Unknown,Transcriptomics,Genomics,Proteomics

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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.

ORGANISM(S): Homo sapiens

SUBMITTER: Charles Spurlock III

PROVIDER: E-GEOD-66261 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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. NaÃ¯ve CD4+ T cells were isolated from buffy coat of four healthy donors using a naÃ¯ve CD4+ T cell isolation kit (Miltenyi, Bergisch-Gladbach, Germany). NaÃ¯ve 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 antiâ??IL-4 (5 Âµg/mL) for Th1, IL-4 (10 ng/mL), IL-2 (10 ng/mL) and anti-IL-12 (5 Âµg/mL) and antiâ??IFN-g (5 Âµg/mL) for Th2. Cells were cultured for six days in Iscoveâ??s modified Dulbecco medium (IMDM) supplemented with 2 mM L-glutamine (PAA Laboratories, Linz, Austria), 10% heat-inactivated FCS (PAA Laboratories, Linz, Austria), 5 ÂµM Î²â??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 manufactureâ??s instruction (Agilent Technologies).

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Expression and functions of long noncoding RNAs during human T helper cell differentiation

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