Comment[ArrayExpressAccession] E-GEOD-46185 MAGE-TAB Version 1.1 Public Release Date 2013-04-19 Investigation Title Genome-wide gene expression profiling revealed a critical role for GATA3 in the maintenance of the Th2 cell identity Comment[Submitted Name] Genome-wide gene expression profiling revealed a critical role for GATA3 in the maintenance of the Th2 cell identity Experiment Description Functionally polarized CD4+ T helper (Th) cells such as Th1, Th2 and Th17 cells are central to the regulation of acquired immunity. However, the molecular mechanisms governing the maintenance of the polarized functions of Th cells remain unclear. GATA3, a master regulator of Th2 cell differentiation, initiates the expressions of Th2 cytokine genes and other Th2-specific genes. GATA3 also plays important roles in maintaining Th2 cell function and in continuous chromatin remodeling of Th2 cytokine gene loci. However, it is unclear whether continuous expression of GATA3 is required to maintain the expression of various other Th2-specific genes. In this report, genome-wide DNA gene expression profiling revealed that GATA3 expression is critical for the expression of a certain set of Th2-specific genes. We demonstrated that GATA3 dependency is reduced for some Th2-specific genes in fully developed Th2 cells compared to that observed in effector Th2 cells, whereas it is unchanged for other genes. Moreover, effects of a loss of GATA3 expression in Th2 cells on the expression of cytokine and cytokine receptor genes were examined in detail. A critical role of GATA3 in the regulation of Th2-specific gene expression is confirmed in in vivo generated antigen-specific memory Th2 cells. Therefore, GATA3 is required for the continuous expression of the majority of Th2-specific genes involved in maintaining the Th2 cell identity. Mock-transfected and GATA3 siRNA-transfected Th2 and Th2-4th cells are profiled for mRNA expression Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Onodera Sasaki Onodera Hosokawa Watanabe Horiuchi Yamashita Tanaka Ogawa Suzuki Nakayama Person First Name Atsushi Tetsuya Atsushi Hiroyuki Yukiko Shu Junji Hitoshi Yasumasa Yutaka Toshinori Person Email geo@ncbi.nlm.nih.gov Person Affiliation Chiba University Person Address Immunology, Chiba University, Chuo-ku Inohana 1-8-1, Chiba, Japan Person Roles submitter Protocol Name P-GSE46185-1 P-GSE46185-5 P-GSE46185-6 P-GSE46185-8 P-GSE46185-2 P-GSE46185-3 P-GSE46185-9 P-GSE46185-4 P-GSE46185-7 Protocol Description Expression values were determined using Affymetrix GeneChip Command Console Software (AGCC) and Console Software (Expression Console). ID_REF = VALUE = MAS5.0 signal intensity Total RNA was labeled using the GeneChip 3' IVT Express Kit cRNA was hybridized to GeneChip Mouse Genome 430 2.0 Arrays from Affymetrix according the manufacturers protocol. Th2-4th cells were collected and treated with control siRNA (AM4635; Applied Biosystems) or Gata3 siRNA (s66482; Applied Biosystems) using the Mouse T cell Nucleofector Kit (Amaxa) according to the manufacturer's protocol. Mock- or Gata3 siRNA-transfected Th2-4th cells were harvested after 24hrs, and stimulated with or without immobilized anti-TCR mAb (H57-597; 3 mg/ml) for 4hrs. Then cells were collected and resuspendet in the Trizol solution (GibcoBRL). Th2 culture cells were collected and treated with control siRNA (AM4635; Applied Biosystems) or Gata3 siRNA (s66482; Applied Biosystems) using the Mouse T cell Nucleofector Kit (Amaxa) according to the manufacturer's protocol. Mock- or Gata3 siRNA-transfected Th2 cells were harvested after 24hrs, and stimulated with or without immobilized anti-TCR mAb (H57-597; 3 mg/ml) for 4hrs. Then cells were collected and resuspendet in the Trizol solution (GibcoBRL). Splenic CD4 T cells were prepared using a magnetic cell sorter (AutoMACS; Miltenyi Biotec) yielding a purity of >98%. Where indicated, cells from C57BL/6 mice were stimulated with immobilized anti-TCR mAb (H57−597; 3 mg/ml) and anti-CD28 mAb under Th2-culture conditions for 2days in vitro. Th2 conditions; 25 U/ml IL-2, 100 U/ml IL-4. After culturing for 3 more days, the cells were harvested. Splenic CD4 T cells were stimulated under Th2 culture conditions for 5 days in vitro. The Th2 cells were further cultured in vitro for another 2 days in the absence of any exogenous cytokines. The cultured CD4 T cells were then restimulated with immobilized anti-TCR mAbs with IL-2 and anti-IL-4 mAbs for 5 days. This cycle was repeated three times. (Th2-4th cells) Trizol extraction of total RNA was performed according to the manufacturer's instructions. GeneChips were scanned using the GeneChip Scanner 3000. Protocol Type normalization data transformation protocol labelling protocol hybridization protocol sample treatment protocol sample treatment protocol growth protocol growth protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name TREATMENT Experimental Factor Type treatment Comment[SecondaryAccession] GSE46185 Comment[GEOReleaseDate] 2013-04-19 Comment[ArrayExpressSubmissionDate] 2013-04-18 Comment[GEOLastUpdateDate] 2013-04-20 Comment[AEExperimentType] transcription profiling by array SDRF File E-GEOD-46185.sdrf.txt