探查p53和Sp1在組蛋白去乙醯酶抑制劑誘導EB病毒再活化過程中調控轉活化子Zta表現之機制

Abstract

在許多與EB病毒相關的疾病組織中，觀察到p53 過量累積的情形。此外，在這些病患的血清中又可偵測到較高力價的抗EB病毒溶裂期產物之抗體，因此本論文欲探討p53 對於誘導EB病毒再活化進入溶裂期過程中所扮演的角色。 在此研究中，本實驗室先前的研究，已證實在去乙醯酶抑制劑 (HDAC inhibitor)引起鼻咽癌上皮細胞株之EB病毒再活化能力的過程中，p53是重要的因子。進一步研究結果顯示，p53在促使EB病毒再活化之重要樞杻的Zta蛋白質表現過程中扮演重要角色。利用基因減弱試驗 (knockdown) 和報導者分析 (reporter assay)，證實p53在活化Zta啟動子的過程扮演關鍵性因子。後續的研究著重於釐清在調控Zta啟動子的分子機制。本論文中，進一步利用site-directed mutagenesis的方法構築一系列片段型和突變型Zta啟動子報導質體，經過報導者分析試驗，定位出足以活化Zta啟動子的重要反應區域，ZID區域。透過DNA親和性沉澱試驗 (DNA affinity precipitation assay, DAPA) 證實p53可以結合於此Zta啟動子重要反應區域，ZID區域。本研究結果亦發現另一轉錄因子──Sp1，同樣可以結合於此特定的DNA區域。此外，進一步利用共同免疫沉澱法 (Co-Immunoprecipitation) 證實p53和Sp1兩個轉錄因子之間具有交互作用的現象。更甚者，使用Sp1的常用抑制劑以及突變型的Sp1作為競爭野生型Sp1的作用，還有Sp1基因減弱試驗，証明Sp1在Zta蛋白質的表現，甚至是Zta啟動子的活化亦扮演要角。 總結，在HDAC抑制劑誘導EB病毒再活化過程中，確認p53和Sp1皆在Zta啟動子層面扮演重要的調控角色，故可以推測p53和Sp1也許以協同調控之方式活化Zta啟動子。本論文研究，試圖窺探p53和Sp1在Zta啟動子上的分子機制，也為p53於病毒和宿主交互關係中提供一個嶄新的視野。

The previous studies reported that p53 usually accumulates in some EBV-infected cells and that high titer antibodies against EBV lytic products are commonly observed in the sera of Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) individuals, suggesting that there is a correlation between p53 and EBV reactivation. Indeed, we have demonstrated that p53 is a critical factor for HDAC inhibitors-induced EBV reactivation in NPC cells. In brief, p53 is especially critical for the expression of Zta transactivator, an immediate early gene which plays vital roles in initiating viral replication. By means of the siRNA approaches and reporter assay, we currently found certain critical events occur on Zta promoter (Zp) which is affected by p53. In this thesis, we try to further reveal the molecular mechanism how p53 regulates the expression of Zta at transcriptional level. Through serial deletion and mutation of the Zp reporter constructs, we defined a specific DNA region, ZID element, which is essential for Zp activation upon HDAC inhibitors treatment. Data from DNA affinity precipitation assay (DAPA) proved the binding of p53 on this specific DNA element, ZID element of Zp. Moreover, we found that another transcription factor, Sp1, also could associate with this specific DNA region. In addition, we demonstrated the interaction between p53 and Sp1 by Co-Immunoprecipitation (Co-IP). Furthermore, using the chemical inhibitor, the dominant negative form of Sp1, Sp1 specific siRNA as well as through genetic manipulation of this putative Sp1 site, we demonstrated the importance of Sp1 for Zp activity. In conclusion, both p53 and Sp1 act as important regulators on Zta promoter in the HDAC inhibitor-induced EBV reactivation. To our knowledge, this is the first report that p53 and Sp1 can interact with each other and bind on Zp promoter to transactivate its activity. This report could let us pry into the molecular behavior of p53 and Sp1 on Zta promoter, and it provides a novel insight into the role of p53 in virus-host interaction.