【作者】 刘春艳；

【导师】 黄百渠； 王丽；

【作者基本信息】 东北师范大学 ， 细胞生物学， 2004， 博士

【摘要】 IL-5主要由Th2类细胞产生，在过敏性哮喘等过敏性疾病的发生过程中起着关键的作用。转录辅因子CBP／p300本身具有组蛋白乙酰转移酶(HAT)活性，参与许多基因的转录调控过程。腺病毒E1A癌蛋白能与CBP／p300蛋白结合并抑制其活性。本文中，我们分析了E1A蛋白在IL-5基因启动子／荧光素酶报告基因表达调控中的作用，结果表明E1A蛋白能抑制PMA／离子霉素激活的和转录因子C／EBPβ介导的IL-5基因启动子／荧光素酶报告基因的活性。而突变体E1AA2-36蛋白因不能与CBP／p300蛋白结合而不能抑制IL-5基因启动子／荧光素酶报告基因的表达。我们发现CBP／p300不仅能促进IL-5启动子报告基因质粒的活性，而且还能提高内源IL-5 mRNA的表达水平。另外，转录辅因子CBP／p300可分别和转录因子C／EBPB、NF-AT和AP-1协同地激活IL-5基因启动子／荧光素酶报告基因的表达。在CBP／p300促进IL-5启动子报告基因表达的过程中CBP／p300的HAT活性发挥了十分重要的作用。通过染色质免疫沉淀实验，我们还发现CBP／p300能够使IL-5启动子处的组蛋白高乙酰化。这样产生一个相对开放的染色质构型，从而促进转录机器与DNA的结合，进而促进转录。另外，CBP／p300还能将促进IL-5基因表达的转录因子C／EBP乙酰化，从而改变其反式激活能力。我们的结果第一次揭示了组蛋白乙酰转移酶CBP／p300参与IL-5基因的表达调控，并对其机制进行了初步的探讨，为深入研究IL-5基因表达调控的机制奠定了基础。更多还原

【Abstract】 Interleukin-5 (IL-5), expressed primarily by type-2 T helper (Th2) cells, plays an essential role in the development of allergic diseases includeing allergic asthma. Histone acetyltransferase CBP/p300 remodels chromatin by acetylating histones resulting in open structure of chromatin and active transcription. Adenovirus protein E1A inhibits the activity of CBP/p300. In this study, we analyzed the effects of E1A on IL-5 gene promoter/luciferase reporter activity. The results showed that El A protein inhibited the activity of PMA/ionomycin-stimulated IL-5 gene promoter/luciferase reporter construct. In contrast, overexpression of the CBP/p300-binding defective El A A2-36 protein did not inhibit IL-5 gene promoter activity. El A protein can modulate CBP/p300 function to activate the transcription of IL-5 gene promoter/luciferase reporter plasmid. Transcriptional coactivator CBP/p300 and transcription factors C/EBPp, NF-AT, and c-Fos synergistically activated IL-5 promoter. Furthermore, we found that ectopic expression of p300 increased endogenous IL-5 mRNA expression. The histone acetyltransferase activity of CBP/p300 was required to activate IL-5 expression. In chromatin immunoprecipitation assay (ChIP), we found that CBP/p300 could acetylate histones on IL-5 promoter, resulting in increased accessibility of the open chromatin to the transcriptional machinery and therefore promoted gene transcription. Moreover, we showed that CBP/p300 acetylated the transcription factor C/EBP, resulting in an increased transactivation activity of the C/EBP. These data demonstrated for the first time that histone acetyltransferase CBP/p300 was involved in the activation of IL-5 gene expression and the HAT activity was important in regulation of IL-5 expression. This study provides further insight into the mechanisms of transcriptional regulation of IL-5 gene.更多还原