Deciphering the modulation of gene expression by type I and II interferons combining 4sU-tagging, translational arrest and in silico promoter analysis.

Mirko Trilling,Nicolás Bellora,Olivia Prazeres Da Costa,Paul Dickinson,Andrzej J Rutkowski,Kevin Robertson,Miranda De Graaf,Caroline C Friedel,Lars Dölken,Peter Ghazal,M Mar Albà

doi:10.1093/nar/gkt589

Abstract

Interferons (IFN) play a pivotal role in innate immunity, orchestrating a cell-intrinsic anti-pathogenic state and stimulating adaptive immune responses. The complex interplay between the primary response to IFNs and its modulation by positive and negative feedback loops is incompletely understood. Here, we implement the combination of high-resolution gene-expression profiling of nascent RNA with translational inhibition of secondary feedback by cycloheximide. Unexpectedly, this approach revealed a prominent role of negative feedback mechanisms during the immediate (≤60 min) IFNα response. In contrast, a more complex picture involving both negative and positive feedback loops was observed on IFNγ treatment. IFNγ-induced repression of genes associated with regulation of gene expression, cellular development, apoptosis and cell growth resulted from cycloheximide-resistant primary IFNγ signalling. In silico promoter analysis revealed significant overrepresentation of SP1/SP3-binding sites and/or GC-rich stretches. Although signal transducer and activator of transcription 1 (STAT1)-binding sites were not overrepresented, repression was lost in absence of STAT1. Interestingly, basal expression of the majority of these IFNγ-repressed genes was dependent on STAT1 in IFN-naïve fibroblasts. Finally, IFNγ-mediated repression was also found to be evident in primary murine macrophages. IFN-repressed genes include negative regulators of innate and stress response, and their decrease may thus aid the establishment of a signalling perceptive milieu.

Highlights

Interferons (IFNs) are soluble factors secreted on infection and capable of interfering with viral replication [1]
As changes in gene expression following a stimulus generally take $3–6 h to become apparent in total RNA for the majority of genes, prolonged translational arrest is required to elucidate the nature of the observed changes. 4sU tagging provides quantitative data on the kinetics of transcriptional changes in a time scale of 30 min [21], thereby detailing the real-time kinetics of transcription factors activity
Differential gene expression caused by both IFNa and IFNg treatment was significantly enhanced on ablation of translation by CHX, revealing a dominant global role of negative feedback loops already within the first hour of treatment

Summary

Introduction

Interferons (IFNs) are soluble factors secreted on infection and capable of interfering ( the name) with viral replication [1]. IFNs lack direct intrinsic anti-viral capabilities and solely act as cytokines in an autocrine and paracrine manner initiating a global anti-infective change in the gene expression profile. IFNs induce many genes that are detrimental to cell survival and cell proliferation so that the expression of IFN-stimulated genes (ISGs) has to be prevented under ‘healthy’ or ‘uninfected’ conditions. A robust defensive state is rapidly initiated to outpace microbial gene expression and replication. Owing to this binary nature, alternating between almost complete shut-off and rapid induction, IFNs have frequently been used as a model system to study stimulus-induced gene expression changes.

Methods

Results

Conclusion