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

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.

The Human Cytomegalovirus pUL145 Isoforms Act as Viral DDB1-Cullin-Associated Factors to Instruct Host Protein Degradation to Impede Innate Immunity

“…By binding to their cognate receptors, IFNs signal via receptor-associated Janus kinases (JAK) to activate signal transducer and activator of transcription (STAT) proteins by phosphorylation (Stark and Darnell, 2012). Activated STAT dimers translocate to the nucleus to regulate the expression of cellular IFN-stimulated genes (ISGs) and IFN-repressed genes (IRepGs) (Megger et al, 2017;Trilling et al, 2013). In the absence of STAT1 or STAT2, the host succumbs a few days after CMV infection, as shown by studies using mouse CMV (MCMV) (Gil et al, 2001;Le-Trilling et al, 2018).…”

Deciphering of the Human Interferon-Regulated Proteome by Mass Spectrometry-Based Quantitative Analysis Reveals Extent and Dynamics of Protein Induction and Repression

“…Beside this two canonical signaling pathways (activating ISRE and GAS) separating type I and III IFNs from type II IFNs, several non-canonical signaling events have been described: for example, IFN-I induce STAT1 homodimers (in this case called alpha activated factor) which elicit an IFNγ-like response ( 18 ) and IFNγ induces STAT2 and IRF9 containing complexes which stimulate ISRE-like responses ( 19 – 22 ). Beneath this receptor proximal signaling events, cross-talk can also be induced down-stream by induction of a second layer of transcription factors (e.g., IRFs): IFNγ strongly induces IRF1 which in turn can enhance genes harboring IRF-E sites.…”

“…A great wealth of IFN-stimulated genes (ISGs) have been described in the past using various techniques. However, we and others have provided evidence that especially IFNγ can also repress the transcription of a considerable number of genes, which we termed IFN-repressed genes (IRepGs) ( 22 ). This finding raises the apparent and relevant question if such a regulation results in an altered protein composition of IFN-exposed cells—especially in humans.…”

Attack, parry and riposte: molecular fencing between the innate immune system and human herpesviruses

“…Depending on cell type, duration, conditions of treatment and IFN concentration, the transcription of several dozen to several hundred genes is rapidly changed. In the past, most effort was spend on the functional analysis of IFN‐induced genes ‐ but IFNs also repress several genes [termed IFN‐repressed genes (IRepGs)] .…”

“…But IFNs also upregulate feed‐back inhibitors, such as Usp18, SOCS and PIAS proteins, to avoid an overshooting innate immune response. In fibroblasts, a global block of down‐stream regulations (by a block of translation) revealed a prevailing role of negative regulation .…”