Transcriptional regulation of interferon γ gene by p300 co-activator

Interferon y (IFN y) is an important immunoreguratory protein and is secreted from CD4, CD8 T cells and NK cells. Expression of IFN y gene is regulated by either DNA-methylation or m-elements of transcriptional factors such as AP-1, NFAT, AP-4, YY-1, ATF and GATA. At a putative ATF binding site in a promoter region of IFN y gene, the position from -59 to -43, is important for the IFN y promoter activity. Our gel-shift competition studies demonstrated that addition of antibodies against ATF-2 caused super-shifts of DNA-protein complexes. In addition, it was clarified that p300 was involved in DNA-protein complexes by in vitro "pull-down" experiments. Moreover, a co-expression of p300 and ATF-2 enhanced the promoter activity of IFN y gene. Thus, these results indicate that p300 and ATF-2 play critical rules in a basal transcription of IFN y gene in Jurkat T cells. Introduction There are many putative transcriptional factor-binding sites such as AP-1, GATA, NFAT and ATF in the promoter of IFN y gene and they play a key role in the basal transcription of IFN y gene (1). The transcriptional co-activator p300 plays critical roles for modulating activities of many transcriptional factors, including CREB, nuclear receptors and myogenic basic helix-loop-helix transcription factors by a direct association (2). p300 has also an intrinsic histone acetyltransferase activity that promotes a chromatin remodeling during an initiation step of transcription. Since it has been reported that p300 is involved in an enhanceosome that is formed in an IFN P promoter (3), it is believed that p300 participates in the immune system. However, it is still unknown whether p300 concerns transcription of IFN y gene. In this study, we analyzed a promoter region of IFN y gene using a promoter assay or a gel-shift assay. Then, we demonstrated that the transcriptional co-activator p300 and ATF-2 promoted transactivation of the human IFN y gene in Jurkat T cells. Results and Discussion Interferon y (IFN y), which belongs to a type II IFN family, is one of the important immunoregulatory cytokines responsible for several immunological effects and diverse activities in an immune system. This cytokine is secreted from CD4, CD8 T cells and NK cells in response to infection of viruses. As a result, a replication of the virus is inhibited and capacity of macrophage is activated. Thus, analyzing an induction mechanism of IFN y gene in detail is important to understand the immune system. At first, we tried to identity a minimal promoter region and an element that was essential for the promoter activity of the human IFN y gene in Jurkat T cells. To identify the minimal promoter activity, we constructed five deletion mutants of the IFN y promoter using the pica geneTM enhancer vector 2 that contained a luciferase gene as a reporter gene. Then, these mutant plasmids were transfected into Jurkat T cells transiently. After 48h, a luciferase activities in each cell were measured using a 90 Nucleic Acids Research Supplement No. I