鑑定梨形鞭毛蟲的ARID/bright同源蛋白質對於cwp1基因的轉錄調控之影響

Abstract

ARID(AT-rich interaction domain)蛋白質家族是真核生物的一種轉錄因子，在許多不同種的真核生物都有它的同源基因，這個家族的蛋白質通常與調控細胞的生長、發育和分化的作用有關，而這個家族的蛋白質和DNA的結合能力，各種ARID蛋白質的專一性都不盡相同，不過大致上都偏好於和AT-rich的序列結合；我們已經在梨形鞭毛蟲的基因組中找到兩個含有ARID 的基因，分別是garid1和garid2，我們首先對於garid1做分析；將AU1標記接到gARID1轉染梨形鞭毛蟲，利用免疫螢光染色可發現gARID1存在於細胞核中。gARID1的訊息RNA量在囊體化後會明顯下降，不過其陽性染色和蛋白質表現量都有明顯增加；EMSA實驗中也發現gARID1會明顯的與cwp1基因啟動子之AT-rich initiator結合，經由突變序列分析，也顯示gARID1的結合序列為AGATC和AATAAAATA，隨後我們也利用ChIP來證明gARID1在細胞內也的確會和cwp1基因的啟動子結合。在老鼠Bright上對DNA結合重要的氨基酸有4個，gARID1有3個，除了Tyr82外，Tyr82在老鼠Bright上是Phe。將此Tyr變成Ala突變，也會使gARID1失去和DNA結合的能力，顯示此氨基酸對DNA結合很重要。由於許多已知的ARID蛋白質可以和DNA minor groove結合，因此我們利用Distamycin A可以與DNA minor groove結合的特性作為競爭劑，也發現gARID也會和DNA minor groove結合。我們也利用螢光酵素基因接上cwp1基因啟動子，如果將其cwp1啟動子上的gARID1結合位置突變，也可以發現螢光酵素和訊息RNA的表現量有明顯的下降；再利用cotransfection assay，可發現gARID1會使螢光酵素和訊息RNA表現量明顯上升，然而如果將其cwp1啟動子上的gARID1結合位置突變，這種轉錄活化則消失。綜合這些結果，我們可以推斷ARID蛋白質家族在演化的過程中，其和DNA結合的特性是被保留的，此外我們也知道gARID1可能是梨形鞭毛蟲調控cwp1基因的一個重要的轉錄活化子。

ARID (AT-rich interaction domain) protein is a transcription factor family in higher eukaryotes that regulates cell proliferation, development, and differentiation. Specificity of DNA binding ability in this family prefers AT-rich sequences, but some ARID family proteins are not sequence-specific DNA-binding proteins or they do not bind AT-rich sequences. We found two genes that encode ARID in Giardia lamblia genome database, garid1 and garid2. We analyzed the function of garid1 first. AU1-tagged gARID1 was found to localize to nuclei. During encystation, gARID1 mRNA level decreased emphatically, but protein level increased. We also found that gARID1 can bind AT-rich initiator of the cwp1 promoter by EMSA. Mutation analysis revealed gARID1 binding sequence was AGATC and AATAAAATA. We used ChIP to demonstrate that gARID1 can bind cwp1 gene promoter in vivo. Four key residues important for DNA binding activity of the mouse Bright are conserved in gARID1 with the exception of Tyr82, which corresponds to Phe in the mouse Bright. Mutation of the Tyr82 to Ala in gARID1 resulted in loss of its DNA binding activity, suggesting the importance of this residue in DNA binding. Experiments using distamycin A, a compound that binds to the minor groove of AT-rich DNA sequences also suggest that gARID1 can bind to the minor groove like other ARID proteins. Mutation of the gARID1 binding site in the cwp1 promoter resulted in a decrease of promoter activity and mRNA levels during vegetative growth and encystation, suggesting that the gARID1 binding sites are positive cis-acting elements of the cwp1 gene promoters in both trophozoites and encysting cells. Using cotransfection assay, we also found overexpession of gARID1 can increase luciferase activity and mRNA levels, while mutation of the gARID1 binding sites of the cwp1 promoter resulted loss of this transactivation. Accordingly, our results suggest DNA binding ability of ARID protein family has been conserved during evolution and gARID1 is an important transcriptional activator in regulation of Giardia cwp1 gene.