梨形鞭毛蟲的E2F同源蛋白質對於Cyst Wall Protein 1和Tymidine kinase基因的轉錄調控之影響

A Novel E2F-related Protein Involved in Transcriptional Regulation of Cyst Wall Protein 1and thymidine kinase Genes in Giardia lamblia

黃昱璋(Yu-Chang Huang)

指導教授 ： 孫錦虹

國立臺灣大學/醫學院/微生物學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.00160

摘要

當真核細胞的細胞週期由G1 phase進入S phase時，許多與細胞生長以及DNA複製相關的基因會在這個時期被活化，這一時期的基因調控主要是由E2F家族轉錄因子所掌控。梨形鞭毛蟲在演化的分類上，為一個非常原始的真核生物。我們在梨形鞭毛蟲的基因組資料庫中進行搜尋，發現一個帶有E2F DNA結合區域的基因，稱之為Giardia e2f1 (ge2f1)。由胺基酸序列分析發現gE2F1帶有兩個E2F DNA結合區域，並且在兩個DNA binding domain中都具有重要的RRXYD motif，與近幾年發現人類的 E2F-7以及E2F-8相似。我發現ge2f1基因的mRNA量在囊體化時期比在滋養體時期下降。將HA標記接到gE2F1轉染梨形鞭毛蟲，利用免疫螢光染色法可發現在囊體化時期及滋養體時期gE2F1都位在於細胞核中。由EMSA實驗發現gE2F1蛋白質可和thymidine kinase的啟動子結合。經由突變序列分析，也發現gE2F1蛋白質的結合序列為TTTCCGCG，與人類的E2F家族蛋白質的結合特性類似，並且gE2F1與DNA結合能力，是不需要DP蛋白質的協助，符合帶有2個DNA 結合區域之E2F蛋白質特色。我也利用螢光酵素基因接上thymidine kinase基因啟動子，再利用cotransfection assay，可發現gE2F1大量表現時會使螢光酵素和訊息RNA表現量明顯上升，顯示gE2F1可以增加thymidine kinase 啟動子活性，因此對於thymidine kinase基因調控上可能為一個轉錄活化子。另外利用ChIP assay發現到gE2F1於in vivo下可以結合到thymidine kinase基因啟動子，也可以結合到cyst wall protein 1 基因啟動子，但是大量表現gE2F1卻讓cwp1的mRNA以及蛋白質表現量降低，並且讓cyst形成受阻，因此在cwp1基因調控當中gE2F1可能為一個轉錄抑制子。由實驗結果我們認為gE2F1蛋白質在梨形鞭毛蟲中，除了會造成DNA合成基因的活化，使之可以順利進入S-phase之外，另一方面也會抑制形成cyst相關的基因。

關鍵字

梨形鞭毛蟲 ； 轉錄調控

並列摘要

The E2F family transcription factors regulate the expression of genes involved in the progression of G1/S transition and DNA replication in higher eukaryotic cells. To understand whether E2F transcription factor is present in the primitive protozoan Giardia lamblia, we searched the Giardia lamblia genome database and found a gene harboring E2F DNA-binding domain (ge2f1). The gE2F1 contains two E2F DNA binding domains and both domains have the RRXYD motif which is conserved in all E2F family proteins. Recently the E2F proteins with two DNA binding-domains were found in human, mouse and plants. gE2F1 may belong to this new E2F subfamily.
Epitope-tagged gE2F1 was found to localize to nuclei. During encystation, the levels of the ge2f1 mRNA and protein decreased significantly. Recombinant gE2F1 specifically bound to DNA replication-related thymidine kinase gene promoter in vitro and in vivo. Mutation analysis revealed that TTTCCGCG sequence was required for the binding of gE2F1 and an A-rich sequence can enhance the binding of gE2F1. gE2F1 contains the important RRXYD motif for DNA binding and its binding sequences are similar to those of the known E2F family factors. Like Two DNA binding domain E2Fs in other eukaryotic systems, gE2F1 binds to DNA without the requirement of DP proteins. To further analyze the role of both DNA binding domains in gE2F1, we mutated each of the RRXYD motif in DNA binding domain. Each mutation resulted in a decrease of gE2F1 DNA binding ability, suggesting that both DNA binding domains in gE2F1 are important for its DNA binding. Using cotransfection assay, we found that overexpession of gE2F1 can increase thymidine kinase promoter activity about 30 fold in the luciferase assay and mutation of the TTTCCGCG sequence in the thymidine kinase promoter abolishs the activation, suggesting that gE2F1 acts as an activator in thymidine kinase gene regulation by binding to the positive cis-acting element TTTCCGCG. Using ChIP assay, we also found that gE2F1 was assciated with cwp1 promoter in vivo. In the gE2F1 constitutively expressed cell line, the levels of the cwp1 mRNA and protein gene decreased significantly. In cotransfection assay, overexpressed gE2F1 decreased cwp1 promoter activity. These results suggest that gE2F1 functions as a repressor in cwp1 gene transcriptional regulation. We also compared the cyst number between the gE2F1 overexpressing cell line and the vector only control. We found that overexpression of gE2F1 decreased the cyst formation in both vegetative growth and encysting culture, suggesting that gE2F1 may decrease cwp1 transcription and influence the cyst formation process. Accordingly, our data suggest that DNA binding ability of E2F protein family has been conserved during evolution and gE2F1 is an important transcriptional activator in regulation of Giardia thymidine kinase gene that is involved in DNA replication. In addition, gE2F1 may also play a role in repressing cwp1 gene that is not relative to DNA replication or cell cycle. It is possible that E2F family transcription factor is also involved in Giardia differentiation.

並列關鍵字

Giardia lamblia ； transcriptional regulation ； E2F

參考文獻

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