DNA crosslinking and recombination-activating genes 1/2 (RAG1/2) are required for oncogenic splicing in acute lymphoblastic leukemia.

Abstract

BACKGROUND:

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 (ERGalt ) 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.

METHODS:

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 ERGalt 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.

RESULTS:

We reported previously unrecognized intron retention events in C-type lectin domain family 12, member A abnormal transcript (CLEC12Aalt ) and chromosome 6 open reading frame 89 abnormal transcript (C6orf89alt ), 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 ERGalt , CLEC12Aalt , and C6orf89alt 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 ERGalt , CLEC12Aalt , and C6orf89alt .

CONCLUSIONS:

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.

• Authors: Hao Zhang, Nuo Cheng, Zhihui Li, Ling Bai, Chengli Fang, Yuwen Li, Weina Zhang, Xue Dong, Minghao Jiang, Yang Liang, Sujiang Zhang, Jianqing Mi, Jiang Zhu, Yu Zhang, Sai-Juan Chen, Yajie Zhao, Xiang-Qin Weng, Weiguo Hu, Zhu Chen, Jinyan Huang, Guoyu Meng
• Journal: Cancer communications (London, England) (Cancer Commun (Lond)) Vol. 41 Issue 11 Pg. 1116-1136 (11 2021) ISSN: 2523-3548 [Electronic] United States
• PMID: 34699692 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2021 The Authors. Cancer Communications published by John Wiley & Sons Australia, Ltd. on behalf of Sun Yat-sen University Cancer Center.

Chemical References

• CLEC12A protein, human
• Homeodomain Proteins
• Lectins, C-Type
• Receptors, Mitogen
• DNA

Topics

• Animals
• Carcinogenesis
• DNA
• Homeodomain Proteins (genetics)
• Humans
• Lectins, C-Type
• Precursor Cell Lymphoblastic Leukemia-Lymphoma
• Receptors, Mitogen
• Recombination, Genetic
• Scattering, Small Angle
• X-Ray Diffraction