A germline HLTF mutation in familial MDS induces DNA damage accumulation through impaired PCNA polyubiquitination
A germline HLTF mutation in familial MDS induces DNA damage accumulation through impaired PCNA polyubiquitination
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DOI：
10.1038/s41375-019-0385-0
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发表时间：
2019-07-01
期刊：
影响因子：
11.4
通讯作者：
Kurokawa, Mineo
中科院分区：
文献类型：
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作者：
Takaoka, Kensuke;Kawazu, Masahito;Kurokawa, Mineo
虽然已经确定了家族性骨髓增生异常综合征（MDS）的几个致病基因，但其遗传格局和分子发病机制尚不完全清楚。为了探索新的驱动基因及其致病意义，我们对来自家族性MDS谱系的4个个体进行了全外显子组序列分析，并鉴定出10个候选单核苷酸变异（C9orf43、CYP7B1、EFHB、ENTPD7、FAM160B2、HELZ2、HLTF、INPP5J、ITPKB和RYK）。敲低筛选显示，下调Hltf可增强小鼠原代骨髓干细胞/祖细胞的集落形成能力。γ - H2AX免疫荧光染色显示，异位表达HLTF E259K的人急性髓性白血病（AML）细胞系DNA损伤增加，而在表达野生型HLTF的细胞中未观察到这种损伤。人类AML细胞中HLTF的沉默也会导致DNA损伤，这表明HLTF E259K是一种功能缺失突变。在分子上，我们发现E259K突变降低了HLTF与泛素偶联酶、甲磺酸敏感2和泛素偶联酶E2N的结合能力，导致HLTF E259K转导的细胞中增殖细胞核抗原（PCNA）的多泛素化受损。总之，我们的研究结果表明，家族性mds相关的HLTF E259K种系突变诱导DNA双链断裂的积累，可能是通过受损的PCNA多泛素化。
Although several causal genes of familial myelodysplastic syndromes (MDS) have been identified, the genetic landscape and the molecular pathogenesis are not totally understood. To explore novel driver genes and their pathogenetic significance, we performed whole-exome sequence analysis of four individuals from a familial MDS pedigree and 10 candidate single-nucleotide variants (C9orf43, CYP7B1, EFHB, ENTPD7, FAM160B2, HELZ2, HLTF, INPP5J, ITPKB, and RYK) were identified. Knockdown screening revealed that Hltf downregulation enhanced colony-forming capacity of primary murine bone marrow (BM) stem/progenitor cells. gamma H2AX immunofluorescent staining assay revealed increased DNA damage in a human acute myeloid leukemia (AML) cell line ectopically expressing HLTF E259K, which was not observed in cells expressing wild-type HLTF. Silencing of HLTF in human AML cells also led to DNA damage, indicating that HLTF E259K is a loss-of-function mutation. Molecularly, we found that an E259K mutation reduced the binding capacity of HLTF with ubiquitin-conjugating enzymes, methanesulfonate sensitive 2 and ubiquitin-conjugating enzyme E2N, resulting in impaired polyubiquitination of proliferating cell nuclear antigen (PCNA) in HLTF E259K-transduced cells. In summary, our results indicate that a familial MDS-associated HLTF E259K germline mutation induces accumulation of DNA double-strand breaks, possibly through impaired PCNA polyubiquitination.