Table2_Generation of somatic de novo structural variation as a hallmark of cellular senescence in human lung fibroblasts.XLSX

Cellular senescence is characterized by replication arrest in response to stress stimuli. Senescent cells accumulate in aging tissues and can trigger organ-specific and possibly systemic dysfunction. Although senescent cell populations are heterogeneous, a key feature is that they exhibit epigenetic changes. Epigenetic changes such as loss of repressive constitutive heterochromatin could lead to subsequent LINE-1 derepression, a phenomenon often described in the context of senescence or somatic evolution. LINE-1 elements decode the retroposition machinery and reverse transcription generates cDNA from autonomous and non-autonomous TEs that can potentially reintegrate into genomes and cause structural variants. Another feature of cellular senescence is mitochondrial dysfunction caused by mitochondrial damage. In combination with impaired mitophagy, which is characteristic of senescent cells, this could lead to cytosolic mtDNA accumulation and, as a genomic consequence, integrations of mtDNA into nuclear DNA (nDNA), resulting in mitochondrial pseudogenes called numts. Thus, both phenomena could cause structural variants in aging genomes that go beyond epigenetic changes. We therefore compared proliferating and senescent IMR-90 cells in terms of somatic de novo numts and integrations of a non-autonomous composite retrotransposons - the so-called SVA elements—that hijack the retropositional machinery of LINE-1. We applied a subtractive and kinetic enrichment technique using proliferating cell DNA as a driver and senescent genomes as a tester for the detection of nuclear flanks of de novo SVA integrations. Coupled with deep sequencing we obtained a genomic readout for SVA retrotransposition possibly linked to cellular senescence in the IMR-90 model. Furthermore, we compared the genomes of proliferative and senescent IMR-90 cells by deep sequencing or after enrichment of nuclear DNA using AluScan technology. A total of 1,695 de novo SVA integrations were detected in senescent IMR-90 cells, of which 333 were unique. Moreover, we identified a total of 81 de novo numts with perfect identity to both mtDNA and nuclear hg38 flanks. In summary, we present evidence for possible age-dependent structural genomic changes by paralogization that go beyond epigenetic modifications. We hypothesize, that the structural variants we observe potentially impact processes associated with replicative aging of IMR-90 cells.

细胞衰老(Cellular Senescence)以应激刺激诱导的复制停滞为典型特征。衰老细胞会在衰老组织中累积，并可引发器官特异性乃至全身性的功能障碍。尽管衰老细胞群体具有异质性，但其关键特征之一是存在表观遗传(Epigenetic)改变。诸如组成型异染色质(constitutive heterochromatin)抑制功能丧失这类表观遗传改变，可引发后续的LINE-1(LINE-1)去抑制现象——这一现象常与细胞衰老或体细胞进化相关联。LINE-1元件可编码逆转录转座(retroposition)机器，其逆转录过程会从自主性与非自主性转座因子(Transposable Elements, TEs)中合成互补DNA(complementary DNA, cDNA)，这些产物可重新整合进入基因组并引发结构变异。
细胞衰老的另一特征是线粒体损伤引发的线粒体功能障碍。结合衰老细胞标志性的受损线粒体自噬(Mitophagy)，这一过程可导致胞质线粒体DNA(Mitochondrial DNA, mtDNA)累积；从基因组层面来看，这会引发线粒体DNA整合进入核DNA(nuclear DNA, nDNA)，最终形成被称为numts(nuclear mitochondrial DNA segments, numts)的线粒体假基因。综上，这两类现象均可在衰老基因组中引发超出表观遗传改变范畴的结构变异。
为此，本研究针对体细胞新发numts以及非自主性复合逆转录转座子——即SVA元件(SVA elements)——的整合情况，对增殖态与衰老态IMR-90细胞系(IMR-90)进行了比较；SVA元件可劫持LINE-1的逆转录转座机器。本研究采用了以增殖态细胞DNA为驱动子、衰老态基因组为测试子的消减动力学富集技术，用于检测新发SVA整合位点的核侧翼序列。结合深度测序(Deep Sequencing)技术，我们获取了IMR-90模型中可能与细胞衰老相关的SVA逆转录转座事件的基因组测序读数。此外，我们通过深度测序或采用AluScan技术(AluScan)富集核DNA后，对增殖态与衰老态IMR-90细胞的基因组进行了比较。
在衰老态IMR-90细胞中共检测到1695处新发SVA整合位点，其中333处为特异性位点。此外，本研究共鉴定出81处新发numts，其序列与线粒体DNA及人类基因组组装版本hg38(hg38)的核侧翼序列完全一致。综上，本研究提供了证据表明，衰老基因组中存在可能由旁系同源化(paralogization)介导的、超出表观遗传修饰范畴的年龄依赖性结构基因组改变。我们推测，本研究中观测到的结构变异可能对IMR-90细胞的复制性衰老相关生物学过程产生影响。