In each of the four ISG clusters (Figure (Figure4B),4B), ISRE was by far the most significantly enriched motif (Supplemental Figure 2). Figure 6 MARA reveals ISRE as the most Vismodegib hedgehog significantly upregulated motif. ISRE motifs are the binding sites for ISGF3 and also IRFs. ISGF3 is activated by IFN-���Cinduced phosphorylation of STAT1 and STAT2. IRFs are transcriptionally induced and are also regulated by phosphorylation (23). We therefore measured the expression of IRF mRNAs. Of the nine IRFs, only IRF1, IRF7, and IRF9 were upregulated by pegIFN-��2b in the liver (Figure (Figure6C).6C). IRF1 is transiently induced at the 4- and 16-hour time points. IRF9 is part of the ISGF3 complex, and its transcriptional activity depends on p-STAT1 and p-STAT2.

IRF7 is upregulated during the entire dosing interval of pegIFN-��2b and could also be involved in ISRE-mediated gene transcription. However, the transcriptional activity of IRF7 depends on serine phosphorylation by IKK-�� (24), a downstream component of cellular sensory pathways that are activated by viral pathogen�Cassociated molecular patterns (PAMPs). Unphosphorylated STAT1 does not prolong ISG induction. The central IFN-���Cinduced signal transducer and transcription factor STAT1 is itself one of the most strongly induced ISGs. Indeed, we found STAT1 mRNA strongly induced in the first 4 days after pegIFN-��2b injection (Supplemental Table 1). STAT1 protein was even upregulated during the entire 1-week dosing interval (Supplemental Figure 1).

The functional significance of the expression of large amounts of U-STAT1 protein is unclear, but, intriguingly, GSK-3 a recent paper described a role of U-STAT1 as an active transcription factor that prolongs gene transcription after dephosphorylation of p-STAT1 (25). In that work, thirty ISGs were found to be upregulated by U-STAT1�Cdriven transcription (25). We therefore hypothesized that U-STAT1 could be involved in the prolonged ISG induction by pegIFN-��2b. However, when we took the list of U-STAT1�Cinduced genes and investigated their expression during the first week of pegIFN-��2b therapy, we did not find them to be overrepresented in clusters 3 and 4 with late and very late induced ISGs, respectively (data not shown). We therefore decided to address the potential of U-STAT1 to induce gene transcription in a more rigorous way. To that end, STAT1-deficient U3A cells (26) were stably transfected with STAT1 wild-type (STAT1-WT) or a mutant STAT1 with a phospho-tyrosine acceptor site at position 701 mutated to phenylalanine (STAT1-Y701F). For both STAT1-WT and STAT1-Y701F, three clones with different STAT1 expression levels were selected.