Procaspase activating compound 1 controls tetracycline repressor-regulated gene expression system.

Chiman Song,Namkyoung Kim,Taebo Sim,Miri Park,Ki-Bong Oh,Jiyeon Lee

doi:10.1042/bsr20180793

Abstract

The tetracycline repressor (TetR)-regulated system is a widely used tool to study gene functions through control of its expression. Various effectors such as tetracycline (Tc) and doxycycline (Dox) quickly induce or shut down gene expression, but reversing gene expression has not been eligible due to long half-lives of such effectors. Here, we found that procaspase activating compound 1 (PAC-1) rapidly reduces transient expression of TetR-regulated green fluorescent protein (GFP) in mammalian cells. Next, we applied PAC-1 to control of expression of transient receptor potential melastatin 7 (TRPM7) protein, whose downstream cellular events can be monitored by cell morphological changes. We observed that PAC-1 quickly reduces TRPM7 expression, consequently affecting cell morphology regulated by TRPM7. The present study demonstrates the first small molecule that efficiently turns off the TetR-regulated gene expression in mammalian cells, thereby precisely regulating the expression level of target gene.

Highlights

Techniques for gene expression regulation are valuable tools to characterize genes in organisms
green fluorescent protein (GFP) mRNA expression levels were measured by Reverse transcription PCR (RT-PCR) analysis at 0, 2, and 8 h after simultaneous treatment with 1 μM Dox and procaspase activating compound 1 (PAC-1) at 0, 10, and 100 μM (Figure 1A)
Residual GFP mRNA expression was observed in the absence of Dox, which may be due to Tc contamination in fetal bovine serum (FBS)

Summary

Introduction

Techniques for gene expression regulation are valuable tools to characterize genes in organisms. Fast on/off switches will enhance the capability of the TetR-regulated system to study biological roles of gene of interest in developmental processes. It is, yet challenging to achieve fast on/off switching due to the long half-life of Tc [17]. A small molecule, GR33076X, is an antagonist acting on the transactivator, tTA in bacterial cells [12]. These are only four antagonists reported to date, so additional efforts are needed to discover novel molecules that rapidly switch on/off target gene expression

Methods

Results

Conclusion