stimulated mouse TSH beta promoter activity 8.5-fold, while each factor alone had a minimal effect, Mutations that abrogated this functional stimulatory effect mapped to the P1 region. Finally, we show that GATA-2 directly interacts with Pit-1 in solution, In summary, these data demonstrate functional synergy and physical interaction between homeobox and zinc finger factors and provide insights into the transcriptional mechanisms of thyrotrope-specific gene expression.