Telomeric DNA breaks in human induced pluripotent stem cells trigger ATR-mediated arrest and telomerase-independent telomere damage repair.

IF 5.3 2区 生物学 Q2 CELL BIOLOGY Journal of Molecular Cell Biology Pub Date : 2024-08-26 DOI:10.1093/jmcb/mjad058
Katrina N Estep, John W Tobias, Rafael J Fernandez, Brinley M Beveridge, F Brad Johnson
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Abstract

Although mechanisms of telomere protection are well-defined in differentiated cells, how stem cells sense and respond to telomere dysfunction, in particular telomeric double-strand breaks (DSBs), is poorly characterized. Here, we report the DNA damage signaling, cell cycle, and transcriptome changes in human induced pluripotent stem cells (iPSCs) in response to telomere-internal DSBs. We engineer human iPSCs with an inducible TRF1-FokI fusion protein to acutely induce DSBs at telomeres. Using this model, we demonstrate that TRF1-FokI DSBs activate an ATR-dependent DNA damage response, which leads to p53-independent cell cycle arrest in G2. Using CRISPR-Cas9 to cripple the catalytic domain of telomerase reverse transcriptase, we show that telomerase is largely dispensable for survival and lengthening of TRF1-FokI-cleaved telomeres, which instead are effectively repaired by robust homologous recombination (HR). In contrast to HR-based telomere maintenance in mouse embryonic stem cells, where HR causes ZSCAN4-dependent extension of telomeres beyond their initial lengths, HR-based repair of telomeric breaks is sufficient to maintain iPSC telomeres at a normal length, which is compatible with sustained survival of the cells over several days of TRF1-FokI induction. Our findings suggest a previously unappreciated role for HR in telomere maintenance in telomerase-positive iPSCs and reveal distinct iPSC-specific responses to targeted telomeric DNA damage.

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人类诱导多能干细胞中的端粒DNA断裂触发ATR介导的阻滞和端粒酶非依赖性的端粒损伤修复。
尽管在分化细胞中端粒保护的机制是明确的,但人们对干细胞如何感知和应对端粒功能障碍知之甚少。特别是,这些细胞中端粒双链断裂(DSBs)的更广泛影响的特征较差。在此,我们报道了人类诱导多能干细胞(iPSC)对端粒内部DSBs的DNA损伤信号、细胞周期和转录组水平变化。我们用可诱导的TRF1-FokI融合蛋白改造了人iPSC,以在端粒处急性诱导DSBs。使用该模型,我们证明TRF1-FokI-DSBs激活ATR依赖性DDR,这导致G2中p53非依赖性细胞周期停滞。使用CRISPR-Cas9来削弱端粒酶的催化结构域,我们发现端粒酶对于TRF1-FokI切割的端粒的存活和延长在很大程度上是可有可无的,相反,它们可以通过强大的同源重组(HR)有效修复。与小鼠胚胎干细胞中基于HR的端粒维持相反,我们既没有发现HR导致端粒延长超过其初始长度的证据,也没有发现ZSCAN4在这一过程中的明显作用。相反,基于HR的端粒断裂修复足以将iPSC端粒维持在正常长度,这与细胞在TRF1-FokI诱导的几天内的持续存活相兼容。我们的研究结果表明,HR在端粒酶阳性的iPSC的端粒维持中的作用以前未被重视,并揭示了不同的iPSC对靶向端粒损伤的特异性反应。
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来源期刊
CiteScore
9.60
自引率
1.80%
发文量
1383
期刊介绍: The Journal of Molecular Cell Biology ( JMCB ) is a full open access, peer-reviewed online journal interested in inter-disciplinary studies at the cross-sections between molecular and cell biology as well as other disciplines of life sciences. The broad scope of JMCB reflects the merging of these life science disciplines such as stem cell research, signaling, genetics, epigenetics, genomics, development, immunology, cancer biology, molecular pathogenesis, neuroscience, and systems biology. The journal will publish primary research papers with findings of unusual significance and broad scientific interest. Review articles, letters and commentary on timely issues are also welcome. JMCB features an outstanding Editorial Board, which will serve as scientific advisors to the journal and provide strategic guidance for the development of the journal. By selecting only the best papers for publication, JMCB will provide a first rate publishing forum for scientists all over the world.
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