Chimeric Transcription Factor Assay

Introduction

In this assay, we investigated whether human cells (the EA.hy926 cell line) receive AHL, a signaling molecule that is synthesized in and exported from E. coli and induce the transcription of atIPT4 and log1 genes to synthesize iP.
AHLs, which stand for [N-]acyl-homoserine lactones, are small signaling molecules and are employed in the bacterial “Quorum Sensing”. Among several kinds of AHLs, 3OC8HSL (C8) was chosen in the assay.
In order to achieve C8-dependent transcription in human cells, a chimeric transcription factor named RelA/NLS/TraR was constructed. This protein is comprised of the transcription activating domain of RelA (a kind of human NF-kB family), nuclear localization signal (NLS), and full-length TraR. This protein can bind to an appropriate enhancer sequence to activate transcription only in the presence of C8 (see below for details).
Isopentenyladenine (iP) is kind of a cytokinin, and we use it as a signal molecule from human to E. coli cells and for the cross-kingdom communication. Cytokinins are the signaling molecules (or Phytohormones) that plants produce and play important roles in cell growth and differentiation. In the case of Arabidopsis thaliana, extracellular iP is received by a transmembrane receptor, AHK4. AHK4 has a histidine kinase activity, and binding of iP to AHK4 triggers auto-phosphorylation of AHK4 and the following histidine-to-aspartate phosphorelay. As a consequence, transcription from target genes is induced and/or repressed so that physiological states of plants are changed. Surprisingly, the histidine kinase activity of AHK4 has shown to be activated depending on iP even in E. coli cells (Suzuki et al. 2001, Lukáš Spíchal et al. 2004). This fact encouraged us to use iP as a signaling molecule in our project (Read AHK4 Assay page).

Summary

　As shown in Fig. 1, two plasmids were introduced into the EA.hy926 cells by electroporation. The CAG promoter (pCAG) constantly expresses RelA/NLS/TraR. When RelA/NLS/TraR binds to C8, this complex binds to the (tra box)₇ sequence and activates transcription from the CMV minimal promoter (CMV min). The (tra box)₇ sequence is the binding site for TraR (and also RelA/NLS/TraR) and works as the transcription enhancer. The core sequence of the (tra box)₇ is “ATGTGCAGATCTGCACAT” and this sequence is repeated seven times. As a result, the atIPT4  and log1 genes are transcribed depending on C8. The atIPT4 and log1 genes are derived from A. thaliana, and their products jointly work as iP synthetase.

IVS (intervining sequence) is one kind of introns and is important for increasing the mRNA stability in eukaryotic cells. IRES (internal ribosome entry site) is an RNA element that allows translation initiation in a cap-independent mannerThe term “polyA” indicates the polyadenylation signal that is important for the nuclear export, translation, and stability of mRNA.

Results

　The transformed cells were treated or not treated with different concentrations of C8. Then, the mRNA level of atIPT4 and log1 was analyzed using quantitative RT-PCR. As shown in Fig. 2, the CMV minimal promoter was activated following the addition of C8.

The term“Cont”means the control cells that are not electroporated, while “EP” means the electroporated cells. the concentrations of C8 used are indicated blow the bars.

Discussion

　We confirmed that the transcription of atIPT4 and log1 genes were induced by C8 addition and the degree of induction depends on C8 concentration.

Protocol

Reference

Petra Neddermann, Cesare Gargioli, Ester Muraglia, Sania Sambuncini, Fabio Bonelli, Raffaele De Francesco, Riccardo cortese (2003) A novel, inducible, eukaryotic gene expression system based on the quorum-sensing transcription facter TraR. EMBO reports VOL 4: 159-165.