An RNA:DNA intermediate of LINE-1 retrotransposition - a novel trigger of cytotoxicity

项目摘要 转座因子在基因组进化中起着重要的作用，但长期以来人们一直认为转座因子在基因组进化中的作用是一个未知数。 发展仍然存在很大争议。事实上，尽管拥有复杂的表观遗传学和后遗传学， TE抑制的转录机制，生殖系以及各种体细胞和癌细胞表达 在其分化过程中至少进行一次TE。然而，TE是否有助于细胞分化， 笼罩在神秘之中过去十年的大量研究表明， 逆转录转座子LINE-1是人类基因组中唯一具有自主活性的转座子， 细胞以及具有破坏的TE的表观遗传抑制的小鼠生殖细胞。此外，我们最近 对胎儿卵母细胞发育保守现象的研究表明， 为小鼠卵母细胞的选择性剔除提供了基础。由于表观遗传机制对TE至关重要， 这些观察结果表明，LINE-1有助于发育， 通过消除细胞分化过程中表观遗传重塑超过其抑制能力的细胞， 重新激活的TE。 LINE-1活性的哪些方面触发细胞毒性？LINE-1编码的ORF 2 p具有两个酶活性， 逆转录酶（RT）和内切酶（EN）是逆转录转座所必需的。生产线-1- 驱动的DNA损伤显然取决于EN活性，但RT活性对细胞毒性的贡献是 未知我们现在已经确定了艾德一个强大的模型系统，用于体内分析LINE-1 RT对 宿主，即小鼠减数分裂前期（MP）胎儿卵母细胞，其在表观遗传学后表达LINE-1 基因组的重组重要的是，LINE-1表达水平在单个卵母细胞之间变化， 与卵母细胞活力相关，导致MP早期50%的卵母细胞损失，在激活前， DNA损伤检查点。此外，RT抑制剂AZT的给药可防止卵母细胞的致死性。 LINE-1逆转录转座的RNA：DNA杂合中间体在触发卵母细胞死亡中的作用 DNA损伤无关的方式。 拟议的研究将确定和量化L1 RNA：DNA杂合体在AZT治疗和未治疗 野生型和L1过表达小鼠的胎儿卵母细胞，并建立了L1 RNAL：DNA杂交在 RNase H2缺陷小鼠。总的来说，该提案将揭示一个以前未知的L1 RT机制- 胎儿卵母细胞的依赖性死亡，这可能导致更深入的探索这一现象，在体细胞和 癌细胞

Project Summary Transposable elements (TEs) play important roles in evolution of genomes, but their long-hypothesized role in development remains highly controversial. Indeed, despite possessing sophisticated epigenetic and post- transcriptional mechanisms of TE repression, the germline as well as various somatic and cancer cells express TEs at least once during their differentiation. However, whether TEs contribute to cell differentiation is shrouded in mystery. Numerous studies over the past decade demonstrated that overexpression of retrotransposon LINE-1, the only autonomously active TE in the human genome, is toxic to cultured human cells as well as to mouse germ cells with disrupted epigenetic repression of TEs. Furthermore, our recent studies of the conserved phenomenon of fetal oocyte aGrition have implicated excessive LINE-1 expression in providing the basis for the selective culling of mouse oocytes. Since epigenetic mechanisms are crucial for TE repression as well as normal development, these observations suggest that LINE-1 contributes to development by eliminating cells whose epigenetic remodeling during cell differentiation exceeds their capacity to restrain reactivated TEs. I Which aspects of LINE-1 activity trigger cytotoxicity? LINE-1-encoded ORF2p has two enzymatic activities, Endonuclease (EN) and Reverse Transcriptase (RT), that are required for retrotransposition. LINE-1- driven DNA damage clearly depends on EN activity, but the contribution of the RT activity to cytotoxicity is unknown. We have now identified a robust model system for the in vivo analysis of LINE-1 RT impact on the host, namely, meiotic prophase (MP) fetal oocytes of mice that express LINE-1 following epigenetic reprogramming of their genomes. Importantly, LINE-1 expression levels vary between individual oocytes and correlate with oocyte viability, leading to the loss of 50% of oocytes in the early MP, before the activation of the DNA damage checkpoint. Furthermore, administration of an RT inhibitor AZT prevents oocyte lethality in the early MP implicating RNA:DNA hybrid intermediates of LINE-1 retrotransposition in triggering oocyte death in a DNA damage-independent manner. I The proposed research will identify and quantify L1 RNA:DNA hybrids in AZT treated and untreated fetal oocytes of wild type and L1 overexpressing mice, and establish a role for L1 RNAL:DNA hybrids in RNase H2 deficient mice. Overall, the proposal will reveal a previously unknown mechanism of L1 RT- dependent death of fetal oocytes which could lead to deeper exploration of this phenomenon in somatic and cancer cells.