陰道滴蟲中Myb3轉錄因子功能分析

Biochemical characterizations of a Myb3 transcription factor in Trichomonas vaginalis

許弘明(Hong-Ming Hsu)

指導教授 ： 戴榮湘

國立臺灣大學/醫學院/微生物學研究所/博士(2013年)

https://doi.org/10.6342/NTU.2013.01959

摘要

陰道滴蟲寄生於人類泌尿生殖係統內，其能量代謝以及致病因子的轉錄表現受宿主環境中的鐵離子調控。此蟲內有400多個含保守性DNA結合區，R2R3，之Myb型轉錄因子。其中，黏附蛋白質ap65-1基因的轉錄活性受鐵誘導，經由Myb1與Myb2 ， 共同調控 。此二轉錄因子以競合方式進入 ap65-1啟動子上Myb1及Myb2共結合之重疊序列，MRE-1/MRE-2r（TAACGATA），與MRE-2f（TAACGA）。本研究進一步確認，Myb3結合於啟動子MRE-1序列（TAACGA），調控ap65-1基因的基礎與受鐵誘導轉錄活性。經實驗觀察，Myb2及Myb3以競結方式進入ap65-1基因啟動子，而其結合趨勢受鐵離子調控。而Myb1之過度表現，同時抑制Myb2與Myb3進入啟動子能力。綜合以上結果推測，陰道滴蟲以三個Myb轉錄因子進入啟動子時機上的差異，來調控ap65-1基因的轉錄活性。這些Myb轉錄因子的R2R3區域包含約100個氨基酸，由六個螺旋序列組成次級結構。去鐵環境下，Myb3少量存在於細胞核，而鐵刺激導致Myb3快速入核：而Myb2入核不受鐵影響。Myb2之核定位訊號亦涵蓋大部份之R2R3區域。Myb3亦利用類似訊號進行入核調控，但仍需要一段富彈性的C端區域，其中包含三個緊鄰的序列因子，分別控制Myb3受鐵刺激後的細胞質滯留、入核與出核。顯示，陰道滴蟲之Myb以R2R3同時控制啟動子序列的選擇及入核時機：唯各自依其內部結構特徵與周邊序列因子， 以控制下游基因適時適量之表現，維持正常生理運作。

關鍵字

陰道滴蟲 ； 鐵離子 ； ap65-1基因 ； Myb3 ； 啟動子結合 ； 核定位訊號 ； 入核與出核

並列摘要

Iron is the key host factor that modulates the energy metabolism and virulence expression of the protozoan parasite, Trichomonas vaginalis. It was demonstrated to regulate gene expression via transcription initiation. Iron-inducible transcription of an adhesion protein, ap65-1, gene in the parasite, was previously demonstrated to involve the Myb1 and Myb2 transcription factors, which share a conserved R2R3 DNA-binding domain, but preferentially bind cis-acting elements, MRE-1/MRE-2r (TAACGATA) and MRE-2f (TATCGT), in the promoter region of the ap65-1 gene. In this study, Myb3 protein was demonstrated to specifically bind a DNA element (TAACGA) in MRE-1 and regulate basal and iron-inducible ap65-1 transcription. Competitive promoter entries of Myb2 and Myb3 were observed when iron concentration in growth medium varied, while promoter entries of both proteins were diminished by overexpressing of Myb1. These results suggest transcriptional activity of the ap65-1 promoter is co-regulated by multiple Myb proteins through differential promoter entry.
Nuclear and cytoplasmic shuttling of Myb3 was further examined. Myb3 was enriched more in the cytoplasm than nucleus, especially when cells were depleted of iron. Iron was found to induce a transient nuclear translocation of Myb3, but not Myb2. A nuclear localization signal（NLS） similar to that of Myb2 was mapped to the highly structured R2R3 DNA-binding domain of Myb3 with a flexible C-terminus Contiguous sequence elements that regulate nuclear translocation at multiple cellular steps, including the cytoplasmic retention, nuclear influx and efflux, were identified within the C-terminal tail. These observations suggest that the R2R3 DNA-binding domain may also serves as a common module for the nuclear translocation of various Myb proteins in the parasite, but each of them may possess intrinsic features for nuclear translocation under specific conditions.

並列關鍵字

Trichomonas vaginalis ； iron ； ap65-1 ； Myb3 ； promoter entry ； nuclear localization signal ； nuclear influx and efflux

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