Abstract
Signal transducers and activators of transcription 5(STAT5) are cytokine induced signaling proteins, which regulate key immunological processes, such as tolerance induction, maintenance of homeostasis, and CD4 T-effector cell differentiation. In this study, transcriptional targets of STAT5 in CD4 T cells were studied by Chromatin Immunoprecipitation (ChIP). Genomic mapping of the sites cloned and 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 105 STAT5 responsive binding sites identified, 94% contained the canonical (IFN-γ activation site) GAS motifs.A number of putative target genes identified here are associated with tumor biology. Here, we identified Fos-related antigen 2 (FRA2) as a transcriptional target of IL-2 regulated STAT5. FRA2 is a basic -leucine zipper (bZIP) motif ‘Fos’ family transcription factor that is part of the AP-1 transcription factor complex and is also known to play a critical role in the progression of human tumours and more recently as a determinant of T cell plasticity. The binding site mapped to an internal intron within the FRA2 gene. The epigenetic architecture of FRA2, characterizes a transcriptionally active promoter as indicated by enrichment for histone methylation marks H3K4me1, H3K4me2, H3K4me3, and transcription/elongation associated marks H2BK5me1 and H4K20me1. FRA2 is regulated by IL-2 in activated CD4 T cells. Consistently, STAT5 bound to GAS sequence in the internal intron of FRA2 and reporter gene assays confirmed IL-2 induced STAT5 binding and transcriptional activation. Furthermore, addition of JAK3 inhibitor (R333) or Daclizumab inhibited the induction in TCR stimulated cells. Taken together, our data suggest that FRA2 is a novel STAT5 target gene, regulated by IL-2 in activated CD4 T cells.
Highlights
Signal transducers and activators of transcription STAT5a and STAT5b are highly homologous proteins that are encoded by two separate genes and are activated by Janus-activated kinases (JAK) downstream of cytokine receptors
We identify one of the Chromatin Immunoprecipitation (ChIP)-cloned sequences to map to an internal intronic region of the Fos-related antigen 2 (FRA2) gene, and show that FRA2 is induced by T –cell receptor activation in human CD4 T cells in an IL-2/JAK3/STAT5 dependent manner
Of the 105 unique cloned inserts isolated from activated CD4 T cells, greater than one third (42%) of the fragments lie within introns of annotated genes, with a larger percentage within intron 1 (13%)
Summary
Signal transducers and activators of transcription STAT5a and STAT5b (collectively called STAT5) are highly homologous proteins that are encoded by two separate genes and are activated by Janus-activated kinases (JAK) downstream of cytokine receptors. Upon activation by cognate JAKs, STAT proteins, dimerize and translocate into the nucleus where they bind to the promoters of genes containing the consensus recognition motif (GAS motif-TTCN3GAA) resulting in the transcriptional regulation of target genes. The target genes identified by STAT5-ChIP differ between cell types and are further influenced by cell treatments and time points studied [6,7]. The range of target genes that STAT5 regulates may differ from one cell to another, from one cell treatment to another as well as being dependant on the time point studied. These studies have begun to provide important mechanistic insights into the regulation of various biological and cellular processes by STAT5
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