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Gene expression of HEK 293 cells transfected with Vector (PCMV), Aire, or MBD-VP16

ABSTRACT: HEK 293 cells were transiently transfected with plasmids expressing Vector only(PCMV), Aire, or MBD-VP16 with the goal of comparing the global gene expression profiles in the Aire and MBD-VP16 groups We used microarrays to detail the global gene expression profile.

ORGANISM(S): Homo sapiens

PROVIDER: GSE52546 | GEO | 2013/11/21

SECONDARY ACCESSION(S): PRJNA229364

REPOSITORIES: GEO

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Gene expression of HEK 293 cells transfected with Vector (PCMV), Aire, or MBD-VP16

Project description:HEK 293 cells were transiently transfected with plasmids expressing Vector only(PCMV), Aire, or MBD-VP16 with the goal of comparing the global gene expression profiles in the Aire and MBD-VP16 groups We used microarrays to detail the global gene expression profile. HEK 293 cells were transiently transfected with plasmids expressing Vector only(PCMV), Aire, or MBD-VP16 and 72 hrs post transfection total RNA was collected for microarray analysis.

2013-11-21 | E-GEOD-52546 | biostudies-arrayexpress

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Project description:The Autoimmune Regulator (AIRE) protein is expressed in thymic medullary epithelial cells, where it promotes the ectopic expression of tissue-restricted antigens needed for efficient negative selection of developing thymocytes. Mutations in AIRE cause APECED syndrome, which is characterized by a breakdown of self-tolerance. The molecular mechanism by which AIRE increases the expression of a variety of different genes remains unknown. Here, we studied AIRE-regulated genes using whole genome expression analysis and chromatin immunoprecipitation. We show that AIRE preferentially activates genes that are tissue-specific and characterized by low levels of initial expression in stably transfected HEK293 cell model and mouse thymic medullary epithelial cells. In addition, the AIRE-regulated genes lack active chromatin marks, such as histone H3 trimethylation (H3K4me3) and acetylation (AcH3), on their promoters. We also show that during activation by AIRE, the target genes acquire histone H3 modifications associated with transcription and RNA polymerase II. In conclusion, our data show that AIRE is able to promote ectopic gene expression from chromatin associated with histone modifications characteristic to inactive genes. We compared the gene expression profiles of HEK293 cell-lines expressing either YFP (NC) or AIRE (AIRE1) proteins using Illumina BeadChips (GSE16877). Based on these results we chose more than 50 genes that where upregulated in the presence of AIRE and used chromatin immunoprecipitation and custom made Nimblegen tiling arrays to study the regions of 200kb up-and downstream of the genes in HEK293 cells expressing either YFP or AIRE protein. The following antibodies where used in the ChIP experiments: H3K4me3, AcH3, H3K27me3, H3, PolII and AIRE.

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Project description:To analyze the impact of Aire on gene expression profile in a model cell line, we used 293T cells and transfected them either with an Aire expression plasmid pCMV-Aire (where mAire is driven by CMV promoter) or with a control plasmid pCMV2B. Total RNA was extracted 48 hours post transfection, processed and used for gene expression profiling by Affymetrix. The data demonstrate that Aire has a very broad impact, effecting (upregulating and downregulating) hundreds of differents genes, however these genes differ dramatically from its targets in medullary epithelial cells. Keywords: transfection 293T cells were seeded on 10cm plates and were grown in DMEM containing 10% FBS under standard TC conditions. Next day, the cells were transfected either by pCMV-Aire (6ug/plate) or empty pCMV (6ug/plate) using Mirus reagent, according to manufacturer's instructions. Total RNA was extracted 48 hrs post transfection by Trizol protocol according to the manufacturer's instructions. 3 biological replicates were used for Aire transfection and 2 replicates for transfection with a control plasmid.

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Homo sapiens

Project description:Gene expression of HEK 293 cells transfected with Vector (PCMV), Aire, or MBD-VP16

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