SiRNA knockdown screening of possible NF-kB p65 interactors influencing IL-1-NF-kB gene expression
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ABSTRACT:
PROVIDER: PRJNA1023945 | ENA |
REPOSITORIES: ENA
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Project description:The transcription factor NF-κB p65 plays a key role in innate and adaptive immunity. It functions as a master regulator of the inflammatory response by inducing the expression of pro-inflammatory genes including secreted cytokines (IL6), chemokines (IL8, CXCL2), and multiple regulatory intracellular proteins (NFKBIA). NF-κB is activated by the so-called canonical, noncanonical and atypical signalling pathways. In the canonical pathway, activation of the Interleukin-1 receptor complex triggers degradation of the cytosolic p65 inhibitor IBα followed by nuclear translocation of p65 where it is directed by unknown pathways to the enhancers and promoters of pro-inflammatory genes. Despite its pleiotropic functions in adaptive and innate immunity or stress reactions, the entire repertoire of p65 interacting factors that contribute to its general or stimulus-specific functions within the nucleus or other cell compartments is still elusive. We identified the p65 interactomes in intact cells by BioID pulldowns under inflammatory condition (+IL-α). Extensive bioinformatics analysis of the proximity labeling data sets revealed several hundred high confidence interactors including multiple novel factors that have not been reported in the literature. We investigated which of our 38 high-confidence interactors have an impact on IL-1 NF-kB gene expression by siRNA screening. We found 5 interesting hits in the context of inflammation which will be further assessed by microarray analysis.
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Project description:Metastasis suppressor 1 (MTSS1) is a 755 amino acid protein found in the cell cytoplasm which binds to actin and promotes cytoskeleton organization and is a known suppressor of lung adenocarcinoma metastasis This study demonstrated that preservation of MTSS1 protein expression in lung adenocarcinoma was associated with a 20% 5-year survival advantage in patients. Furthermore, overexpression of MTSS1 was found to reduce metastasis and disease progression in an in-vivo orthotopic lung adenocarcinoma mouse model. Nuclear factor kappa B (NF-kB), an important nuclear transcription factor, has been shown to be constitutively active in lung adenocarcinoma and strongly associated with the development of metastasis. The NF-kB RelA/p65 subunit is involved in NF-kB heterodimer formation and subsequent nuclear translocation leading to activation of NF-kB responsive gene transcription. Phosphorylation and acetylation of p65 are critical post-translational modifications required for NF-kB activation. In this study, we demonstrate that MTSS1 expression leads to decreased NF-κB mediated gene transcription through inhibition of p65 phosphorylation. These findings uncover a novel mechanism through which MTSS1 may regulate lung adenocarcinoma metastasis by impairment of NF-κB regulated gene transcription.
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Project description:To explore the effects on the NF-kB transcriptome after knockdown of RPS3 in stimulated T cells, we performed microarray assays comparing RPS3-silenced versus NS-treated samples on the same chip. p65-silenced versus NS-treated samples were used as a control. Keywords: time series design Jurkat cells transfected with control siRNA versus RPS3 or p65-siRNA were stimuated with TCR for 1, 3, or 6 hours for a total of 6 arrays.
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Project description:To explore the effects on the NF-kB transcriptome after knockdown of RPS3 in stimulated T cells, we performed microarray assays comparing RPS3-silenced versus NS-treated samples on the same chip. p65-silenced versus NS-treated samples were used as a control. Keywords: time series design
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Project description:Aberrant regulation of NF-kB pathway is believed to be a major event contributing to malignant transformation and progression of prostate cancer (PCa). P65 consists of a DNA-binding and dimerization domain (RHD), nuclear localization signal (NLS) and transactivation domains (TA1 and TA2). The c-terminal 30 amino acids (TA1 domain) comprise the most important transactivation domain and NF-kB transactivation may be regulated by multiple phosphorylation in this domain. This p536 is located in TA1 domain and is conserved in human, mouse, chicken. We previously discovered that p536 is present in majority of PCa and associated with ERG expression indicating that ERG can significantly enhance phosphorylation of p65 at Ser536 in vivo. We have successfully generated a dominant negative (DN) p65 which has been mutated (S536A) such that it cannot be phosphorylated at Ser 536 and cannot carry out Ser536 phosphorylation dependent functions. We also generated two mutants S536D and S536E which are the phosphomimetic mutant that resembles phospho-p65 and can be detected by anti-phospho-p65 antibody. We carried out microarray studies and discovered a set of p536 regulated genes compared with wild type p65 regulated gene set.
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Project description:The inflammatory gene response requires activation of the protein kinase TAK1, but it is currently unknown how TAK1-derived signals coordinate transcriptional programs in the genome. We determined the genome-wide binding of the TAK1-controlled NF-?B subunit p65 in relation to active enhancers and promoters of transcribed genes by ChIP-seq experiments. Out of 35,000 active enhancer regions, 410 H3K4me1-positive enhancers show interleukin (IL)-1-induced H3K27ac and p65 binding. Inhibition of TAK1, IKK2 or depletion of p65 blocked inducible enhancer activation and gene expression. As exemplified by the CXC chemokine cluster located on chromosome 4, the TAK1-p65 pathway also regulates the recruitment kinetics of the histone acetyltransferase CBP, of NF-?B p50 and of AP-1 transcription factors to both, promoters and enhancers. This study provides a high resolution view of epigenetic changes occurring during the IL-1 response and allows the first genome-wide identification of a novel class of inducible p65 NF-?B-dependent enhancers in epithelial cells. RNA-seq of KB cells either untreated or treated with IL-1 alpha
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Project description:The inflammatory gene response requires activation of the protein kinase TAK1, but it is currently unknown how TAK1-derived signals coordinate transcriptional programs in the genome. We determined the genome-wide binding of the TAK1-controlled NF-κB subunit p65 in relation to active enhancers and promoters of transcribed genes by ChIP-seq experiments. Out of 35,000 active enhancer regions, 410 H3K4me1-positive enhancers show interleukin (IL)-1-induced H3K27ac and p65 binding. Inhibition of TAK1, IKK2 or depletion of p65 blocked inducible enhancer activation and gene expression. As exemplified by the CXC chemokine cluster located on chromosome 4, the TAK1-p65 pathway also regulates the recruitment kinetics of the histone acetyltransferase CBP, of NF-κB p50 and of AP-1 transcription factors to both, promoters and enhancers. This study provides a high resolution view of epigenetic changes occurring during the IL-1 response and allows the first genome-wide identification of a novel class of inducible p65 NF-κB-dependent enhancers in epithelial cells. ChIP-seq in KB cells with 5 different antibodies under different treatment conditions
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