ChIP-seq analysis to understand the mechanistic role of NCoR1 as a tolerogenic program suppressor in dendritic cells
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ABSTRACT: We found in our study that NCoR1 knock down dendritic cells (DCs) develop tolerogenic behavior upon activation and these cells have the potential to modulate T helper cell differentiation toward Treg phenotype. Here to understand the mechanism of this process we performed NCoR1 ChIP-seq in wild type DCs before and after CpG ligand activation and transcriptome analysis of NCoR1 KD DCs to identify the direct and indirect target genes. We also predicted PU.1 as the recruting factor for NCoR1 in DCs based on motif enrichment and later on validated that by performing PU.1 ChIP-seq in control and NCoR1 KD DCs.
Project description:Purpose: The goals of this study to dissect mechanism by which NCoR1 regulate expression of diiferent genes at transcriptional level in CD8a+ Dendritic cells. Methods: mRNA expression of Control(Empty) and knockdown (NCoR1+/−) before and after CpG and pIC activation were generated by deep sequencing, in duplicate, using Illumina NextSeq 500 . The sequence reads that passed quality filters were analyzed at the transcript level with two methods:TopHat followed by Cufflinks. Results: we mapped raw reads to the mouse genome (build mm10) with TopHat. Differential expression study was carried out using Cufflinks v2.2.1 and list were filtered using fold change cut-off of 2 and q value <0.05. Conclusions: We found in our study that NCoR1 knock down dendritic cells (DCs) develop tolerogenic behavior upon activation and these cells have the potential to modulate T helper cell differentiation toward Treg phenotype. Here to understand the mechanism of this process we performed NCoR1 ChIP-seq in wild type DCs before and after CpG ligand activation and transcriptome analysis of NCoR1 KD DCs to identify the direct and indirect target genes. We also predicted PU.1 as the recruting factor for NCoR1 in DCs based on motif enrichment and later on validated that by performing PU.1 ChIP-seq in control and NCoR1 KD DCs.
Project description:In this study, we focused on demonstrating the metabolic aspect of NCoR1 in the regulation of dendritic cells (DCs) functionality. We report that NCoR1 deficient tolerogenic DCs meet their anabolic requirements through enhanced glycolysis and OxPhos, supported by FAO-driven oxygen consumption. Furthermore, individual and dual inhibition of glycolysis through HIF-1α inhibitor (KC7F2) and fatty acid oxidation through CPT1 inhibitor (etomoxir) significantly impaired the secretion of tolerogenic cytokines. To validate the same at the global mRNA level we did bulk RNA-seq to determine the differentially expressed genes in control and NCoR1 KD 6h CpG activated DCs with and without inhibitor treatment.
Project description:RNA-seq was performed to understand the role of NCoR1 in CD8α+ DCs. MutuDC cell line was transduced with empty and Ncor1 shrna lentiviral particles and cells were prepared in unstimulated, 2hr and 6hr (IFNg, pIC and CpG+pIC+IFNg) stimulation condition.
Project description:Dendritic cell (DC) activation and function are underpinned by profound changes in cellular metabolism. Several studies indicate that the ability of DCs to promote tolerance is dependent on catabolic metabolism. Yet the contribution of AMP-activated kinase (AMPK), a central energy sensor promoting catabolism, to DC tolerogenicity remains unknown. Here, we show that AMPK activation renders human monocyte-derived DCs tolerogenic as evidenced by an enhanced ability to drive differentiation of regulatory T cells, a process dependent on increased RALDH activity. This is accompanied by a several metabolic changes, including increased breakdown of glycerophospholipids, enhanced mitochondrial fission-dependent fatty acid oxidation, and upregulated glucose catabolism. This metabolic rewiring is functionally important as we found interference with these metabolic processes to reduce to various degrees AMPK-induced RALDH activity as well as the tolerogenic capacity of moDCs. Altogether, our findings reveal a key role for AMPK signaling in shaping DC tolerogenicity, and suggest AMPK as target to direct DC-driven tolerogenic responses in therapeutic settings.
Project description:Immature dendritic cells (DCs) that are incubated with TLR-ligands during in vitro differentiation develop a tolerogenic phenotype. Especially an early encounter of isolated monocytes with the Toll-like receptor 7/8 ligand R848 mediates a switch of pro-inflammatory to immunosupressive properties. We were strongly interested in the identification of factors that are involved in the differentiation of these tolerogenic cells, thus we performed gene expression analysis.
Project description:We use tolDCs from syngeneic (C57BL6) and allogeneic (BALB/c) mice to show potent renoprotective abilities in a ischemia reperfusion AKI model. Dendritic cells were derived from mice bone marrow and cultured for 7 days with GM-CSF and IL-4. Tolerogenic DCs were cultured in the same DC media but with the addition of VitD3 and IL10. LPS was added on day 6 and cells harvested for use or analysis on day 7.
Project description:ChIP-seq of mouse embryonic fibroblast-adipose like cell line 3T3-L1 to identify binding sites of NCoR1 and SMRT following induction of differentiation, and RNA Pol-II after SMRT knock down