TALEN-mediated enhancer knockout influences TNFAIP3 gene expression and mimics a molecular phenotype associated with systemic lupus erythematosus.
TALEN-mediated enhancer knockout influences TNFAIP3 gene expression and mimics a molecular phenotype associated with systemic lupus erythematosus.
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DOI：
10.1038/gene.2016.4
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发表时间：
2016-04
影响因子：
5
通讯作者：
Gaffney PM
中科院分区：
文献类型：
--
作者：
Wang S;Wen F;Tessneer KL;Gaffney PM
连锁不平衡对特定疾病相关易感性变体的功能表征提出了重大挑战。精确的基因组编辑技术为应对这一挑战提供了新的机会。作为概念验证，我们采用TALEN介导的基因组编辑来特异性破坏TT>A增强子区域，以模拟在缺乏其他连锁不平衡相关变体的同基因HEK 293 T细胞系中系统性狼疮相关易感基因TNFAIP 3中鉴定的候选致病变体。TT>A增强子的靶向破坏通过长距离DNA环破坏了其与TNFAIP 3启动子的相互作用，从而降低了TNFAIP 3基因表达。TNFAIP 3 mRNA及其编码蛋白A20的缺失损害了TNFα诱导的受体介导的NF-κB信号下调;这是自身免疫的标志。结果表明，TT>A增强子变体有助于因果关系，并且独立于其他变体发挥功能以破坏TNFAIP 3表达。此外，我们相信这种方法可以在未来独立地检查其他候选因果变量。
Linkage disequilibrium poses a major challenge to the functional characterization of specific disease-associated susceptibility variants. Precision genome editing technologies have provided new opportunities to address this challenge. As proof-of-concept, we employed TALEN-mediated genome editing to specifically disrupt the TT>A enhancer region to mimic candidate causal variants identified in the systemic lupus erythematosus-associated susceptibility gene, TNFAIP3, in an isogenic HEK293T cell line devoid of other linkage disequilibrium-associated variants. Targeted disruption of the TT>A enhancer impaired its interaction with the TNFAIP3 promoter by long-range DNA looping, thereby reducing TNFAIP3 gene expression. Loss of TNFAIP3 mRNA and its encoded protein, A20, impaired TNFα-induced receptor-mediated downregulation of NF-κB signaling; a hallmark of autoimmunity. Results demonstrate that the TT>A enhancer variants contribute to causality and function independently of other variants to disrupt TNFAIP3 expression. Further, we believe this approach can be implemented to independently examine other candidate casual variants in the future.