The KRAS Promoter Responds to Myc-associated Zinc Finger and Poly(ADP-ribose) Polymerase 1 Proteins, Which Recognize a Critical Quadruplex-forming GA-element
The KRAS Promoter Responds to Myc-associated Zinc Finger and Poly(ADP-ribose) Polymerase 1 Proteins, Which Recognize a Critical Quadruplex-forming GA-element
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DOI：
10.1074/jbc.m110.101923
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发表时间：
2010-07-16
影响因子：
4.8
通讯作者：
Xodo, Luigi E.
中科院分区：
文献类型：
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作者：
Cogoi, Susanna;Paramasivam, Manikandan;Xodo, Luigi E.
鼠KRAS启动子在转录起始位点上游含有对转录必需的富含G的核酸酶超敏元件（GA元件）。下拉和染色质免疫沉淀试验表明，该GA元件是由Myc相关的锌指（MAZ）和聚（ADP-核糖）聚合酶1（PARP-1）蛋白结合。这些蛋白质对转录至关重要，因为当它们被短发夹RNA敲低时，转录被下调。当PARP-1的聚（ADP-核糖基）化活性被3，4-二氢-5-[ 4-（1-哌啶基）丁氧基]-1（2 H）异喹啉酮抑制时，情况也是如此。我们发现，MAZ特异性结合的双链体和四链体构象的GA-元素，而PARP-1显示的特异性仅为G-四链体。基于荧光共振能量转移熔解和聚合酶终止测定，我们看到MAZ稳定KRAS四链体。当GA元件中的折叠能力被特定的G -> T或G -> A点突变废除时，KRAS转录被下调。相反，胍改性的酞菁，其特异性地与KRAS G-四链体相互作用并稳定KRAS G-四链体，将启动子活性提高一倍以上。总的来说，我们的数据支持小鼠KRAS的转录机制，涉及MAZ，PARP-1和启动子GA元件中的四链体构象变化。
The murine KRAS promoter contains a G-rich nuclease hypersensitive element (GA-element) upstream of the transcription start site that is essential for transcription. Pulldown and chromatin immunoprecipitation assays demonstrate that this GA-element is bound by the Myc-associated zinc finger (MAZ) and poly(ADP-ribose) polymerase 1 (PARP-1) proteins. These proteins are crucial for transcription, because when they are knocked down by short hairpin RNA, transcription is down-regulated. This is also the case when the poly(ADP-ribosyl) ation activity of PARP-1 is inhibited by 3,4-dihydro-5-[ 4-(1-piperidinyl) butoxyl]-1(2H) isoquinolinone. We found that MAZ specifically binds to the duplex and quadruplex conformations of the GA-element, whereas PARP-1 shows specificity only for the G-quadruplex. On the basis of fluorescence resonance energy transfer melting and polymerase stop assays we saw that MAZ stabilizes the KRAS quadruplex. When the capacity of folding in the GA-element is abrogated by specific G -> T or G -> A point mutations, KRAS transcription is down-regulated. Conversely, guanidine-modified phthalocyanines, which specifically interact with and stabilize the KRAS G-quadruplex, push the promoter activity up to more than double. Collectively, our data support a transcription mechanism for murine KRAS that involves MAZ, PARP-1 and duplex-quadruplex conformational changes in the promoter GA-element.