Genome surveillance through repression of intronless mobile elements by the HUSH complex

All life forms defend their genome against DNA invasion. Eukaryotic cells recognize incoming DNA and limit transcription through repressive chromatin modifications. The human silencing hub (HUSH) complex transcriptionally represses long interspersed element-1 retrotransposons (L1s) and retroviruses through histone H3 Lys9 trimethylation (H3K9me3). How HUSH recognizes and initiates silencing of these invading genetic elements is unknown. Here, we show that HUSH is able to recognize and transcriptionally repress a broad range of long, intronless transgenes. Intron insertion into HUSH-repressed transgenes counteracts repression, even in the absence of intron splicing. HUSH binds transcripts from the target locus, prior to and independent of H3K9me3 deposition, and target transcription is essential for both initiation and propagation of HUSH-mediated H3K9me3. Genomic data reveals how HUSH binds and represses a subset of endogenous intronless genes generated through retrotransposition of cellular mRNAs. Therefore, intronless cDNA, a hallmark of reverse transcription, provides a versatile means to distinguish invading retroelements from host genes and allows HUSH to protect the genome from ‘non-self’ DNA, despite no prior exposure to the invading element. Our findings reveal the existence of a genome surveillance system and explain how it provides immediate protection against newly acquired elements while avoiding inappropriate repression of host genes. UV-crosslinked RNA immunoprecipitation followed by next generation sequencing (UV-RIPseq) to identify RNA interactome of Periphilin. ChIPseq to identify methylation changes over lentiviral reporter and CRISPR/Cas9-modified endogenous locus.

所有生命形式均会对DNA入侵展开基因组防御。真核细胞可识别侵入性DNA，并通过抑制性染色质修饰限制其转录。人类沉默枢纽复合物（human silencing hub, HUSH）可通过组蛋白H3赖氨酸9三甲基化（histone H3 Lys9 trimethylation, H3K9me3），对长散在核元件-1逆转录转座子（long interspersed element-1 retrotransposons, L1s）及逆转录病毒实施转录沉默。目前HUSH如何识别这些入侵遗传元件并启动其沉默的机制仍不明晰。本研究证实，HUSH能够识别并转录沉默一系列广泛的长片段无内含子转基因。即便在不存在内含子剪接的情况下，将内含子插入经HUSH沉默的转基因中，仍可抵消沉默效应。HUSH可在H3K9me3沉积之前且独立于该修饰的情况下，结合靶位点的转录本；且靶位点的转录对于HUSH介导的H3K9me3的起始与传播均至关重要。基因组数据分析揭示了HUSH如何结合并沉默由细胞mRNA逆转录转座产生的一类内源性无内含子基因亚群。由此可见，作为逆转录标志性特征的无内含子cDNA，可作为一种通用标志物，区分入侵性逆转录元件与宿主基因；这使得HUSH能够在未预先接触入侵元件的情况下，保护基因组免受“非己”DNA的侵扰。本研究发现揭示了一类基因组监视系统的存在，并阐释了该系统如何在避免对宿主基因产生不当沉默的同时，为基因组提供针对新获得元件的即时防御。本研究采用紫外交联RNA免疫沉淀结合高通量测序（UV-crosslinked RNA immunoprecipitation followed by next generation sequencing, UV-RIPseq）技术，鉴定Periphilin的RNA互作组；同时通过染色质免疫沉淀测序（chromatin immunoprecipitation sequencing, ChIPseq）分析慢病毒报告基因及CRISPR/Cas9修饰的内源性位点的甲基化变化。