Global functional profiling of human ubiquitome identifies E3 ubiquitin ligase DCST1 as a novel negative regulator of Type-I interferon signaling.

Sajith Nair,Manoj N Krishnan,Pradeep Bist,Neha Dikshit

doi:10.1038/srep36179

Abstract

Type I interferon (IFN-I) mediated innate immune response controls virus infections by inducing the expression of interferon stimulated genes (ISGs). Although ubiquitination plays key roles in immune signaling regulation, a human genome-wide understanding of the role of E3 ubiquitin ligases in interferon mediated ISG induction is lacking. Here, we report a genome-wide profiling of the effect of ectopic expression of 521 E3 ubiquitin ligases and substrate recognition subunits encoded in the human genome (which constitutes 84.4% of all ubiquitination related genes encoded in the human genome, hereafter termed Human Ubiquitome) on IFNβ mediated induction of interferon stimulated DNA response element (ISRE) driven reporter activity. We identified 96 and 42 genes of the human ubiquitome as novel negative and positive regulators of interferon signaling respectively. Furthermore, we characterized DCST1 as a novel E3 ubiquitin ligase negatively regulating interferon response. Ectopic expression and gene silencing of DCST1 respectively attenuated and increased ISRE reporter activity. DCST1 regulated Type I interferon signaling by interacting with and promoting ubiquitination-mediated degradation of STAT2, an essential component of antiviral gene induction. In summary, this study provided a systems level view on the role of human ubiquitination associated genes in Type I interferon response.

Highlights

The type-I interferon (IFN-I) family of cytokines (e.g., IFNα/β) is essential for controlling RNA viruses[1,2,3]
The high-throughput screening assay used a green fluorescent protein (GFP) based transcriptional reporter driven by human interferon stimulated DNA response element (ISRE) DNA element, in human HEK293T cells stimulated with IFNβ, a widely used in vitro system to model IFN-I signaling
We systematically investigated the effect of ectopic expression of DCST1 on the stability of major known protein components of IFN-I signaling such as IFNAR1, IFNAR2, TYK2, Janus kinase 1 (JAK1), STAT1, STAT2 and interferon regulatory factor 9 (IRF9)

Summary

Introduction

The type-I interferon (IFN-I) family of cytokines (e.g., IFNα/β) is essential for controlling RNA viruses[1,2,3]. The phosphatase, TC45 mediated dephosphorylation of STAT1 and Skp1-Cullin1-HOS-Roc[1] ubiquitin ligase controlled downregulation of IFNAR1 are known negative regulatory mechanisms attenuating IFN-I signaling[21,22]. Ubiquitination is well known to regulate several aspects of innate immune antiviral signaling, our understanding on the role of ubiquitination related processes involved in IFN-I signaling is less comprehensive[19,20,21,22,23]. The human genome encodes for around 617 genes involved in ubiquitination, comprising both the E3 ubiquitin ligases and substrate recognition subunits, together referred to hereafter as the human ubiquitome[24] Given their pivotal role in diverse cellular functions, it is highly likely that several members of the human ubiquitome might have unidentified roles in IFN-I signaling. There has not been any systematic genome-wide study to profile the role of the human ubiquitome in IFN-I signaling, through targeted experimental approaches

Methods

Results

Conclusion