Expression and functions of long noncoding RNAs during human T helper cell differentiation
ABSTRACT: To improve our understanding of lncRNA expression in T cells, we used whole genome sequencing (RNA-seq) to identify lncRNAs expressed in human T cells and those selectively expressed in T cells differentiated under TH1, TH2, or TH17 polarizing conditions. The majority of these lineage-specific lncRNAs are co-expressed with lineage-specific protein-coding genes. These lncRNAs are predominantly intragenic with co-expressed protein-coding genes and are transcribed in sense and antisense orientations with approximately equal frequencies. Further, genes encoding TH lineage specific mRNAs are not randomly distributed across the genome but are highly enriched in the genome in genomic regions also containing genes encoding TH lineage-specific lncRNAs. Our analyses also identify a cluster of antisense lncRNAs transcribed from the RAD50 locus that are selectively expressed under TH2 polarizing conditions and co-expressed with IL4, IL5 and IL13 genes. Depletion of these lncRNAs via selective siRNA treatment demonstrates the critical requirement of these lncRNAs for expression of the TH2 cytokines, IL-4, IL-5 and IL-13. Collectively, our analyses identify new lncRNAs expressed in a TH lineage specific manner and identify a critical role for a cluster of lncRNAs for expression of genes encoding TH2 cytokines. Human peripheral blood mononuclear cells (PBMC) were cultured under TH1, TH2, and TH17 polarizing conditions. TH1, TH2, and TH17 primary and effector cultures were isolated and poly(A)+ and total RNA sequencing performed.
Project description: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
Project description: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. Examination of Ezh2 binding in Th1 and Th2 cells.
Project description:Transcriptional analysis of human T cells differentiated in 4 T Helper context ( Th0, Th1, Th2 and Th17) in the presence or not of Interferon alpha We analyzed the transcriptomic profiles of 4 human naives T cells diferentiated in Th0, Th1, Th2 and Th17 in the presence or not of Interferon Alpha. Microarray analyses were performed in 2 time points : 1/ after Day 5 of polarization (= Day5); 2/ after Day 5+ four hours of re-stimulation (=Day 5+ 4H restim) in 3 different donors.
Project description:We found binding of the remodeling protein BRG1 was programmed by lineage and activation signals. BRG1 binding was positively correlated with gene activity at protein-coding and miRNA genes. BRG1 binding was found at promoters and distal regions, including known and novel distal regulatory elements. Distal BRG1 binding correlated with expression, and novel distal sites possessed enhancer activity, suggesting a general role for BRG1 in long-distance gene regulation. Together, these findings suggest BRG1 interprets differentiation and activation signals and plays a causal role in gene regulation, chromatin structure, and cell fate. Our findings indicate BRG1 binding is a useful marker for identifying cis-regulatory regions in protein-coding and miRNA genes. Compare BRG1 binding in T helper subsets genome wide; Naïve, resting Th1, resting Th2, Stimulated Th1, Stimulated Th2, Stimulated Th17, compared to input DNA as negative control
Project description:Differentiation of naive CD4 T cells into type 2 helper (Th2) cells is accompanied by chromatin remodeling and increased expression of a set of Th2-specific genes including those encoding Th2 cytokines. IL-4-mediated STAT6 activation induces high levels of transcription of GATA3, a master regulator of Th2 cell differentiation, and enforced expression of GATA3 induces Th2 cytokine expression. However, it remains unclear whether the expression of other Th2-specific genes is induced directly by GATA3. A genome-wide unbiased ChIP-seq analysis revealed that GATA3 bound to 1,279 genes selectively in Th2 cells, and 101 genes in both Th1 and Th2 cells. Simultaneously, we identified 26 highly Th2-specific STAT6-dependent inducible genes by a DNA microarray analysis-based three-step selection processes, and among them 17 genes showed GATA3 binding. We assessed dependency on GATA3 for the transcription of these 26 Th2-specific genes, and 10 genes showed increased transcription in a GATA3-dependent manner while 16 genes showed no significant responses. The transcription of the 16 GATA3-nonresponding genes was clearly increased by the introduction of an active form of STAT6, STAT6VT. Therefore, although GATA3 has been recognized as a master regulator of Th2 cell differentiation, many Th2-specific genes are not regulated by GATA3 itself but in collaboration with STAT6. Th1 and Th2 subsets are profiled for mRNA expression Examination of GATA3-binding and 3 different histone modifications in Th1 and Th2 cells.
Project description:Follicular T helper cells (Tfh) provide critical help to B cells for germinal center (GC) formation. Mutations affecting SLAM-associated Protein (SAP) prevent GC formation due to defective T-B cell interactions, yet effects on Tfh cell differentiation remain unclear. We describe the in vitro differentiation of functionally-competent “Tfh-like” cells that expressed Interleukin-21, Tfh markers, and Bcl6, and rescued GC formation in SAP-deficient hosts better than other T helper (Th) cells. SAP-deficient Tfh-like cells appeared virtually indistinguishable from wildtype, yet failed to support GCs in vivo. Interestingly, both Tfh-like and in vivo-derived Tfh cells could produce effector cytokines in response to polarizing conditions. Moreover, Th1, Th2 and Th17 cells could be reprogrammed to obtain Tfh characteristics. ChIP-Seq analyses revealed positive epigenetic markings on Tbx21, Gata3 and Rorc in Tfh-like and ex vivo Tfh cells, and Bcl6 in other Th cell populations, supporting the concept of plasticity between Tfh and other Th cells. Epigenetic modifications were evaluated in ChIP-Seq studies by profiling histone H3K4 and H3K27 trimethylation marks between Th1, Th2, Th17, Tfh-like, and CXCR5+PD-1+ sorted Tfh-like in vitro generated T helper cell populations and ex vivo derived Tfh cells.
Project description:Naive CD4+ CD62L+ CD25- T cells were differentiated under TH1 and TH2 conditions for 7 days, restimulated with anti-CD3 and anti-CD28 for 24h and sorted for IFN-gamma (TH1) and IL-4 (TH2) production using cytokine secretion assays.
Project description:In this work we present an analytical strategy to systematically identify early regulators by combining gene regulatory networks (GRN) with GWAS. We hypothesized that early regulators in T-cell associated diseases could be found by defining upstream transcription factors (TFs) in T-cell differentiation. Time series expression and DNA methylation profiling of T-cell differentiation identified several upstream TFs, of which TFs involved in Th1/2 differentiation were most enriched for disease associated SNPs identified by GWAS. Naïve CD4+ T cells were isolated from buffy coat of four healthy donors using a naïve CD4+ T cell isolation kit (Miltenyi, Bergisch-Gladbach, Germany). Naïve CD4+ T cells were stimulated with plate-bound anti-CD3 (500 ng/mL), soluble anti-CD28 (500 ng/mL), in the presence of IL-12 (5 ng/mL), IL-2 (10 ng/mL) and anti–IL-4 (5 µg/mL) for Th1, IL-4 (10 ng/mL), IL-2 (10 ng/mL) and anti-IL-12 (5 µg/mL) and anti–IFN-g (5 µg/mL) for Th2. Cells were cultured for six days in Iscove’s modified Dulbecco medium (IMDM) supplemented with 2 mM L-glutamine (PAA Laboratories, Linz, Austria), 10% heat-inactivated FCS (PAA Laboratories, Linz, Austria), 5 µM β–mercaptoethanol (Sigma-Aldrich, St. Louis, Missouri, USA) and 50 ug/mL gentamicin (Sigma-Aldrich, St. Louis, Missouri, USA). Cells were cultured for 6 days and then re-stimulated with plate-bound anti-CD3 and soluble anti-CD28 in the presence of corresponding polarizing cytokines and antibodies for another 2 days (Zhang et al. 2013). RNA was extracted using a miRneasy Mini Kit (Qiagen). The RNA concentrations were analysed with NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies). For the gene expression microarray analysis, The cRNA was prepared using a Low Input QuickAmp Labeling Kit. For Th1 and Th2 cells the gene expression microarray analysis was performed using SurePrint G3 Human Gene Expression 8x60K microarray kit, according to the manufacture’s instruction (Agilent Technologies).
Project description:Microarray analyses were performed to compare gene expression in cultured mouse Th9, Th2 and Treg cells and resting versus activated Th9 cells. Three replicates were analyzed for each culture condition; Th9 unstim, Th2 unstim, Treg unstim, Th9 stim
Project description:Host defense against diverse pathogens involves the recruitment and differentiation of CD4+ T effector subsets including T helper 1 (Th1), Th2, Th17 and induced regulatory T (Treg) cells. Surface phenotype studies have revealed subset-specific surface markers for the identification and purification of human primary CD4+ T effector subsets. In the present study, we aimed to characterize the mRNA and large intergenic non-coding RNA (lincRNA) expression differences between human primary CD4+ T effector subsets and identify potential subset-specific genes. To achieve this goal, mRNA and lincRNA microarray profiling of flow cytometry-sorted human primary Th1, Th2, Th17 and Treg cells was performed. Principal component and pathway analyses revealed subset-specific gene expression patterns. A Th2-specific lincRNA, GATA3-AS1, also termed FLJ45983, was identified in primary immune cells and tissues, as well as in in vitro polarized CD4+ T effector subsets. Further analysis showed that GATA3-AS1 was a potential diagnostic marker in allergy, a Th2-associated disease. This first systematic genome-wide analysis of gene expression differences between primary CD4+ T effector subsets may help to identify novel regulatory protein-coding genes and lincRNAs regulating CD4+ T cell subset differentiation, as well as potential diagnostic markers. As an example, we identified a GATA3-associated Th2-specific marker lincRNA GATA3-AS1. Gene expression microarray analysis of flow-cytometry sorted human primary naïve CD4+ T cells, CD4+ T central memory cells, Th1, Th2, Th17 and Treg cells from buffy coat of four healthy controls Gene expression microarray analysis was performed using SurePrint G3 Human Gene Expression 8X60K microarray.