Bidirectional reporter assay using HAL promoter and TOPFLASH improves specificity in high-throughput screening of Wnt inhibitors.

Abstract:

: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.

journal_name

Biotechnol Bioeng

authors

Yamaguchi K,Zhu C,Ohsugi T,Yamaguchi Y,Ikenoue T,Furukawa Y

doi

10.1002/bit.26394

subject

Has Abstract

pub_date

2017-12-01 00:00:00

pages

2868-2882

issue

12

eissn

0006-3592

issn

1097-0290

journal_volume

114

pub_type

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