Shanghai Institute of Hematology, State Key Laboratory of Medical Genomics, National Research Center for Translational Medicine, Rui-Jin Hospital, School of Medicine and School of Life Sciences and Biotechnology, Shanghai JiaoTong University, Shanghai, 200025, P. R. China.
Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan, 610044, P. R. China.
Cancer Commun (Lond). 2021 Nov;41(11):1116-1136. doi: 10.1002/cac2.12234. Epub 2021 Oct 26.
Abnormal alternative splicing is frequently associated with carcinogenesis. In B-cell acute lymphoblastic leukemia (B-ALL), double homeobox 4 fused with immunoglobulin heavy chain (DUX4/IGH) can lead to the aberrant production of E-26 transformation-specific family related gene abnormal transcript (ERG ) and other splicing variants. However, the molecular mechanism underpinning this process remains elusive. Here, we aimed to know how DUX4/IGH triggers abnormal splicing in leukemia.
The differential intron retention analysis was conducted to identify novel DUX4/IGH-driven splicing in B-ALL patients. X-ray crystallography, small angle X-ray scattering (SAXS), and analytical ultracentrifugation were used to investigate how DUX4/IGH recognize double DUX4 responsive element (DRE)-DRE sites. The ERG biogenesis and B-cell differentiation assays were performed to characterize the DUX4/IGH crosslinking activity. To check whether recombination-activating gene 1/2 (RAG1/2) was required for DUX4/IGH-driven splicing, the proximity ligation assay, co-immunoprecipitation, mammalian two hybrid characterizations, in vitro RAG1/2 cleavage, and shRNA knock-down assays were performed.
We reported previously unrecognized intron retention events in C-type lectin domain family 12, member A abnormal transcript (CLEC12A ) and chromosome 6 open reading frame 89 abnormal transcript (C6orf89 ), where also harbored repetitive DRE-DRE sites. Supportively, X-ray crystallography and SAXS characterization revealed that DUX4 homeobox domain (HD)1-HD2 might dimerize into a dumbbell-shape trans configuration to crosslink two adjacent DRE sites. Impaired DUX4/IGH-mediated crosslinking abolishes ERG , CLEC12A , and C6orf89 biogenesis, resulting in marked alleviation of its inhibitory effect on B-cell differentiation. Furthermore, we also observed a rare RAG1/2-mediated recombination signal sequence-like DNA edition in DUX4/IGH target genes. Supportively, shRNA knock-down of RAG1/2 in leukemic Reh cells consistently impaired the biogenesis of ERG , CLEC12A , and C6orf89 .
All these results suggest that DUX4/IGH-driven DNA crosslinking is required for RAG1/2 recruitment onto the double tandem DRE-DRE sites, catalyzing V(D)J-like recombination and oncogenic splicing in acute lymphoblastic leukemia.
异常剪接与癌发生密切相关。在 B 细胞急性淋巴细胞白血病（B-ALL）中，双同源盒 4 融合免疫球蛋白重链（DUX4/IGH）可导致 E-26 转化特异性家族相关基因异常转录物（ERG）和其他剪接变体的异常产生。然而，这一过程的潜在分子机制仍难以捉摸。在这里，我们旨在了解 DUX4/IGH 如何引发白血病中的异常剪接。
采用差异内含子保留分析鉴定 B-ALL 患者中新型 DUX4/IGH 驱动的剪接。X 射线晶体学、小角度 X 射线散射（SAXS）和分析超速离心用于研究 DUX4/IGH 如何识别双 DUX4 反应元件（DRE）-DRE 位点。进行 ERG 生物发生和 B 细胞分化测定以表征 DUX4/IGH 交联活性。为了检查 RAG1/2 是否需要 DUX4/IGH 驱动的剪接，进行了邻近连接测定、共免疫沉淀、哺乳动物双杂交表征、体外 RAG1/2 切割和 shRNA 敲低测定。
我们之前报道了 C 型凝集素结构域家族 12 成员 A 异常转录物（CLEC12A）和染色体 6 开放阅读框 89 异常转录物（C6orf89）中的未被识别的内含子保留事件，这两个转录物也含有重复的 DRE-DRE 位点。X 射线晶体学和 SAXS 表征支持 DUX4 同源结构域（HD）1-HD2 可能二聚形成哑铃形反式构型以交联两个相邻的 DRE 位点。DUX4/IGH 介导的交联受损会破坏 ERG、CLEC12A 和 C6orf89 的生物发生，从而显著减轻其对 B 细胞分化的抑制作用。此外，我们还观察到 DUX4/IGH 靶基因中罕见的 RAG1/2 介导的重组信号序列样 DNA 编辑。支持性的是，在白血病 Reh 细胞中 shRNA 敲低 RAG1/2 一致地损害了 ERG、CLEC12A 和 C6orf89 的生物发生。
所有这些结果表明，DUX4/IGH 驱动的 DNA 交联对于 RAG1/2 招募到双串联 DRE-DRE 位点、催化 V(D)J 样重组和急性淋巴细胞白血病中的致癌剪接是必需的。
