B 細胞淋巴瘤基因啟動子區域中 DNA 四股結構之構型間轉換的單分子研究
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2018
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B 細胞淋巴瘤基因 2 (簡稱 Bcl-2) 在人體內是一負責控制細胞凋亡的前致癌基因，其過表達現象和人體多種癌症皆有高度的關聯性。在 Bcl-2 的啟動子 P1 區域上有一富含 G、C 的 39 個鹼基對的序列被指出會參與此基因的基因表達，名為 Pu39，且其在特定的鹽離子濃度與 pH 值下會形成特殊的 DNA 二級結構 G-quadruplex (G4結構)，此結構可以抑制基因轉錄進而阻止癌細胞增生。
先前 Yang 團隊使用 Pu39 的縮短版序列 midG4 和 Pu30 鑑定出兩種不同的 G4 結構，且認為這兩種結構會互相轉換，但並沒有找到相關的直接證據。在本篇研究中，我們的目標物為完整版的 Pu39 序列，並且使用單分子螢光共振能量轉移光譜技術觀察即時的 G4 結構互相轉換的動態學與動力學。
我們發現 midG4 和 Pu30 兩種結構會經由一未知的中間狀態進行互相轉換，有趣的是互相轉換的現象來自於 Pu39 上的第六組連續 G 片段，表示第六組連續 G 片段對於基因的調節與表達而言相當重要，然而這組片段在先前 Yang 團隊的實驗中皆是被去除的。此外我們也在 Pu39 序列上發現了先前的研究團隊沒有鑑定出來的G4 結構 (3456G4)。
B-cell Lymphoma 2 (Bcl-2) is a proto-oncogene that is responsible for cell apoptosis. The overexpression of Bcl-2 gene is highly correlated to human cancer of many kinds. A 39-nucleotide (nt) GC-rich region (Pu39) in promoter P1 of Bcl-2 gene is believed to be involved in the gene modulation. This 39nt GC-rich region may form multiple G-quadruplex (G4) structures at specific cation concentration and pH value, stabilizing this G4 structure can inhibit gene transcription and potentially suppress the cancer cell formation. Previous studies have shown that this 39nt GC-rich region might fold into two different G4 conformations, among them, Bcl-2midG4 sequence fold into hybrid conformation and Bcl-2Pu30 sequence form parallel conformation. Interestingly, an interconversion between these two structures was proposed but no direct experimental evidence was shown. These two different interchangeable G4s in Pu39 may be important for the regulation of transcription, as each G4 is likely to be recognized by different proteins leading to different gene modulation. We applied single-molecule fluorescence resonance energy transfer (smFRET) spectroscopy to directly reveal the interconversion dynamics between Pu39 G4 states. We found that the interconversion between these two conformational states undergoes a stepwise mechanism through one unidentified intermediated state. Interestingly, the 6th tandem G-sequence, which was usually believed to be unimportant and truncated in the previous studies, modulate the mechanism of interconversion. Moreover, an additional G4 state was also found in the Pu39 configuration.
B-cell Lymphoma 2 (Bcl-2) is a proto-oncogene that is responsible for cell apoptosis. The overexpression of Bcl-2 gene is highly correlated to human cancer of many kinds. A 39-nucleotide (nt) GC-rich region (Pu39) in promoter P1 of Bcl-2 gene is believed to be involved in the gene modulation. This 39nt GC-rich region may form multiple G-quadruplex (G4) structures at specific cation concentration and pH value, stabilizing this G4 structure can inhibit gene transcription and potentially suppress the cancer cell formation. Previous studies have shown that this 39nt GC-rich region might fold into two different G4 conformations, among them, Bcl-2midG4 sequence fold into hybrid conformation and Bcl-2Pu30 sequence form parallel conformation. Interestingly, an interconversion between these two structures was proposed but no direct experimental evidence was shown. These two different interchangeable G4s in Pu39 may be important for the regulation of transcription, as each G4 is likely to be recognized by different proteins leading to different gene modulation. We applied single-molecule fluorescence resonance energy transfer (smFRET) spectroscopy to directly reveal the interconversion dynamics between Pu39 G4 states. We found that the interconversion between these two conformational states undergoes a stepwise mechanism through one unidentified intermediated state. Interestingly, the 6th tandem G-sequence, which was usually believed to be unimportant and truncated in the previous studies, modulate the mechanism of interconversion. Moreover, an additional G4 state was also found in the Pu39 configuration.
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Bcl-2, 前致癌基因, Pu39, DNA 二級結構, G4 結構, 單分子, 螢光共振能量轉移, B-cell Lymphoma 2, Pu39, proto-oncogene, G-quadruplex, interconversion, single-molecule, fluorescence resonance energy transfer, smFRET