Project description:Gene regulation by cytokine-activated STAT transcription factors requires serine phosphorylation within the transactivation domain (TAD). STAT1 and STAT3 TAD phosphorylation was reported to occur upon promoter binding by an unknown kinase. Here we show that the Mediator CDK8 module phosphorylates S727 of the STAT1 TAD in the interferon (IFN) signaling pathway as well as the TADs of other STATs. Microarray analysis reveals that CDK8-mediated STAT1 TAD phosphorylation positively or negatively regulates over 40% of IFN-gamma-responsive genes, and RNA polymerase II occupancy correlates with gene expression changes. This selective regulation occurs despite CDK8 occupancy and STAT1 S727 phosphorylation at both S727 phosphorylation-dependent and -independent IFN-gamma target genes. Independently of its role as STAT1 S727 kinase CDK8 acts as a positive regulator of IFN-gamma responses. These data reveal a dual input of CDK8 in STAT1-controlled transcription and propose a key role for CDK8 in TAD phosphorylation of other STATs during cytokine responses. STAT1 WT and STAT1 S727A mouse fibroblasts were treated with siRNA to CDK8 (siCdk8 smart pool, On Target Plus, Dharmacon) and control siRNA (siCtrl) and stimulated with IFN-gamma for 4 h or left untreated. Total RNA from three independent experiments for each treatment and each genotype was isolated from cells using Trizol reagent (Invitrogen) following the manufactures protocol and used for expression analysis using Agilent Whole Mouse Genome Microarrays, 8x60K. Standard protocols for labeling and hybridization were followed. In brief, fluorescent cRNA was generated using Low Input Quick Amp Labeling Kit (Agilent). The amplified cyanine 3-labeled cRNA samples were then purified using SV Total RNA Isolation System (Promega) and hybridized to microarray slides. Microarray slides were washed and scanned with an Agilent Scanner. Note: The outlier array #10 [SA.CDK.gamma.R3] was removed from subsequent analysis and its processed data was not provided. However, its raw data file has been linked as a supplementary file at the foot of the Series record.

Project description:Intracellular pathogens including the apicomplexan and opportunistic parasite Toxoplasma gondii profoundly modify their host cells in order to establish infection. We have shown previously that intracellular T. gondii inhibit up-regulation of regulatory and effector functions in murine macrophages (MΦ) stimulated with interferon (IFN)-γ, which is the cytokine crucial for controlling the parasites’ replication. Using genome-wide transcriptome analysis we show herein that infection with T. gondii leads to global unresponsiveness of murine macrophages to IFN-γ. More than 61% and 89% of the transcripts, which were induced or repressed by IFN-γ in non-infected MΦ, respectively, were not altered after stimulation of T. gondii-infected cells with IFN-γ. These genes are involved in a variety of biological processes, which are mostly but not exclusively related to immune responses. Analyses of the underlying mechanisms revealed that IFN-γ-triggered nuclear translocation of STAT1 still occurred in Toxoplasma-infected MΦ. However, STAT1 bound aberrantly to oligonucleotides containing the IFN-γ-responsive gamma-activated site (GAS) consensus sequence. Conversely, IFN-γ did not induce formation of active GAS-STAT1 complexes in nuclear extracts from infected MΦ. Mass spectrometry of protein complexes bound to GAS oligonucleotides showed that T. gondii-infected MΦ are unable to recruit non-muscle actin to IFN-γ-responsive DNA sequences, which appeared to be independent of stimulation with IFN-γ and of STAT1 binding. IFN-γ-induced recruitment of BRG-1 and acetylation of core histones at the IFN-γ-regulated CIITA promoter IV, but not β-actin was diminished by >90% in Toxoplasma-infected MΦ as compared to non-infected control cells. Remarkably, treatment with histone deacetylase inhibitors restored the ability of infected macrophages to express the IFN-γ regulated genes H2-A/E and CIITA. Taken together, these results indicate that Toxoplasma-infected MΦ are unable to respond to IFN-γ due to disturbed chromatin remodelling, but can be rescued using histone deacetylase inhibitors. Comparison of 4 different RNA pools with a 2-Color-Loop Design including 10 microarrays: [1] T. gondii infected and IFN-gamma treated, [2] T. gondii infected and untreated, [3] Non-infected and IFN-gamma treated, and [4] Non-infected and untreated.

Project description:Qi2013 - IL-6 and IFN crosstalk model This model [BIOMD0000000544] describes the crosstalk between IFN-gamma and IL-6 induced signalling; it aims to outline mechanisms and factors that may control the interaction between both signalling pathways, discussing a role of heterodimer formation in signalling dysfunction. To account for the possibility of different IFNR and gp130 binding sites for STAT1 and STAT3, model 1 [BIOMD0000000543] assumes that there is no competition between STAT1 and STAT3 for the receptor complexes (includes two extra reactions). The reverse of this is true in model 2 [BIOMD0000000544] where it generally is assumed that there is competition between STAT1 and STAT3 for the receptor complexes. This model is described in the article: Elucidating the crosstalk mechanism between IFN-gamma and IL-6 via mathematical modelling. Qi YF, Huang YX, Wang HY, Zhang Y, Bao YL, Sun LG, Wu Y, Yu CL, Song ZB, Zheng LH, Sun Y, Wang GN, Li YX. BMC Bioinformatics 2013; 14: 41 Abstract: BACKGROUND: Interferon-gamma (IFN-gamma) and interleukin-6 (IL-6) are multifunctional cytokines that regulate immune responses, cell proliferation, and tumour development and progression, which frequently have functionally opposing roles. The cellular responses to both cytokines are activated via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. During the past 10 years, the crosstalk mechanism between the IFN-gamma and IL-6 pathways has been studied widely and several biological hypotheses have been proposed, but the kinetics and detailed crosstalk mechanism remain unclear. RESULTS: Using established mathematical models and new experimental observations of the crosstalk between the IFN-gamma and IL-6 pathways, we constructed a new crosstalk model that considers three possible crosstalk levels: (1) the competition between STAT1 and STAT3 for common receptor docking sites; (2) the mutual negative regulation between SOCS1 and SOCS3; and (3) the negative regulatory effects of the formation of STAT1/3 heterodimers. A number of simulations were tested to explore the consequences of cross-regulation between the two pathways. The simulation results agreed well with the experimental data, thereby demonstrating the effectiveness and correctness of the model. CONCLUSION: In this study, we developed a crosstalk model of the IFN-gamma and IL-6 pathways to theoretically investigate their cross-regulation mechanism. The simulation experiments showed the importance of the three crosstalk levels between the two pathways. In particular, the unbalanced competition between STAT1 and STAT3 for IFNR and gp130 led to preferential activation of IFN-gamma and IL-6, while at the same time the formation of STAT1/3 heterodimers enhanced preferential signal transduction by sequestering a fraction of the activated STATs. The model provided a good explanation of the experimental observations and provided insights that may inform further research to facilitate a better understanding of the cross-regulation mechanism between the two pathways. This model is hosted on BioModels Database and identified by: BIOMD0000000544. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

Dataset Information

Homo sapiens

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets