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Bidirectional reporter assay using HAL promoter and TOPFLASH improves specificity in high‐throughput screening of Wnt inhibitors
Author(s) -
Yamaguchi Kiyoshi,
Zhu Chi,
Ohsugi Tomoyuki,
Yamaguchi Yuko,
Ikenoue Tsuneo,
Furukawa Yoichi
Publication year - 2017
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.26394
Subject(s) - wnt signaling pathway , reporter gene , enhancer , biology , signal transduction , microbiology and biotechnology , gene , transcription factor , gene expression , biochemistry
Constitutive activation of Wnt signaling plays an important role in colorectal and liver tumorigenesis. Cell‐based assays using synthetic TCF/LEF (T‐cell factor/lymphoid enhancer factor) reporters, as readouts of β‐catenin/TCF‐dependent transcriptional activity, have contributed greatly to the discovery of small molecules that modulate Wnt signaling. In the present study, we report a novel screening method, called a bidirectional dual reporter assay. Integrated transcriptome analysis identified a histidine ammonia‐lyase gene ( HAL ) that was negatively regulated by β‐catenin/TCF‐dependent transcriptional activity. We leveraged a promoter region of the HAL gene as another transcriptional readout of Wnt signaling. Cells stably expressing both an optimized HAL reporter and the TCF/LEF reporter enabled bidirectional reporter activities in response to Wnt signaling. Increased HAL reporter activity and decreased TCF/LEF reporter activity were observed simultaneously in the cells when β‐catenin/TCF7L2 was inhibited. Notably, this method could decrease the number of false positives observed when screening an inhibitor library compared with the conventional TCF/LEF assay. We found that Brefeldin A, a disruptor of the Golgi apparatus, inhibited the Wnt/β‐catenin signaling pathway. The utility of our system could be expanded to examine other disease‐associated pathways beyond the Wnt/β‐catenin signaling pathway.