Double-stranded RNA (dsRNA), a frequent byproduct of virus infection, is recognized by Toll-like receptor 3 (TLR3) to mediate innate immune response to virus infection. TLR3 signaling activates the transcription factor IRF-3 by its Ser/Thr phosphorylation, accompanied by its dimerization and nuclear translocation. It has been reported that the Ser/Thr kinase TBK-1 is essential for TLR3-mediated activation and phosphorylation of IRF-3. Here we report that dsRNA-activated phosphorylation of two specific tyrosine residues of TLR3 is essential for initiating two distinct signaling pathways. One involves activation of TBK-1 and the other recruits and activates PI3 kinase and the downstream kinase, Akt, leading to full phosphorylation and activation of IRF-3. When PI3 kinase is not recruited to TLR3 or its activity is blocked, IRF-3 is only partially phosphorylated and fails to bind the promoter of the target gene in dsRNA-treated cells. Thus, the PI3K-Akt pathway plays an essential role in TLR3-mediated gene induction.

The positive regulatory machinery in the microRNA (miRNA) processing pathway is relatively well characterized, but negative regulation of the pathway is largely unknown. Here we show that a complex of nuclear factor 90 (NF90) and NF45 proteins functions as a negative regulator in miRNA biogenesis. Primary miRNA (pri-miRNA) processing into precursor miRNA (pre-miRNA) was inhibited by overexpression of the NF90 and NF45 proteins, and considerable amounts of pri-miRNAs accumulated in cells coexpressing NF90 and NF45. Treatment of cells overexpressing NF90 and NF45 with an RNA polymerase II inhibitor, ␣-amanitin, did not reduce the amounts of pri-miRNAs, suggesting that the accumulation of pri-miRNAs is not due to transcriptional activation. In addition, the NF90 and NF45 complex was not found to interact with the Microprocessor complex, which is a processing factor of primiRNAs, but was found to bind endogenous pri-miRNAs. NF90-NF45 exhibited higher binding activity for pri-let-7a than pri-miR-21. Of note, depletion of NF90 caused a reduction of pri-let-7a and an increase of mature let-7a miRNA, which has a potent antiproliferative activity, and caused growth suppression of transformed cells. These findings suggest that the association of the NF90-NF45 complex with pri-miRNAs impairs access of the Microprocessor complex to the pri-miRNAs, resulting in a reduction of mature miRNA production.MicroRNAs (miRNAs) constitute a class of noncoding small RNAs that function as repressors for eukaryotic gene regulation by binding to the 3Ј untranslated regions of target mRNAs (2). This binding causes mRNA cleavage or translational inhibition of the mRNA, depending upon the degree of complementarity. The lengths of miRNAs are 21 to 23 nucleotides (nt), and over 500 miRNAs have been discovered in mammals. miRNAs regulate the expression of a large number of genes (38) that are involved in cell proliferation, apoptosis, hematopoietic differentiation, viral infection, and tumorigenesis (4,5,7,22,26,32,39,45).In mammals, miRNA genes are transcribed by RNA polymerase II as primary miRNAs (pri-miRNAs) (36). These primiRNAs are processed into precursor miRNAs (pre-miRNAs) by the Microprocessor complex (8,13,20,31,33). Another complex comprised of exportin-5 and RanGTP transports the pre-miRNAs from the nucleus to the cytoplasm (3, 40, 58). In the cytoplasm, Dicer, a cytoplasmic RNase III enzyme, cleaves the pre-miRNAs to approximate 22-nt mature miRNA duplexes with 2-nt 3Ј overhangs (14,24,28). One strand of the duplex is incorporated into the RNA-induced silencing complex (12,19,29,41,51). The single strand of RNA guides the RNA-induced silencing complex to the target mRNA with sequence complementarity, which leads either to mRNA cleavage or to translational repression (12,24,41,44).The Microprocessor complex, which cleaves pri-miRNA to pre-miRNA during miRNA biogenesis, is comprised of a nuclear RNase III enzyme, Drosha, and its cofactor, DGCR8 (8,13,20). In addition to the Microprocessor complex, excessively expressed Drosha forms ...

The biological actions of interferons (IFNs) 1 require the activation of immediate early genes, which are mediated by the Stat family of transcription factors (1). Type 1 interferons (IFN␣/␤) binding to their cell surface receptors initiate a set of events that leads to tyrosine phosphorylation of Stat1 and Stat2. Tyrosine-phosphorylated Stat1 and Stat2 heterodimerize through their Src homology 2 domains and translocate to the nucleus where they bind to interferon regulatory factor 9 (IRF9) to form the transcription complex ISGF3. ISGF3 binds to an interferon-stimulated response element (ISRE) that is present in many IFN␣/␤-stimulated genes. Alternatively, tyrosine-phosphorylated Stat1 can form homodimers and bind to a ␥ interferon activation sequence (GAS), an enhancer in the promoter of genes that do not require the participation of Stat2 or IRF9.Binding of ISGF3 to cellular genes containing ISREs is accompanied by changes in the chromatin structure of interferonstimulated genes (ISGs) (2). Cells incubated with IFN␣ show altered DNase I sensitivity surrounding the TATA box region as well as the ISRE of ISGs, suggesting that gene activation occurs as a result of chromatin remodeling (2). Consistent with this observation, both Stat1 and Stat2 have been shown to interact with the histone acetylases CBP/p300 and GCN5 resulting in increased association of the acetylated histone H3 with the promoter of the IFN␣/␤-stimulated gene ISG54 (3-6).From these observations one would predict that incubation of cells with HDAC inhibitors would increase IFN-activated gene expression, and this is indeed the case when one examines IFN␥-stimulated expression of the MHC-II (7). However, in contrast to IFN␥, interleukin-3-activated Stat5-dependent genes are inhibited in cells exposed to TSA (8), suggesting that in certain contexts HDAC activity may be required for Stat-dependent gene activation. Interestingly, other genes in which expression is activated by interleukin-3 in a Stat5-independent manner are unaffected by TSA. To examine the role of HDAC activity in the expression of genes regulated by Stat1 and Stat2, we have assayed for the expression of several RNAs in which genes are controlled by these transcription factors. To our surprise the actions of TSA on IFN␣/␤-stimulated gene expression were relatively selective. Whereas some IFN␣/␤-stimulated genes in which expression is regulated by an ISRE were strongly suppressed by TSA, others showed a more modest inhibition. Furthermore, genes in which activation required a GAS element, such as IRF-1, were not altered by TSA treatment. HDAC activity appears to be required to recruit RNA polymerase II (Pol II), but not Stat1 or Stat2, to the promoter of TSA-sensitive genes. The effects of TSA seem to be mediated either directly or indirectly by IRF9. Interestingly, if Pol II is already bound to the promoter and there is basal expression of the gene in the absence of IFN␣/␤ (i.e. IRF-1), then the suppressive actions of TSA are not observed. These data suggest a novel function for IRF9 ...

Allelic exclusion of antigen-receptor genes is ensured primarily by monoallelic locus activation upon rearrangement and subsequently by feedback inhibition of continued rearrangement. Here, we demonstrated that the basic helix-loop-helix protein, E47, promoted T cell receptor beta (TCRbeta) gene rearrangement by directly binding to target gene segments to increase chromatin accessibility in a dosage-sensitive manner. Feedback signaling abrogated E47 binding, leading to a decline in accessibility. Conversely, enforced expression of E47 induced TCRbeta gene rearrangement by antagonizing feedback inhibition. Thus, the abundance of E47 is rate limiting in locus activation, and feedback signaling downregulates E47 activity to ensure allelic exclusion.

We isolated several clones from a matchmaker twohybrid system human lymphocyte cDNA library using an automodification domain of poly(ADP-ribose) synthetase (PARS) as a probe. A DNA sequence (V1 kbp) of the clone was identical to part of the Oct-1 DNA sequence. We then constructed either a His-tagged or GST fusion protein of the inserted cDNA from the clone and the fusion protein was shown to interact with PARS by far-Western blot analysis and coprecipitation with affinity resin. Furthermore, the His-tagged Oct-1/POU-homeo fusion protein interacted weakly with the octamer motif of the DRa promoter and the addition of PARS fusion protein greatly increased the DNA binding activity. These results suggest that PARS interacts with Oct-1 and stabilizes the binding of Oct-1 to the octamer motif.z 1998 Federation of European Biochemical Societies.