Homogeneous reporter system enables quantitative functional assessment of multiple transcription factors

Abstract

We developed a high-content reporter system that allows quantitative assessment of activities of multiple transcription factors (TFs) in a eukaryotic cell. The system comprises a library of reporter constructs that are evaluated according to their transcription rates. All reporters produce essentially identical messages that are subjected to 'processing', which generates a spectrum of distinguishable fragments that are analyzed quantitatively. The homogeneity of the reporter library afforded inherently uniform detection conditions for all reporters and provided repeatability, accuracy and robustness of assessment. We showed that this technology can be used to identify pathways transmitting cell responses to inducers, and that the profile of TF activities generated using this system represents a stable and sustained cell signature that clearly distinguishes different cell types and pathological conditions. This technology provides a framework for functional characterization of signal transduction networks through profiling activities of multiple TFs.

Central role of liver in anticancer and radioprotective activities of Toll-like receptor 5 agonist

“…(A) Schematic illustration of the in vivo FACTORIAL assay used to profile changes in the activities of transcription factors in the mouse liver following in vivo CBLB502 or LPS treatment. Two weeks after library transfection, mice were injected with CBLB502 (5 μg per mouse), LPS (10 μg per mouse), or PBS, and RTU activities in total liver RNA samples were isolated 1 h later and analyzed for the RTU activity as previously described (18). Description of transcription factors listed in the table can be found in www.attagene.com/cis-1-list.…”

“…Mice were allowed to recover after transfection for 2 wk and then were injected (s.c.) with CBLB502 (5 μg per mouse), LPS (10 μg per mouse), or PBS (eight mice per group). RTU activities were evaluated in total liver RNA samples collected 1 h later using multiplex RT-PCR detection with RTU-specific primers (18). Changes in transcriptional factor activities were calculated by normalizing the mean activities of RTUs in the livers of mice treated with CBLB502 or LPS to those in PBS-treated mice.…”

“…To identify perturbations in signaling pathways in the mouse liver in response to TLR5 activation, we used a multiplexed reporter system (the FACTORIAL) enabling a simultaneous assessment of multiple transcription factor families within cells (18). As shown schematically in Fig.…”

“…Our focus was, foremost, on identification of the tissues and cells that are direct primary responders to CBLB502 and, subsequently, on analysis of the molecular effects of CBLB502 in primary responders (e.g., induction of signaling pathways, production of bioactive factors). These goals were met by (i) analyzing tissue specificity of TLR5 response by using NF-κB reporter mice that enable detection of organ sites responding to CBLB502, (ii) an in vivo adaptation of the recently developed FACTORIAL assay (18) to monitor the response of multiple transcription factors to CBLB502 simultaneously, and (iii) a surgical procedure for temporary occlusion of blood circulation through the liver to test the biological effects of factors secreted by liver cells in response to CBLB502. This work defined the liver as the major primary target organ of CBLB502, leading to activation of several prosurvival and immunoregulatory signaling pathways, as well as dramatic changes in the spectrum of secreted factors and immune cell content.…”

Accounting for all the factors

“…Now, researchers at Attagene Inc. have published in Nature Methods an approach to simultaneously report on the activity of a panel of several dozen different transcription factors in cells. 1 Genomics and transcriptomics service providers think the tool could have utility in toxicology studies while complementing existing techniques for profiling transcriptional activity within cells.…”