IRF2BP2 Modulates the Crosstalk between Glucocorticoid and TNF Signaling

Here we have analyzed the role of interferon regulatory factor-2 binding protein-2 (IRF2BP2) in glucocorticoid and tumor necrosis factor alpha (TNF) signaling. We used ChIP-seq to analyze chromatin binding of IRF2BP2 in glucocorticoid (dexamethasone, dex) and vehicle treated HEK293 cells expressing GR (HEK293-GR). Furthermore, we used RNA-seq to analyze how silencing of IRF2BP2 modulates transcriptional responses to dex treatment in HEK293-GR cells, and dex, TNF and co-treatment (dex and TNF, DT) in A549 cells. Overall design: For ChIP-seq, HEK293-GR cells were grown on steroid-depleted medium for two days and treated with dex or vehicle for one hour. Cells were fixed with 1% (v/v) formaldehyde, chromatin was fragmented to ~200-400 bp by sonication, chromatin immunoprecipitation was done using anti-IRF2BP2 antibody and libraries were prepared using NEBNext Ultra II kit (New England Biolabs). Two biological replicates of both conditions were sequenced using Illumina HiSeq 2000 at EMBL GeneCore (Heidelberg, Germany). For RNA-seq, HEK293-GR or A549 cells were cultured in steroid-depleted media and transfected with siRNA against IRF2BP2 (siIRF2BP2, 20nM, Dharmacon, ON-TARGETplus SMARTpool, L-007177-02-0005) or control siRNA (siNON, Dharmacon, non-targeting pool) using Lipofectamine RNAiMAX (Invitrogen). After three days, cells were treated with dexamethasone (dex, 100 nM), Tumor Necrosis Factor alpha (TNF, 10 ng/ml), or vehicle (EtOH, equal amount of ethanol) for six hours. RNA from three biological replicates was extracted using RNeasy Plus Mini kit (Qiagen) and mRNA isolated using NEBNext Poly(a) mRNA Magnetic Isolation Module (New England Biolabs). Libraries were prepared using NEBNext Ultra II kit (New England Biolabs) and sequenced with HiSeq 2000 at EMBL GeneCore (Heidelberg, Germany).