Regulation of NF-κB activity through lysine monomethylation of p65

NF-κB is a key activator of inflammatory and immune responses with important pathological roles in cancer, heart disease, and autoimmune diseases. Transcriptional activity of NF-κB is regulated by different posttranslational modifications. Here, we report a novel mechanism of NF-κB regulation through lysine monomethylation by SET9 methyltransferase. Set9 specifically methylates p65 at lysine 37. Both TNFα and IL-1β treatments induced methylation of p65. Methylated p65 is restricted to the nucleus and this modification regulates the promoter binding of p65. Moreover, Set9 mediated methylation of p65 is required for the expression of a subset of NF-κB target genes in response to TNFα stimulation

EHMT1 Protein Binds to Nuclear Factor-κB p50 and Represses Gene Expression

Transcriptional homeostasis relies on the balance between positive and negative regulation of gene transcription. Methylation of histone H3 lysine 9 (H3K9) is commonly correlated with gene repression. Here, we report that a euchromatic H3K9 methyltransferase, EHMT1, functions as a negative regulator in both the NF-κB- and type I interferon-mediated gene induction pathways. EHMT1 catalyzes H3K9 methylation at promoters of NF-κB target genes. Moreover, EHMT1 interacts with p50, and, surprisingly, p50 appears to repress the expression of type I interferon genes and genes activated by type I interferons by recruiting EHMT1 to catalyze H3K9 methylation at their promoter regions. Silencing the expression of EHMT1 by RNA interference enhances expression of a subset NF-κB-regulated genes, augments interferon production, and augments antiviral immunity

miR-146a controls the resolution of T cell responses in mice

T cell responses in mammals must be tightly regulated to both provide effective immune protection and avoid inflammation-induced pathology. NF-κB activation is a key signaling event induced by T cell receptor (TCR) stimulation. Dysregulation of NF-κB is associated with T cell–mediated inflammatory diseases and malignancies, highlighting the importance of negative feedback control of TCR-induced NF-κB activity. In this study we show that in mice, T cells lacking miR-146a are hyperactive in both acute antigenic responses and chronic inflammatory autoimmune responses. TCR-driven NF-κB activation up-regulates the expression of miR-146a, which in turn down-regulates NF-κB activity, at least partly through repressing the NF-κB signaling transducers TRAF6 and IRAK1. Thus, our results identify miR-146a as an important new member of the negative feedback loop that controls TCR signaling to NF-κB. Our findings also add microRNA to the list of regulators that control the resolution of T cell responses

Identification and Characterization of MAVS, a Mitochondrial Antiviral Signaling Protein that Activates NF-κB and IRF3

SummaryViral infection triggers host innate immune responses through activation of the transcription factors NF-κB and IRF3, which coordinately regulate the expression of type-I interferons such as interferon-β (IFN-β). Herein, we report the identification of a novel protein termed MAVS (mitochondrial antiviral signaling), which mediates the activation of NF-κB and IRF3 in response to viral infection. Silencing of MAVS expression through RNA interference abolishes the activation of NF-κB and IRF3 by viruses, thereby permitting viral replication. Conversely, overexpression of MAVS induces the expression of IFN-β through activation of NF-κB and IRF3, thus boosting antiviral immunity. Epistasis experiments show that MAVS is required for the phosphorylation of IRF3 and IκB and functions downstream of RIG-I, an intracellular receptor for viral RNA. MAVS contains an N-terminal CARD-like domain and a C-terminal transmembrane domain, both of which are essential for MAVS signaling. The transmembrane domain targets MAVS to the mitochondria, implicating a new role of mitochondria in innate immunity

Transcriptomic analysis of the role of RasGEF1B circular RNA in the TLR4/LPS pathway

Abstract Circular RNAs (circRNAs) have recently emerged as a large class of novel non-coding RNA species. However, the detailed functional significance of the vast majority of them remains to be elucidated. Most functional characterization studies targeting circRNAs have been limited to resting cells, leaving their role in dynamic cellular responses to stimuli largely unexplored. In this study, we focus on the LPS-induced cytoplasmic circRNA, mcircRasGEF1B, and combine targeted mcircRasGEF1B depletion with high-throughput transcriptomic analysis to gain insight into its function during the cellular response to LPS stimulation. We show that knockdown of mcircRasGEF1B results in altered expression of a wide array of genes. Pathway analysis revealed an overall enrichment of genes involved in cell cycle progression, mitotic division, active metabolism, and of particular interest, NF-κB, LPS signaling pathways, and macrophage activation. These findings expand the set of functionally characterized circRNAs and support the regulatory role of mcircRasGEF1B in immune response during macrophage activation and protection against microbial infections

Inhibition of Euchromatic Histone Methyltransferase 1 and 2 Sensitizes Chronic Myeloid Leukemia Cells to Interferon Treatment

<div><p>Background</p><p>H3K9 methylation is one of the essential histone post-translational modifications for heterochromatin formation and transcriptional repression. Recently, several studies have demonstrated that H3K9 methylation negatively regulates the type I interferon response.</p><p>Results</p><p>We report the application of EHMT1 and EHMT2 specific chemical inhibitors to sensitize CML cell lines to interferon and imatinib treatments. Inhibition of EHMT1 and EHMT2 with BIX01294 enhances the cytotoxicity of IFNα2a in four CML cell lines, K562, KCL22, BV173 and KT1 cells. Chromatin immunoprecipitation assay shows that BIX01294 treatment enhances type I interferon response by reducing H3K9me2 at the promoters of interferon-stimulated genes. Additionally, BIX01294 treatment augments IFNα2a- and imatinib-mediated apoptosis in CML cell lines. Moreover, our data suggest that the expression level of EHMT1 and EHMT2 inversely correlates with the type I interferon responsiveness in CML cell lines.</p><p>Conclusions</p><p>Our study sheds light on the role of EHMT1 and EHMT2 as potential targets in improving the efficacy of standard treatments of CML.</p></div

BIX01294 slightly enhance IFNα2a-induced anti-proliferation in non-CML cells.

<p>Jurkat (<b>A</b>), HeLa (<b>B</b>) and HaCat (<b>C</b>) cells were cultured with various concentrations of BIX01294 and IFNα2a as indicated. After four days, cell proliferation was measured with a MTT assay. Results represent the mean ± SD in quadruplicate experiments.</p

BIX01294 enhances the expressions of ISGs in K562 cells.

<p>(<b>A</b>) K562 cells were incubated with 2.5 µM BIX01294 for 24 hours. The cells were then treated with various concentrations of IFNα2a as indicated. After two hours of IFNα2a stimulation, the expression of various ISGs was measured with RT-qPCR. Error bars represent the variation range of duplicate experiments. *: p<0.05, **: p<0.01. (<b>B</b>) K562 cells were incubated with 2.5 µM BIX01294 for 24 hours. The cells were then treated with IFNβ or IFNγ for two hours. The expression of <i>IFIT2</i> and <i>IFIT3</i> was measured with RT-qPCR. Error bars represent the variation range of duplicate experiments. *: p<0.05.</p

UNC06398 inhibits the proliferation of K562 cells and potentiates the expression of ISGs.

<p>(<b>A</b>) K562 cells were cultured with various concentrations of UNC0638 and IFNα2a as indicated. After four days, cell proliferation was measured with a MTT assay. Results represent the mean ± SD in quadruplicate experiments. (<b>B</b>) K562 cells were incubated with 5 µM UNC0638 for 24 hours followed with various concentrations of IFNα2a stimulation as indicated. After two hours of IFNα2a stimulation, the expression of various ISGs was measured with RT-qPCR. Error bars represent the variation range of duplicate experiments. *: p<0.05, **: p<0.01. (<b>C</b>) Whole cell extracts or total RNA were generated from K562 cells infected with control or lentiviruses carrying EHMT1- or EHMT2-specific shRNAs (left). EHMT1 or EHMT2 protein levels were analyzed by immunoblotting using indicated antibodies while mRNA levels were measured with RT-qPCR. Error bars represent the variation range of duplicate experiments. The same cells were stimulated with 1000 IU/ml IFNα2a for two hours (right). The expression of various ISGs was measured with RT-qPCR<b>.</b> Error bars represent the variation range of duplicate experiments. **: p<0.01. (<b>D</b>) K562 cells as in (<b>C</b>) were cultured with various concentrations of IFNα2a as indicated. After four days, cell proliferation was measured with a MTT assay. Results represent the mean ± SD in quadruplicate experiments.</p