A common atopy‐associated variant in the Th2 cytokine locus control region impacts transcriptional regulation and alters SMAD3 and SP1 binding
A common atopy‐associated variant in the Th2 cytokine locus control region impacts transcriptional regulation and alters SMAD3 and SP1 binding
复制标题
Th2 细胞因子基因座控制区中常见的特应性相关变异影响转录调控并改变 SMAD3 和 SP1 结合
作者：
Kretschmer A;Möller G;Laumen H;Toerne C;Schramm K; Weidinger S
研究背景由Th 2细胞介导的以IL 4、IL 5和IL 13为特征的2型免疫应答在特应性疾病中起主要的致病作用。在全基因组关联研究（GWAS）中，人类Th 2细胞因子基因座的单核苷酸多态性，特别是DNA修复基因RAD 50内的基因座控制区，含有几个RAD 50 DNA酶1-超敏位点（RHS），与特应性性状密切相关。IL 13的功能变体已被深入研究，然而尚未鉴定出IL 13非依赖性RAD 50信号的致病变体。本研究旨在表征特异性相关的多态性rs 2240032位于人RHS 7顺式调节活性和转录factors.MethodsDifferential结合的功能影响，通过电泳迁移率变动分析（EMSAs）与Jurkat T细胞核提取物的结合进行了分析。使用质谱法（LC-MS/MS）鉴定差异结合因子。报告载体构建携带的主要或次要等位基因的rs 2240032进行了测试，用于调节转录活性在Jurkat和HeLa cells.ResultsThe变体rs 2240032影响转录活性和等位基因特异性结合SMAD 3，SP1，和其他推定的蛋白质复合物的合作伙伴。我们进一步证明，rs 2240032是位于RHS 7亚基，它本身包括阻遏活性，并可能是重要的微调转录regulation within this region.ConclusionThe人类RHS 7 critically有助于基因转录的调节，和常见的特应性相关的多态性rs 2240032影响转录活性和转录因子结合。
BackgroundType 2 immune responses directed by Th2 cells and characterized by the signature cytokines IL4, IL5, and IL13 play major pathogenic roles in atopic diseases. Single nucleotide polymorphisms in the human Th2 cytokine locus in particular in a locus control region within the DNA repair geneRAD50, containing severalRAD50DNase1‐hypersensitive sites (RHS), have been robustly associated with atopic traits in genome‐wide association studies (GWAS). Functional variants inIL13have been intensely studied, whereas no causative variants for theIL13‐independentRAD50signal have been identified yet. This study aimed to characterize the functional impact of the atopy‐associated polymorphism rs2240032 located in the human RHS7 oncis‐regulatory activity and differential binding of transcription factors.MethodsDifferential transcription factor binding was analyzed by electrophoretic mobility shift assays (EMSAs) with Jurkat T‐cell nuclear extracts. Identification of differentially binding factors was performed using mass spectrometry (LC‐MS/MS). Reporter vector constructs carrying either the major or minor allele of rs2240032 were tested for regulating transcriptional activity in Jurkat and HeLa cells.ResultsThe variant rs2240032 impacts transcriptional activity and allele‐specific binding of SMAD3, SP1, and additional putative protein complex partners. We further demonstrate that rs2240032 is located in an RHS7 subunit which itself encompasses repressor activity and might be important for the fine‐tuning of transcription regulation within this region.ConclusionThe human RHS7 critically contributes to the regulation of gene transcription, and the common atopy‐associated polymorphism rs2240032 impacts transcriptional activity and transcription factor binding.
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