Abstract
Abstract The proximal promoter controls transcription of IFN-γ in T cells, which is central for defense against many pathogens. By performing EMSA and supershift assays on nuclear extracts of TB-stimulated PBMC, we found that CREB, cAMP response element modulator (CREM), ATF-1, ATF-2, and c-Jun bound to the IFN-γ proximal promoter. Microaffinity purification with a biotinylated IFN-γ proximal promoter, followed by Western blotting, showed that only CREB, ATF-2 and c-Jun bound to the proximal promoter, and this was confirmed in live T cells by chromatin immunoprecipitation. Coimmunoprecipitation of nuclear extracts of TB-stimulated PBMC indicated interaction of these transcription factors. Introduction of a plasmid expressing a dominant-negative ATF-2 peptide into TB-stimulated T-cells inhibited IFN-γ secretion by >90% and reduced expression of CREB, ATF-2 and c-Jun. Nucleofection of siRNA specific for ATF-2 decreased expression of CREB, c-Jun and ATF-2 in T cells. TB-stimulated PBMC from tuberculosis patients, known to have reduced IFN-γ production, were found to have low levels of CREB, ATF-2 and c-Jun, but not CREM, ATF-1 and c-Fos, compared to PBMC from healthy donors. We conclude that: CREB, ATF-2 and c-Jun bind to the IFN-γ proximal promoter;ATF-2 contributes to expression of CREB and c-Jun;CREB, ATF-2 and c-Jun contribute to T-cell production of IFN-γ in response to TB. (NIH AI063514)
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