Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

A module-based translational strategy shows a central role for S100A4 in allergy

ABSTRACT: The involvement of thousands of genes complicates the identification of clinically relevant candidate genes in common diseases. We hypothesized that genes co-regulated with a key gene in allergy, IL13, would form a module that could help to discover novel candidate genes. We identified a Th2 cell module by siRNA mediated knock down of 25 putative IL13-regulating transcription factors (TFs) followed by expression profiling. Human CD4+ T cells (hBP CD4+ T cells, 2W-200, Lonza, Vallensbak Strand, Denkmark) were nucleofected either with nucleofection buffer, 1 ÂµM human on target plus SMART pool siRNA against ELK1, GATA3, NFATC3, MAF, NFKB1, JUN, STAT3 (Dharmacon, Lafayette, CO) or non-targeting siRNA using the AMAXA nucleofection program U-014. Six hours after then nucleofection cells were washed, activated and polarized towards Th2. The CD4+ cells were activated with plate bound anti-CD3 (500ng / ml for coating of the plate), with soluble anti-CD28 (500ng / ml) and with IL-2 (17ng /ml, all purchased from R&D). Th2 polarization was induced with anti-IL-12 (5Âµg/ml) and IL-4 (10ng / ml). For RT-PCR and microarray analyisis the cells were harvested 12 hours of polarization and total RNA was extracted.

ORGANISM(S): Homo sapiens

SUBMITTER: SÃ¶ren Bruhn

PROVIDER: E-GEOD-46333 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

A generally applicable translational strategy identifies S100A4 as a candidate gene in allergy.

Bruhn Sören S Fang Yu Y Barrenäs Fredrik F Gustafsson Mika M Zhang Huan H Konstantinell Aelita A Krönke Andrea A Sönnichsen Birte B Bresnick Anne A Dulyaninova Natalya N Wang Hui H Zhao Yelin Y Klingelhöfer Jörg J Ambartsumian Noona N Beck Mette K MK Nestor Colm C Bona Elsa E Xiang Zou Z Benson Mikael M

Science translational medicine 20140101 218

The identification of diagnostic markers and therapeutic candidate genes in common diseases is complicated by the involvement of thousands of genes. We hypothesized that genes co-regulated with a key gene in allergy, IL13, would form a module that could help to identify candidate genes. We identified a T helper 2 (TH2) cell module by small interfering RNA-mediated knockdown of 25 putative IL13-regulating transcription factors followed by expression profiling. The module contained candidate genes ...[more]

PMID: 24401939

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Project description:Several versions of SWI/SNF complexes, BAF and PBAF, have been described based on unique subunit composition. We find that T cell development in the thymus and lymphoid periphery is largely normal in the absence of the PBAF-specific component BAF180. However, BAF180-deficient Th2 cells express high levels of the immunoregulatory cytokine IL-10. BAF180 binds directly to regulatory elements in the Il-10 locus but is replaced by BAF250 BAF complexes in the absence of BAF180, resulting in increased histone acetylation and CBP recruitment to the IL-10 locus. These results demonstrate that BAF180 is a repressor of IL-10 transcription in Th2 cells and suggest that the differential recruitment of different SWI/SNF subtypes can have direct consequences on chromatin structure and gene transcription. mouse primary T helper lymphocytes, naïve and Th2 cells, resting and stimulated, comparison of wild-type and BAF180/Pbrm1 deficient cells RNA from T helper cells was compared in the presence (WT) and absence (KO) of the Pbrm1/BAF180 gene. Comparison was made in 2 T helper subsets (Naive and Th2), each under 2 condtions (resting and stimulated), in triplicate (A,B,C). Resting naïve CD4+ T cells (N) were purified to 95% purity from the spleens and lymph nodes of 4-6 week old Balb/c mice (WT, or BAF180 cKO) using CD4+CD62L+ T cell Purification Kit II per manufacturer’s instructions (Miltenyi). Stimulated Naive cells (S) were prepared from resting naive cells by stimulation for 24 hours on anti-CD3 (1ug/ml), anti-CD28 (2 ug/ml) coated plates at 1-2 x 106/ml. Resting Th2 cells (2U) were prepared from resting naive cells by plating onto anti-CD3 (1ug/ml), anti-CD28 (2 ug/ml) coated plates at 1-2 x 106/ml in the presence of 10ng/ml IL-4, 10ug/ml anti-IFN-gamma. IL-2 (100U/ml) was added 24 hours later. Cultures were expanded in IL-2 (100U/ml) three days after initial culture. Stimulated Th2 cells (2S) were prepared from resting Th2 cells, stimulated with PMA (50ng/ml) and Ionomycin (500ng/ml) for 1.5 hours. RNA was harvested from 3 replicates of the primary Th cells under each condition. RNA was isolated using RNAeasy Kit (Qiagen), Quality and quantity of the total RNA was checked with the Agilent 2100 bioanalyzer using RNA 6000 Nano chips. RNA was labeled using the standard Illumina protocol and Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791) Biotin labeled cRNA was hybridized to Illumina’s Sentrix MouseRef-8,v2 Expression BeadChips.

Dataset Information

A module-based translational strategy shows a central role for S100A4 in allergy

Publications

A generally applicable translational strategy identifies S100A4 as a candidate gene in allergy.

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