The HMG-I/Y-related protein p8 binds to p300 and Pax2 trans-activation domain-interacting protein to regulate the trans-activation activity of the Pax2A and Pax2B transcription factors on the glucagon gene promoter
The HMG-I/Y-related protein p8 binds to p300 and Pax2 trans-activation domain-interacting protein to regulate the trans-activation activity of the Pax2A and Pax2B transcription factors on the glucagon gene promoter
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DOI：
10.1074/jbc.m201657200
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发表时间：
2002-06-21
影响因子：
4.8
通讯作者：
Iovanna, JL
中科院分区：
文献类型：
--
作者：
Hoffmeister, A;Ropolo, A;Iovanna, JL
p8是一种核DNA结合蛋白，其被鉴定是因为其表达在响应于几种应激时被强烈激活。生物化学和生物物理学研究表明，尽管弱序列同源性p8是一个HMG-I/Y样蛋白，这表明p8可能参与转录调控。这里报告的结果强烈支持这一假设。使用下拉的方法，我们发现，p8与一般的共激活p300相互作用。我们还发现，类似于HMG蛋白，p300能够乙酰化重组p8在体外，虽然这种修饰的意义仍有待确定。然后，通过双杂交系统的筛选，使用p8作为诱饵，使我们能够确定Pax 2反式激活结构域相互作用蛋白（PTIP）作为p8的另一个合作伙伴。瞬时转染研究表明，PTIP是胰高血糖素基因启动子上Pax 2A和Pax 2B的反式激活活性的强抑制剂，选择胰高血糖素基因启动子作为模型是因为它是Pax 2A和Pax 2B转录因子的靶点。通过在产生胰高血糖素的InRIG 9细胞中共转染p8完全消除了这种作用，表明p8与PTIP的结合阻止了胰高血糖素基因启动子的抑制。这在不表达胰高血糖素的NIH 3 T3成纤维细胞中未观察到。最后，p8的表达增强了p300对胰高血糖素基因启动子的Pax 2A和Pax 2B反式激活的作用。这些观察结果表明，在胰高血糖素产生细胞中，p8是Pax 2A和Pax 2B激活胰高血糖素基因启动子的正辅因子，通过募集p300辅因子来增加Pax 2A和Pax 2B活性，并通过结合Pax 2相互作用蛋白PTIP来抑制其抑制。
p8 is a nuclear DNA-binding protein, which was identified because its expression is strongly activated in response to several stresses. Biochemical and biophysical studies revealed that despite a weak sequence homology p8 is an HMG-I/Y-like protein, suggesting that p8 may be involved in transcription regulation. Results reported here strongly support this hypothesis. Using a pull-down approach, we found that p8 interacts with the general co-activator p300. We also found that, similar to the HMG proteins, p300 was able to acetylate recombinant p8 in vitro, although the significance of such modification remains to be determined. Then a screening by the two-hybrid system, using p8 as bait, allowed us to identify the Pax2 trans-activation domain-interacting protein (PTIP) as another partner of p8. Transient transfection studies revealed that PTIP is a strong inhibitor of the trans-activation activities of Pax2A and Pax2B on the glucagon gene promoter, which was chosen as a model because it is a target of the Pax2A and Pax2B transcription factors. This effect is completely abolished by co-transfection of p8 in glucagon-producing InRIG9 cells, indicating that p8 binding to PTIP prevents inhibition of the glucagon gene promoter. This was not observed in NIH3T3 fibroblasts that do not express glucagon. Finally, expression of p8 enhances the effect of p300 on Pax2A and Pax2B transactivation of the glucagon gene promoter. These observations suggest that in glucagon-producing cells p8 is a positive cofactor of the activation of the glucagon gene promoter by Pax2A and Pax2B, both by recruiting the p300 cofactor to increase the Pax2A and Pax2B activities and by binding the Pax2,interacting protein PTIP to suppress its inhibition.