Gideon Schreiber Lab

Interferon regulatory factor 1 (IRF1) plays a pivotal role in interferon (IFN) signaling. Here, we dissect the impact of IRF1 on gene transcription regulation in HeLa cells, by targeted knockout (KO) or overexpression of IRF1. IRF1 KO partially diminished IFN-γ but not IFN-β induced gene regulation. IRF1 KO did show a homeostatic role in basal transcript abundance, including increasing the abundance of antiviral gene transcripts, apparently through increased expression of other IRF genes. IRF1 overexpression induced potent antiviral protection, which is mediated by secretion of type I IFN proteins, particularly of IFN-α subtypes, which expression is driven by IRF1. This paracrine effect was confirmed by transcriptomics, cytokine profiling, and mass spectrometry. Surprisingly, antiviral protection was observed also in JAK1 KO or ruxolitinib-treated cells but not in type I IFN receptor KO cells, suggesting the involvement of noncanonical signaling pathways. Hierarchical clustering of RNA-seq data revealed distinct IFN-independent gene clusters activated or repressed by IRF1, including pathways related to adaptive immunity and T cell function. Using protein-binding microarrays and predictive modeling, we generated an energy-normalized binding matrix for IRF1, enabling sequence-specific prediction of promoter-binding affinities beyond classical consensus motifs. This approach allows estimation of IRF1-binding potential across diverse genomic contexts as validated for the IFIT2 gene promoter by a reporter assay. Evaluating the biological significance of our study, we show that IRF1 abundance varies by 10,000-fold between cell lines, with positive correlations of IRF1 with the abundance of gene transcripts involved in antiviral and immune-driving activities.

For drugs to be active they have to reach their targets. Within cells this requires crossing the cell membrane, and then free diffusion, distribution, and availability. Here, we explored the in-cell diffusion rates and distribution of a series of small molecular fluorescent drugs, in comparison to proteins, by microscopy and fluorescence recovery after photobleaching (FRAP). While all proteins diffused freely, we found a strong correlation between pKa and the intracellular diffusion and distribution of small molecule drugs. Weakly basic, small-molecule drugs displayed lower fractional recovery after photobleaching and 10- to-20-fold slower diffusion rates in cells than in aqueous solutions. As, more than half of pharmaceutical drugs are weakly basic, they, are protonated in the cell cytoplasm. Protonation, facilitates the formation of membrane impermeable ionic form of the weak base small molecules. This results in ion trapping, further reducing diffusion rates of weakly basic small molecule drugs under macromolecular crowding conditions where other nonspecific interactions become more relevant and dominant. Our imaging studies showed that acidic organelles, particularly the lysosome, captured these molecules. Surprisingly, blocking lysosomal import only slightly increased diffusion rates and fractional recovery. Conversely, blocking protonation by N-acetylated analogues, greatly enhanced their diffusion and fractional recovery after FRAP. Based on these results, N-acetylation of small molecule drugs may improve the intracellular availability and distribution of weakly basic, small molecule drugs within cells.

Janus kinases (JAKs) bind to class I and II cytokine receptors, activating signaling and regulating gene transcription through signal transducer and activator of transcription (STAT) proteins. Type I interferons (IFNs) require the JAK members TYK2 and JAK1, which bind to the receptor subunits IFNAR1 and IFNAR2, respectively. We investigated the role of JAKs in regulating IFNAR signaling activity. Synthetic IFNARs in which the extracellular domains of IFNAR1 and IFNAR2 are replaced with nanobodies had near-native type I IFN signaling, whereas the homomeric variant of IFNAR2 initiated much weaker signaling, despite harboring docking sites for JAKs and STATs. Cells with JAK1 and TYK2 knockout (KO) showed residual signaling, suggesting partial complementation by the remaining JAKs, particularly when they were overexpressed. Live-cell micropatterning experiments confirmed the promiscuous binding of JAK1, JAK2, and TYK2 to IFNAR1 and IFNAR2, and their recruitment correlated with their relative cellular abundances. However, each JAK had a different efficacy in inducing cross-phosphorylation and downstream signaling. JAK binding was also promiscuous for other cytokine receptors, including IFN-L1, IL-10Rβ, TPOR, and GHR, but not for EPOR, which activated different downstream signaling pathways. These findings suggest that competitive binding of JAKs to cytokine receptors together with the varying absolute and relative abundances of the JAKs in different cell types can account for the cell typedependent signaling pleiotropy of cytokine receptors.