7A). As we expected, IFN-�� treatment of U3A cells transfected with STAT1-WT induced ISGs. In contrast, we observed no ISG induction in U3A cells transfected with STAT1-Y701F (Figure (Figure7B7B and Supplemental Figure 3). These results do not support a role for U-STAT1 in prolonged ISG expression. Figure 7 U-STAT1 does not clearly induce ISGs. Ongoing gene transcription and lower mRNA decay rates both contribute to prolonged expression of ��late�� ISGs. Since ISRE seems to be the main TFBS in all transcription clusters and U-STAT1 was not able to induce ISGs, we next hypothesized that the genes belonging to the late ISG clusters might show prolonged expression due to lower mRNA degradation rates, since such a mechanism was recently proposed to play an important role in temporal expression patterns of genes induced by TNF-�� (27).

Decay of mRNAs can be regulated by specific microRNA recognition sequences present in the 3�� untranslated regions (UTRs) of mRNAs (28). We therefore analyzed our transcriptome datasets for specific binding sites of microRNAs to test whether the four ISG clusters defined by our unbiased clustering approach (Figure (Figure4)4) have distinct microRNA binding sites in their 3��UTRs. However, we could not identify biologically meaningful microRNA binding patterns that would predict or explain the differences in decay rates of the four clusters (data not shown). We also analyzed the decay rates of mRNAs experimentally in IFN-���Ctreated Huh7 cells by inhibition of gene transcription with actinomycin D.

Relative to GAPDH mRNA, early ISGs (RSAD2, USP18) showed faster mRNA decay, while the late ISGs (IFI27, LGALS3BP) decayed more slowly than GAPDH (Figure (Figure8A).8A). However, a delayed mRNA decay rate cannot readily explain the expression peaks at later time points such as those observed in cluster 4 genes (Figure (Figure3).3). We therefore also analyzed the transcription of representative early (RSAD2, USP18) and late (IFI27, LGALS3BP) ISGs using a nuclear run-on assay. Nuclei were isolated from Huh7 cells after 1, 2, 4, 16, and 24 hours of stimulation with 1,000 IU/ml IFN-�� and were then incubated with biotin-labeled UTP for 45 minutes. The newly transcribed mRNA was purified on streptavidin beads and quantified by quantitative PCR (qPCR). We found a markedly prolonged transcription of late versus early ISGs (Figure (Figure88B).

Figure 8 Late ISGs show a more prolonged transcriptional induction and a slower mRNA degradation rate than early ISGs in vitro. We conclude that the temporal expression patterns of ISGs are determined by the duration of gene transcription as well as by different mRNA decay rates. Discussion Arguably, no other cytokine has been used in clinical medicine Dacomitinib more extensively than recombinant (peg)IFN-��.