Analysis of MUTYH alternative transcript expression, promoter function, and the effect of human genetic variants
Analysis of MUTYH alternative transcript expression, promoter function, and the effect of human genetic variants
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DOI：
10.1002/humu.23709
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发表时间：
2019-04-01
期刊：
影响因子：
3.9
通讯作者：
Plotz, Guido
中科院分区：
文献类型：
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作者：
Koeger, Nicole;Brieger, Angela;Plotz, Guido
人类 DNA 修复基因 MUTYH 包含三个替代的第一外显子，其突变缺失会导致结直肠息肉病和癌症易感性。为了分析人类中发现的替代转录和基因改变的影响，我们建立了支持转录和剪接的基于细胞的小基因实验模型，并彻底验证了其功能。我们鉴定了高度保守的启动子区域并将其在小基因中灭活，并且还引入了六种人类变体。此外，还讨论了 CpG 岛甲基化和特定转录因子对 MUTYH 转录的潜在贡献。这些发现将调控作用归因于启动子中的三个保守基序：M4 基序、转录因子 IIB 识别元件和 GC 盒。此外，数据显示，三种患者变异损害了 MUTYH 表达，因此有可能引起致病作用。我们没有发现 CpG 岛甲基化的生物学相关贡献或 DNA 损伤直接转录激活的证据。除了深入了解 MUTYH 转录的调控之外，这项工作还提供了一个功能性 MUTYH 小基因实验系统，适合作为分析患者变异的诊断工具，以及启动子的功能图谱，也可以促进人类变异的致病性分类。
The human DNA repair gene MUTYH, whose mutational loss causes a colorectal polyposis and cancer predisposition, contains three alternative first exons. In order to analyze alternative transcription and the effect of genetic alterations found in humans, we established a cell-based minigene experimental model supporting transcription and splicing and thoroughly verified its functionality. We identified highly conserved promoter areas and inactivated them in the minigene, and also introduced six human variants. Moreover, the potential contribution of CpG island methylation and specific transcription factors on MUTYH transcription was addressed. The findings allowed to attribute regulatory roles to three conserved motifs in the promoter: an M4 motif, a transcription factor IIB recognition element, and a GC box. Moreover, the data showed that three patient variants compromised MUTYH expression and therefore have the potential to cause pathogenic effects. We did not find evidence for a biologically relevant contribution of CpG island methylation or a direct transcriptional activation by DNA damage. Besides insight into the regulation of MUTYH transcription, the work therefore provides a functional MUTYH minigene experimental system suitable as a diagnostic tool for analyzing patient variants, and a functional map of the promotor that also can facilitate pathogenicity classifications of human variants.