A Parallel G Quadruplex-Binding Protein Regulates the Boundaries of DNA Elimination Events of Tetrahymena thermophila.
A Parallel G Quadruplex-Binding Protein Regulates the Boundaries of DNA Elimination Events of Tetrahymena thermophila.
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DOI：
10.1371/journal.pgen.1005842
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发表时间：
2016-03
期刊：
影响因子：
4.5
通讯作者：
Chalker DL
中科院分区：
文献类型：
--
作者：
Carle CM;Zaher HS;Chalker DL
富含鸟嘌呤 (G) 的 DNA 在体外很容易形成四链四链体，但它们参与基因组调控的证据有限。我们已经鉴定出一种四链体结合蛋白 Lia3，它控制嗜热四膜虫种系限制的内部消除序列 (IES) 的边界。这种纤毛虫体细胞基因组的分化需要切除数千个 IES，通过小 RNA 指导的异染色质形成来去除。在缺乏 LIA3 (ΔLIA3) 的细胞中，由特定富含 G 的多嘌呤束包围的 IES 的切除受到损害且不精确，而没有此类控制序列的 IES 的去除不受影响。我们发现含有这些多嘌呤束的寡核苷酸形成了与 Lia3 特异性结合的平行 G-四链体结构。 Lia3 结合 G-四链体 DNA 并控制特定 G-束位点 DNA 消除的准确性，这一发现揭示了四链体结构调节染色体组织的未被认识的潜力。非规范 DNA 结构，包括四链鸟嘌呤四链体 (G4 DNA)，在体外很容易观察到，但要最终证明它们在细胞内的调节重要性特别具有挑战性。我们发现了一种 G4 DNA 结合蛋白 Lia3，它专门调节嗜热四膜虫中的程序性 DNA 消除事件。这种纤毛虫从每个发育中的体细胞核中删除了近三分之一的种系基因组。这些基因组删除事件必须准确，因为切除的数千个 DNA 区域位于基因和/或其启动子附近，因此异常切除可能会改变基因表达。当我们敲除编码 Lia3 的基因时，我们发现切除区域的边界对于侧翼富含 G (5'-AAAAAGGGGG-3') 边界控制序列的基因座子集来说是异质的。当我们测试Lia3是否结合这个序列时，我们发现该序列本身形成G4 DNA，并且只有当该序列采用这种构象时，Lia3才会结合。我们的研究结果表明，Lia3 结合 G4 DNA，并且编码 Lia3 的基因的缺失扰乱了两侧是这种四链体形成 DNA 的切除基因座的边界，这提供了令人信服的证据，表明这种非规范 DNA 结构在这些细胞的发育过程中发挥着关键作用。
Guanine (G)-rich DNA readily forms four-stranded quadruplexes in vitro, but evidence for their participation in genome regulation is limited. We have identified a quadruplex-binding protein, Lia3, that controls the boundaries of germline-limited, internal eliminated sequences (IESs) of Tetrahymena thermophila. Differentiation of this ciliate’s somatic genome requires excision of thousands of IESs, targeted for removal by small-RNA-directed heterochromatin formation. In cells lacking LIA3 (ΔLIA3), the excision of IESs bounded by specific G-rich polypurine tracts was impaired and imprecise, whereas the removal of IESs without such controlling sequences was unaffected. We found that oligonucleotides containing these polypurine tracts formed parallel G-quadruplex structures that are specifically bound by Lia3. The discovery that Lia3 binds G-quadruplex DNA and controls the accuracy of DNA elimination at loci with specific G-tracts uncovers an unrecognized potential of quadruplex structures to regulate chromosome organization. Non-canonical DNA structures, including four-stranded Guanine quadruplexes (G4 DNA), have been observed readily in vitro, but their regulatory importance within cells has been particularly challenging to demonstrate conclusively. We have discovered a G4 DNA binding protein, Lia3, that specifically regulates programmed DNA elimination events in Tetrahymena thermophila. This ciliate deletes nearly one-third of its germline genome from each developing somatic nucleus. These genomic deletion events must be accurate as the thousands of DNA regions excised are located near genes and/or their promoters, thus aberrant excision may alter gene expression. When we knocked out the gene encoding Lia3, we found that the boundaries of the excised regions were heterogeneous for a subset of loci that are flanked by G-rich (5’-AAAAAGGGGG-3’) boundary controlling sequences. When we tested whether Lia3 bound this sequence, we discovered that the sequence itself formed G4 DNA and that Lia3 bound only when the sequence adopted this conformation. Our findings that Lia3 binds G4 DNA and that deletion of the gene encoding Lia3 perturbs the boundaries of the excised loci which are flanked by this quadruplex-forming DNA provides compelling evidence that this non-canonical DNA structure has a critical role during development of these cells.