Constitutive and inducible protein/DNA interactions of the interferon‐γ promoter in <i>in vivo</i> CD45RA and CD45RO T helper subsets

Abstract

Abstract Interferon‐γ (IFN‐γ) is a key cytokine of T lymphocytes with major regulatory functions in the immune system. To determine and compare protein/DNA interactions at the native IFN‐γ locus in T cells, we analyzed the human IFN‐γ promoter by ligation‐mediated polymerase chain reaction (LM‐PCR) techniques. Accordingly, Jurkat T cells and primary CD45RA and CD45R0 CD4 + T cell subsets isolated from peripheral blood using immunomagnetic beads were cultured and analyzed by LM‐PCR. Constitutive and inducible protein/DNA interactions of the IFN‐γ promoter in vivo were detected in all T cells tested. Interestingly, an inducible footprint between − 183 and − 196 was consistently observed in Jurkat T cells and CD45RA and CD45R0 T helper subsets upon stimulation with phorbol 12‐myristate 13‐acetate + phytohemagglutinin (PMA + PHA) that was highly sensitive to treatment with corticosteroids. This novel target site, denoted the C‐site, was shown by several criteria, including cell distribution studies, stimulation experiments, supershift assays, and cross‐competition electrophoretic mobility shift assays to bind the transcription factor AP‐1. Mutation of the C‐site that prevented AP‐1 binding to this site was sufficient strikingly to reduce inducible promoter activity in primary CD45R0 T cells. In summary, the data demonstrate that IFN‐γ gene transcription in primary T cells is regulated in vivo at the level of constitutive and inducible protein/DNA interactions. We propose a model where basal transcription is maintained by binding of various transcription factors to the IFN‐γ promoter, whereas PMA + PHA‐inducible IFN‐γ transcription in CD45R0 T cells is associated with binding of AP‐1 to the C‐site.