P169 Identication of FRA2 as a novel target gene, regulated by IL-2 in T cells

Abstract

Introduction Signal transducers and activators of transcription 5 (STAT5) is a key signaling protein and therefore critically regulates genes involved in cell differentiation, proliferation and survival. In this study, chromatin immunoprecipitation (ChIP) was used to identify novel STAT5 target genes in human CD4+ T cells. One of STAT5 binding sites mapped to the FRA2 gene which is a basic-leucine zipper (bZIP) motif ‘Fos’ family transcription factor that is part of the ‘activating protein 1’ (AP-1) transcription factor complex. FRA2 is known to play a critical role in the progression of human tumors and cancers. Overexpression of FRA2 has been identified in various human cancers, such as salivary gland tumors, breast cancer, colorectal cancer, adult T-cell leukemias and cutaneous T cell leukemias. In the immune system, FRA2 is involved in IL-4 gene regulation and is involved in CD4 T-helper2 (TH2) cell differentiation. Previously, we and others have shown that STAT5 promotes Th2 cells differentiation by regulating a number of key genes involved in this lineage specification process and the identification of FRA2 as a STAT5-regulated gene here is consistent with this theme. Methods Chromatin immunoprecipitation was used to identify STAT5 target genes in human CD4+ T cells. Thirty STAT5 binding sites were confirmed as bonafide target sites by performing independent ChIP-PCR. Fifteen genes were validated for regulation by IL-2 using qRT-PCR. Results Using ChIP, this study identified 192 putative STAT5 responsive binding sites in CD4+ T cells. Genomic mapping of the STAT5 binding sites identified in this study revealed the striking observation that the majority of STAT5-binding sites mapped to intergenic (>50 kb upstream) or intronic, rather than promoter proximal regions. Of the 192 STAT5 responsive binding sites identified in CD4+ T cells, 87% contained canonical (IFN-gactivation site) GAS motifs, TTCN3GAA and/or TTN5AA. Bioinformatic analyses, using Gene Ontology programmes to annotate and functionally classify the genes associated with binding sites, predicted cell cycle control, cell structure, cell adhesion and motility, transport, and metabolism as the most common functions for putative STAT5-regulated genes, in addition to previously known functions such as cell differentiation, proliferation, signal transduction, apoptosis and development. Additionally, several target-genes were identified, whose aberrant functions are associated with malignant transformation of cells, consistent with the frequent dysregulation of STAT5 noted in various cancers. The FRA2 gene was selected due to its obvious role in T cell differentiation as well as cancers and tumors. The STAT5 binding site in the FRA2 gene maps to an internal intron within the gene and corresponds toa region that is enriched for the activating marks, H3K4me1, H3K4me3mark and the active enhancer mark H3K27Ac. Conclusion FRA2 is regulated by IL-2 in T cells.