利用 CRISPR Cas9 绘制 LINE-1 启动子的无偏蛋白质组图谱。

IF 4.7 2区 生物学 Q1 GENETICS & HEREDITY Mobile DNA Pub Date : 2021-08-23 DOI:10.1186/s13100-021-00249-9
Erica M Briggs, Paolo Mita, Xiaoji Sun, Susan Ha, Nikita Vasilyev, Zev R Leopold, Evgeny Nudler, Jef D Boeke, Susan K Logan
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引用次数: 0

摘要

背景:自主性逆转录因子长穿插元件-1(LINE-1)通过复制和粘贴机制,利用 RNA 中间体(逆转录)进行调动。在整个人类进化过程中,基因组中积累了约 500,000 个 LINE-1 序列。这些序列中的大部分属于祖先的 LINE-1 亚家族,包括 L1PA2-L1PA7,已经无法再移动。只有一小部分属于L1Hs亚家族的LINE-1序列(约80至100个拷贝)是完整的,仍能进行逆转录。虽然LINE-1在大多数细胞中处于沉默状态,但关于癌细胞中LINE-1的失调仍存在许多问题:在这里,我们对CRISPR Cas9 gRNA进行了优化,以特异性靶向L1Hs 5'UTR启动子的调控序列。我们发现了三种对L1Hs更具特异性的gRNA,它们与较早的LINE-1序列(L1PA2-L1PA7)的结合有限。我们还采用了C-BERST方法(dCas9-APEX2 Biotinylation at genomic Elements by Restricted Spatial Tagging)来鉴定癌细胞中的LINE-1转录调节因子。我们的LINE-1 C-BERST筛选发现了已知的和新的LINE-1转录调控因子,包括CTCF、YY1和DUSP1:结论:我们对 gRNA 特异性的优化和评估以及 C-BERST 方法的应用为研究 LINE-1 在癌症中的调控机制提供了一种工具。此外,我们还发现双重特异性蛋白磷酸酶 DUSP1 是 LINE-1 转录的新型调控因子。
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Unbiased proteomic mapping of the LINE-1 promoter using CRISPR Cas9.

Background: The autonomous retroelement Long Interspersed Element-1 (LINE-1) mobilizes though a copy and paste mechanism using an RNA intermediate (retrotransposition). Throughout human evolution, around 500,000 LINE-1 sequences have accumulated in the genome. Most of these sequences belong to ancestral LINE-1 subfamilies, including L1PA2-L1PA7, and can no longer mobilize. Only a small fraction of LINE-1 sequences, approximately 80 to 100 copies belonging to the L1Hs subfamily, are complete and still capable of retrotransposition. While silenced in most cells, many questions remain regarding LINE-1 dysregulation in cancer cells.

Results: Here, we optimized CRISPR Cas9 gRNAs to specifically target the regulatory sequence of the L1Hs 5'UTR promoter. We identified three gRNAs that were more specific to L1Hs, with limited binding to older LINE-1 sequences (L1PA2-L1PA7). We also adapted the C-BERST method (dCas9-APEX2 Biotinylation at genomic Elements by Restricted Spatial Tagging) to identify LINE-1 transcriptional regulators in cancer cells. Our LINE-1 C-BERST screen revealed both known and novel LINE-1 transcriptional regulators, including CTCF, YY1 and DUSP1.

Conclusion: Our optimization and evaluation of gRNA specificity and application of the C-BERST method creates a tool for studying the regulatory mechanisms of LINE-1 in cancer. Further, we identified the dual specificity protein phosphatase, DUSP1, as a novel regulator of LINE-1 transcription.

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来源期刊
Mobile DNA
Mobile DNA GENETICS & HEREDITY-
CiteScore
8.20
自引率
6.10%
发文量
26
审稿时长
11 weeks
期刊介绍: Mobile DNA is an online, peer-reviewed, open access journal that publishes articles providing novel insights into DNA rearrangements in all organisms, ranging from transposition and other types of recombination mechanisms to patterns and processes of mobile element and host genome evolution. In addition, the journal will consider articles on the utility of mobile genetic elements in biotechnological methods and protocols.
期刊最新文献
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