Project description:Auto Immune REgulator (AIRE) protein expression in HEK293 cells Three cell-lines where used in the study: HEK293-NC; control cell-line expressing yellow fluorescent protein (YFP) HEK-AIRE1, HEK-AIRE2; two different clone-derived Autoimmune regulator protein expressing cell-lines HEK-AIRE-D312A; cell line expressing Autoimmune regulator protein with D312A mutation in the first PHD finger With all cell-lines three independent experiments were performed.
Project description:Methylated DNA immunoprecipitation-sequencing of HEK293 cells (HEK293-CT) and HEK293 cells stably over-expressing the BAHD1 gene (HEK-BAHD1)
Project description:Comparison of methylome of HEK293-CT cells and HEK293 cells stably over-expressing the BAHD1 gene (HEK-BAHD1) We used BS-seq to identify genomic regions differentially methylated upon overexpression of the chromatin repressor BAHD1 in HEK293 cells.
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.
