Unknown,Transcriptomics,Genomics,Proteomics

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The polycomb protein Ezh2 regulates differentiation and plasticity of CD4 T helper type-1 and type-2 cells.

ABSTRACT: Following antigen encounter by CD4 T cells, polarizing cytokines induce the expression of master regulators that control differentiation. Inactivation of the histone methyltransferase Ezh2 was found to specifically enhance T-helper (Th)1 and Th2 cell differentiation and plasticity. Ezh2 directly bound and facilitated correct expression of Tbx21 and Gata3 in differentiating Th1 and Th2 cells, accompanied by substantial tri-methylation at lysine 27 of histone 3 (H3K27-Me3). In addition, Ezh2 deficiency resulted in spontaneous generation of discrete IFN-γ and Th2 cytokine-producing populations in non-polarizing cultures, and under these conditions IFN-γ expression was largely dependent on enhanced expression of the transcription factor Eomesodermin. In vivo, Loss of Ezh2 caused increased pathology in a model of allergic asthma and resulted in progressive accumulation of memory phenotype Th2 cells. This study establishes a functional link between Ezh2 and transcriptional regulation of lineage-specifying genes in terminally differentiated CD4 T cells. Wild type and Ezh2 knock out unpolarized Th cells, Th1 cells and Th2 cells are profiled for mRNA expression

ORGANISM(S): Mus musculus

SUBMITTER: Atsushi Onodera

PROVIDER: E-GEOD-50729 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Gene expression profiling of repeatedly activated compared to recently activated Th1 cells to identify genes that play a role in chronic inflammatory disorders and may qualify as diagnostic or therapeutic targets; ; Upon activation under appropriate costimulatory conditions, naive T helper (Th) cells differentiate into Th2 or Th17 cells, each characterized by the expression of specific effector cytokines. In response to a repeated stimulation with antigen, Th cells develop a stable memory for the expression of those cytokines as well as for other secreted or membrane-associated factors. The stable memory for the expression of proinflammatory effector functions may explain the resistance of Th effector cells to conventional immunosuppressive therapy, and the inability of immunosuppression to cure chronic inflammation. The imprinting of the functional memory is based on epigenetic modifications and expression of distinct transcription factors. In this project, we compare the transcriptomes of once and repeatedly activated murine Th1 cells, to identify genes that induce and maintain the functional memory and control the persistence of pathogenic memory Th1 cells. This in turn might help to discriminate pathogenic versus protective cells in immunopathology and present novel targets for the diagnosis and therapy of chronic inflammatory disease. Experiment Overall Design: Genes differentially expressed in once versus four times stimulated Th1 cells. In vitro polarization of murine naÃ¯ve DO11.10 T cells towards Th1 direction (5 ng/ml recombinant murine IL-12, 5 Î¼g/ml anti-IL-4 antibody) with antigenic stimulation (ova323-339 and irradiated splenic APCs). The transcriptional profiles of resting one week old Th1 (Th1 1w) cells and resting 4 week old Th1 (Th1 4w) cells were compared using Affymetrix Murine Genome (MG) U74V2A GeneChip arrays. Experiment Overall Design: 10 Âµg of total RNA from each cell sample was reverse transcribed using T7-(d)T24 primer and SuperScript II reverse transcriptase Experiment Overall Design: cDNA extraction with a PhaseLock gel (Eppendorf), and precipitation with ethanol and ammonium acetate Experiment Overall Design: Biotinylated cRNA was transcribed with the MEGAscript high yield transcription kit (Ambion), fragmented, and the hybridization cocktail was prepared (15 Âµg fragmented biotin-labeled cRNA spiked with Eukaryotic Hybridization control) Experiment Overall Design: probes were subsequently hybridised with the GeneChip U74Av2 for 16 hrs at 45 oC Experiment Overall Design: after washing the hybridisation signals were visualised by staining with streptavidin-phycoerythrin and amplification with an anti-streptavidin antibody Experiment Overall Design: TH1_1w_C1; TH1_1w_C2; TH1_1w_C3TH1_4w_C1; TH1_4w_C2; TH1_4w_C3 Experiment Overall Design: 1w: 1 week in culture; 4w: 4 weeks in culture; C1-3: Culture or Experiment No.

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The polycomb protein Ezh2 regulates differentiation and plasticity of CD4 T helper type-1 and type-2 cells.

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