GSK3ß-SCFFBXW7α mediated phosphorylation and ubiquitination of IRF1 are required for its transcription-dependent turnover.

IRF1 (Interferon Regulatory Factor-1) is the prototype of the IRF family of DNA binding transcription factors. IRF1 protein expression is regulated by transient up-regulation in response to external stimuli followed by rapid degradation via the ubiquitin-proteasome system. Here we report that DNA bound IRF1 turnover is promoted by GSK3ß (Glycogen Synthase Kinase 3ß) via phosphorylation of the T181 residue which generates a phosphodegron for the SCF (Skp-Cul-Fbox) ubiquitin E3-ligase receptor protein Fbxw7α (F-box/WD40 7). This regulated turnover is essential for IRF1 activity, as mutation of T181 results in an improperly stabilized protein that accumulates at target promoters but fails to induce RNA-Pol-II elongation and subsequent transcription of target genes. Consequently, the anti-proliferative activity of IRF1 is lost in cell lines expressing T181A mutant. Further, cell lines with dysfunctional Fbxw7 are less sensitive to IRF1 overexpression, suggesting an important co-activator function for this ligase complex. As T181 phosphorylation requires both DNA binding and RNA-Pol-II elongation, we propose that this event acts to clear 'spent' molecules of IRF1 from transcriptionally engaged target promoters.

Genome-wide Identification of IRF1 Binding Sites Reveals Extensive Occupancy at Cell Death Associated Genes.

IRF1 is a transcription factor involved in interferon signaling and has been shown to harbor tumor suppressor activity. In order to comprehensively identify pathways regulated by IRF1, we used chromatin immunoprecipitation followed by massive-parallel sequencing (ChIP-seq) to evaluate the gene targets of IRF1 genome-wide. We identified 17,416 total binding events in breast cancer cells. Functional categorization of the binding sites after IFN-gamma (interferon-gamma) treatment determined that 'apoptosis' or 'cell death' is the most enriched target process. Motif discovery analysis of the chromosomal regions bound by IRF1 identified a number of unique motifs correlated with apoptosis, DNA damage and immune processes. Analysis of GEO transcriptome data from IRF1-transduced cells or IFN-gamma treated fibroblasts indicates that IRF1-bound targets in IFN-treated cells are associated with a positive transcriptional response. Many of the enriched target genes from the expression analysis are associated with apoptosis. Importantly, this data indicates that a significant function of IRF1 is the regulation of anti-cancer apoptotic pathways and this reinforces IRF1's role as a tumor suppressor.