LSD1 collaborates with EZH2 to regulate expression of interferon-stimulated genes.

Histone methylation is a complicate and dynamic epigenetic modification that regulates gene transcription, chromosomal structure and cell differentiation. Here, we discovered the interaction between the H3K4 demethylase, lysine specific demethylase 1 (LSD1, an important component of CoREST repressor complex) and the H3K27 methyltransferase, enhancer of zeste homolog 2 (EZH2, an essential component of PRC2). Immuno-precipitation and GST-pull down assay were performed to observe the interaction between the proteins. The MCF-7 cells were cultured and transfected with the siRNA. The mRNA and proteins were examined by using the real-time polymerase chain reaction (RT-PCR) and western blot assay, respectively. HPLC and LC-MS/MS analysis were performed to purify the proteins. RT-PCR-based quantitative ChIP analysis were performed. LSD1 interacts with histone modification protein EZH2 in MCF-7 cells. LSD1 and EZH2 target a few common genes. LSD1 knockdown and EZH2 knockdown affect protein expression. LSD1 knockdown and EZH2 knockdown affect the proteins involving in IFN signaling pathway. LSD1 and EZH2 modify histone methylation at IRF9 gene locus. We systematically analyzed the proteins that are affected by either LSD1 or EZH2 knockdown with proteomic approaches and identified that the interferon pathway and some other pathways are commonly affected. The interaction between LSD1 and EZH2 stabilizes the binding of LSD1 to the promoter region of IRF9, which is a key transcription factor of the interferon pathway. In conclusion, our study revealed that the coordination between histone demethylases and methyl-transferases might serve as a double lock system to suppress the expression of interferon stimulated genes.

Interferon regulatory factor 9 protects against cardiac hypertrophy by targeting myocardin.

Pathological cardiac hypertrophy is a major risk factor for heart failure. In this study, we identified interferon regulatory factor 9 (IRF9), a member of the IRF family, as a previously unidentified negative regulator of cardiac hypertrophy. The level of IRF9 expression was remarkably elevated in the hearts from animals with aortic banding-induced cardiac hypertrophy. IRF9-deficient mice exhibited pronounced cardiac hypertrophy after pressure overload, as demonstrated by increased cardiomyocyte size, extensive fibrosis, reduced cardiac function, and enhanced expression of hypertrophy markers, whereas transgenic mice with cardiac-specific overexpression of murine IRF9 exhibited a significant reduction in the hypertrophic response. Mechanistically, IRF9 competes with p300 for binding to the transcription activation domain of myocardin, a coactivator of serum response factor (SRF). This interaction markedly suppresses the transcriptional activity of myocardin because IRF9 overexpression strongly inhibits the ability of myocardin to activate CArG box-dependent reporters. These results provide compelling evidence that IRF9 inhibits the development of cardiac hypertrophy by suppressing the transcriptional activity of myocardin in the heart.

Beta interferon-mediated activation of signal transducer and activator of transcription protein 1 interferes with Rickettsia conorii replication in human endothelial cells.

Infection of the endothelial cell lining of blood vessels with Rickettsia conorii, the causative agent of Mediterranean spotted fever, results in endothelial activation. We investigated the effects of R. conorii infection on the status of the Janus kinase (JAK)-signal transducer and activator of transcription protein (STAT) signaling pathway in human microvascular endothelial cells (HMECs), the most relevant host cell type, in light of rickettsial tropism for microvascular endothelium in vivo. R. conorii infection induced phosphorylation of STAT1 on tyrosine 701 and serine 727 at 24, 48, and 72 h postinfection in HMECs. Employing transcription profile analysis and neutralizing antibodies, we further determined that beta interferon (IFN-ß) production and secretion are critical for STAT1 activation. Secreted IFN-ß further amplified its own expression via a positive-feedback mechanism, while expression of transcription factors interferon regulatory factor 7 (IRF7) and IRF9, implicated in the IFN-ß-STAT1 feedback loop, was also induced. Metabolic activity of rickettsiae was essential for the IFN-ß-mediated response(s) because tetracycline treatment inhibited R. conorii replication, IFN-ß expression, and STAT1 phosphorylation. Inclusion of IFN-ß-neutralizing antibody during infection resulted in significantly enhanced R. conorii replication, whereas addition of exogenous IFN-ß had the opposite inhibitory effect. Finally, small interfering RNA-mediated knockdown further confirmed a protective role for STAT1 against intracellular R. conorii replication. In concert, these findings implicate an important role for IFN-ß-mediated STAT1 activation in innate immune responses of vascular endothelium to R. conorii infection.

Ribavirin potentiates interferon action by augmenting interferon-stimulated gene induction in hepatitis C virus cell culture models.

UNLABELLED: The combination of pegylated interferon (PEG-IFN) and ribavirin is the standard treatment for chronic hepatitis C. Our recent clinical study suggests that ribavirin augments the induction of interferon-stimulated genes (ISGs) in patients treated for hepatitis C virus (HCV) infection. In order to further characterize the mechanisms of action of ribavirin, we examined the effect of ribavirin treatment on ISG induction in cell culture. In addition, the effect of ribavirin on infectious HCV cell culture systems was studied. Similar to interferon (IFN)-α, ribavirin potently inhibits JFH-1 infection of Huh7.5.1 cells in a dose-dependent manner, which spans the physiological concentration of ribavirin in vivo. Microarray analysis and subsequent quantitative polymerase chain reaction assays demonstrated that ribavirin treatment resulted in the induction of a distinct set of ISGs. These ISGs, including IFN regulatory factors 7 and 9, are known to play an important role in anti-HCV responses. When ribavirin is used in conjunction with IFN-α, induction of specific ISGs is synergistic when compared with either drug applied separately. Direct up-regulation of these antiviral genes by ribavirin is mediated by a novel mechanism different from those associated with IFN signaling and intracellular double-stranded RNA sensing pathways such as RIG-I and MDA5. RNA interference studies excluded the activation of the Toll-like receptor and nuclear factor κB pathways in the action of ribavirin. CONCLUSION: Our study suggests that ribavirin, acting by way of a novel innate mechanism, potentiates the anti-HCV effect of IFN. Understanding the mechanism of action of ribavirin would be valuable in identifying novel antivirals.

Genetic redundancy in human cervical carcinoma cells: identification of cells with "normal" properties.

Although it is generally assumed that cancer arises from a singular cell, a tumor must be considered as a dynamic and emergent biological structure, whose organizing principle is determined by genetic and epigenetic modifications, occurring variably in response to microenvironmental selection conditions. As previously shown, HPV-positive cervical carcinoma cells have lost their ability to induce IFN-beta upon TNF-alpha treatment. However, regarding cancer as a non-linear system, which may, even in the absence of an apparent selection pressure, fluctuate between different "metastable" phenotypes, we demonstrate that TNF-alpha mediated IFN-beta induction is not irreversibly disturbed in all cells. Using the IFN-beta sensitive Encephalomyocarditis virus (EMCV) as a tool to monitor antiviral activity in long-term established malignant HeLa cells, rare IFN-beta expressing clones were rescued from a population of non-responsive and EMCV-sensitive cells. Antiviral activity was mediated by the re-expression of IRF-1 and p48 (IRF-9), both key regulatory molecules normally found to be suppressed in cervical carcinoma cells. Upon inoculating of selected clones into immunocompromised animals, a reduced or even an absence of tumorigenicity of initially highly malignant cells could be discerned. These data indicate that both the absence of interferon signaling and the ability to form tumors were reversed in a minority of cells. We provide a paradigm for the existence of innate genetic redundancy mechanisms, where a particular phenotype persists and can be isolated without application of drugs generally changing the epigenetic context.

Genes transcriptionally modulated by interferon alpha2a correlate with the cytokine activity.

BACKGROUND AND OBJECTIVES: Interferon alpha2a (IFNalpha2a) mediates important antiviral, antiproliferative and immunomodulatory responses and is employed in the treatment of human diseases, including chronic myelogenous leukemia. Here, we report the IFNalpha2a-dependent expression profiles of three malignant cell lines derived from liver, lymphocytes and muscle. DESIGN AND METHODS: The experiments were performed in the presence of cycloheximide, thus our results exclusively reflect direct transcriptional modulation. The short exposure time i.e. 5 hours evidences only the early events, excluding the effects of complex phenotypic changes on the expression. RESULTS: Our findings indicate that IFNalpha2a rapidly up-regulates the expression of STAT1, STAT2 and ISGF3G genes. This activity should result in the amplification of the cellular response to the cytokine. Moreover, IFNalpha2a directly modulates the expression of: (i) important transcriptional factors, e.g. IRF1 and IRF7 which control pivotal cellular events, and (ii) enzymes involved in the IFNalpha2a-dependent antiviral and apoptotic response. Interestingly, we showed that the cytokine induces transcriptional expression of Sjögren's syndrome antigen A1, a protein involved in several autoimmune diseases. INTERPRETATION AND CONCLUSIONS: The observed changes induced by IFNalpha2a could be related to the development of autoimmune syndromes observed during IFNalpha2a treatment. A number of genes transcriptionally regulated by the cytokine have been identified for the first time; these might represent additional effectors of IFNalpha2a activity.