Long noncoding RNA LINP1: scaffolding non-homologous end joining.
Long noncoding RNA LINP1: scaffolding non-homologous end joining.
复制标题
DOI：
10.1038/cddiscovery.2016.59
复制
发表时间：
2016
影响因子：
7
通讯作者：
Sieber OM
中科院分区：
文献类型：
--
作者：
Sakthianandeswaren A;Liu S;Sieber OM
非同源末端连接 (NHEJ) 是哺乳动物细胞中 DNA 双链断裂 (DSB) 修复的主要途径 1，但也参与抗原受体基因的组装和端粒维护。 2, 3 NHEJ 功能丧失可能导致染色体不稳定，促进恶性转化，特别是当其他细胞保护措施受到损害时。遗传性 NHEJ 缺陷是放射敏感性严重联合免疫缺陷的基础。 NHEJ 的启动是通过 Ku70-Ku80 异二聚体与 DNA 断裂的两端结合，然后招募 DNA 依赖性蛋白激酶 (DNA-PKcs) 的催化亚基形成并列的 DNA-PK 全酶，将末端连接到突触复合体中。粘性平 DNA 末端可以通过 XFL-XRCC4-DNA 连接酶 IV 复合物直接连接，但在许多情况下，DNA 末端含有受损碱基或 DNA 主链糖，需要在连接前进行处理。末端加工可能涉及核酸酶，例如Artemis、DNA聚合酶和多核苷酸激酶以及其他酶。 4, 5 《自然结构与分子生物学》，Zhang 等人。 6 最近报道在非同源末端连接 (NHEJ) 途径 1 (LINP1) 中发现了一种称为 lncRNA 的长非编码 RNA (lncRNA)，它作为与 Ku70-Ku80 和 DNA-PKcs 相互作用的 RNA 支架，促进 NHEJ 修复（图 1）。使用癌症基因组图谱和癌细胞系百科全书 (CCLE) 的 RNA-seq 数据与其他乳腺癌亚型相比，LINP1 被确定在三阴性乳腺癌 (TNBC) 中过度表达。在阿霉素（TNBC 的化疗药物）治疗后，LINP1 敲除增强了 TNBC 细胞系的细胞凋亡，而在雌激素受体阳性乳腺癌 (ER+ BC) 细胞系中，LINP1 过表达且 LINP1 检测不到，可保护这些细胞免受阿霉素诱导的细胞凋亡。使用 RNA Pull-down、CHART 和 RNA-IP 检测，Zhang 等人。图6证明LINP1与Ku70-Ku80和DNA-PKcs相互作用。具体而言，LINP1 转录物通过其 5' 区域与 Ku80 结合，并通过其 3' 区域与 DNA-PKcs 结合。 TNBC 细胞中的 LINP1 敲低导致彗星、γ-H2AX 焦点和 NHEJ 报告基因检测中 DNA 断裂修复减少。相反，ER+ BC 细胞中 LINP1 的过度表达会增加 NHEJ 活性。对 LINP1、Ku80 或 DNA-PKcs 敲低照射后染色质相关 NHEJ 复合物的分析表明，Ku70-Ku80 对 DSB 的识别导致 LINP1 和 DNA-PKcs 的募集，其中 LINP1 增强了 Ku80-DNA-PKcs 相互作用。确定 Ku80 和 DNA-PKcs 中介导 LINP1 相互作用的蛋白质结构域、LINP1 结合是否受 Ku70-Ku80 或 DNA-PKcs 的翻译后修饰和构象状态调节或影响，以及这对其他 NHEJ 组件的影响程度，将会很有趣。 EGF 信号传导被确定为 LINP1 表达的正调节因子。 LINP1 水平与原发性乳腺癌和 CCLE 细胞系中的 EGFR 表达相关。在 CCLE 细胞系中，LINP1 表达与 EGF 下游转录因子 c-Jun 和 c-Fox（均为 AP1 转录因子）相关，表明通过 RAS-MEK-JNK 途径进行调节。因此，EGF 处理在 TNBC 细胞中诱导 LINP1，但在 ER+ BC 细胞中则不然，并且 TNBC 细胞系中的 LINP1 表达可以被 EGFR、MEK 和 JNK 抑制剂抑制。 ENCODE ChIP-seq 数据表明 TNBC 细胞中 LINP1 启动子处 c-Jun 或 c-Fos 富集，经 ChIP-qPCR 验证，EGF 刺激后结合增加。 LINP1 启动子中的 AP1 结合位点是……
Non-homologous end joining (NHEJ) is a principal pathway of DNA double-strand break (DSB) repair in mammalian cells, 1 but is also involved in assembly of antigen receptor genes and telomere maintenance. 2, 3 Loss of NHEJ function can result in chromosome instability promoting malignant transformation, in particular when other cellular safeguards are compromised. Inherited NHEJ defects underlie radiosensitive severe combined immunodeficiency. NHEJ is initiated by binding of Ku70-Ku80 heterodimers to both ends of a DNA break, followed by recruitment of catalytic subunits of DNA-dependent protein kinase (DNA-PKcs) to form juxtaposed DNA-PK holoenzymes which join the ends in a synaptic complex. Cohesive blunt DNA ends can be joined directly by the XFL-XRCC4-DNA ligase IV complex, but in many cases DNA ends contain damaged bases or DNA backbone sugars that require processing before ligation. End processing may involve nucleases such as Artemis, DNA polymerases and polynucleotide kinase among other enzymes. 4, 5 In Nature Structural & Molecular Biology, Zhang et al. 6 recently reported the discovery of a long noncoding RNA (lncRNA) termed lncRNA in non-homologous end joining (NHEJ) pathway 1 (LINP1), that serves as an RNA scaffold interacting with Ku70-Ku80 and DNA-PKcs, promoting NHEJ repair (Figure 1). LINP1 was identified as overexpressed in triple-negative breast cancers (TNBCs) when compared with other breast cancer subtypes using RNA-seq data from The Cancer Genome Atlas and the Cancer Cell Line Encyclopedia (CCLE). LINP1 knockdown enhanced apoptosis in TNBC cell lines following doxorubicin treatment, a chemotherapy drug for TNBC, while overexpression of LINP1 in an estrogen-receptor positive breast cancer (ER+ BC) cell line with undetectable LINP1 protected these cells from doxorubicin-induced apoptosis. Using RNA pull-down, CHART and RNA-IP assays, Zhang et al. 6 demonstrated that LINP1 interacted with Ku70-Ku80 and DNA-PKcs. Specifically, LINP1 transcript bound to Ku80 via its 5′ region, and to DNA-PKcs via its 3′ region. LINP1 knockdown in TNBC cells resulted in reduced DNA break repair in comet, γ-H2AX foci and NHEJ reporter assays. Conversely, overexpression of LINP1 in ER+ BC cells increased NHEJ activity. Analyses of chromatin-associated NHEJ complexes post-irradiation with LINP1, Ku80 or DNA-PKcs knockdown indicated that recognition of DSBs by Ku70-Ku80 resulted in recruitment of LINP1 and DNA-PKcs, with LINP1 enhancing the Ku80-DNA-PKcs interaction. It will be interesting to determine the protein domains in Ku80 and DNA-PKcs mediating LINP1 interaction, whether LINP1 binding is regulated by or affects post-translational modifications and conformation states of Ku70-Ku80 or DNA-PKcs, and to what extent this impacts on other NHEJ components. EGF signaling was identified as a positive regulator of LINP1 expression. LINP1 levels were correlated with EGFR expression in primary breast cancers and CCLE cell lines. In CCLE cell lines, LINP1 expression was associated with the EGF downstream transcription factors c-Jun and c-Fox (both AP1 transcription factors), suggesting regulation via the RAS-MEK-JNK pathway. Accordingly, EGF treatment induced LINP1 in TNBC cells, but not in ER+ BC cells and LINP1 expression in TNBC cell lines could be repressed with EGFR, MEK and JNK inhibitors. ENCODE ChIP-seq data indicated enrichment of c-Jun or c-Fos at the LINP1 promoter in TNBC cells, validated by ChIP-qPCR with increased binding following EGF stimulation. An AP1-binding site in the LINP1 promoter was …