Interferon (IFN)-gamma, a cytokine produced by T-lymphocytes, plays an important role in host self-defense against viral infection and intracellular pathogens, cell growth and differentiation, and regulation of immunological responses. The biological activities of IFN-gamma are mediated, at least in part, by changes in cellular proteins and enzymes. This includes indoleamine 2,3-dioxygenase (IDO), an important enzyme in the metabolism of tryptophan. IDO is induced strongly by IFN-gamma but poorly by IFN-alpha or -beta and its has been implicated in the antiproliferative action of IFN-gamma on tumor cells, and inhibition of intracellular pathogens, e.g., Toxoplasma gondii, Chlamydia psittaci, etc. A cDNA clone (called C5-4) isolated in our laboratory, corresponding to an IFN-gamma- inducible mRNA, was identified as a clone for IDO. This provides a tool to study the biochemical mechanism involved in the differential regulation of IDO gene expression by IFN-gamma as against IFN-alpha and -beta. Earlier studies showed that IFN-gamma induced the transcription of the C5-4 (IDO) gene, but de novo synthesis of some protein(s) was required, indicating the involvement of a novel pathway. Genomic DNA clones corresponding to C5- 4/IDO gene have been isolated and analyzed. A 155 nucleotide 5'-flanking fragment of the IDO gene conferred inducibility to a reporter gene by IFN- gamma but not (or very poorly) by IFN-alpha2, indicating that this fragment contained an IFN-gamma-responsive sequence (IGRS) which responded poorly, if at all, to IFN-alpha. Studies are planned to delineate the IFN-gamma- responsive cis-acting element(s) of the IDO gene and determine the distinction from the IFN(-alpha)-stimulated-responsive element (called ISRE) found in IFN-alpha/beta-inducible cellular genes. For example, deletion analyses and in vivo competition experiments will be carried out to identify the IGRS and determine (a) whether an ISRE homolog is important for the IGRS response to IFN-gamma; (b) whether ISRE from an IFN- alpha/beta-inducible gene would compete for the response of IGRS to IFN- gamma; and (c) whether the response of IGRS to IFN-gamma may involve factor(s) which serve(s) as activator(s) or neutralize some inhibitor of IDO gene expression. The IGRS element(s) will be used to identify IFN- gamma-induced or modified nuclear factor(s) that interact(s) with the IGRS element(s) by mobility shift experiments, methylation interference and DNase footprinting analyses, etc.; and we will determine whether any IFN- gamma-induced changes in DNA-binding factors are dependent on de novo protein synthesis, which would allow the identification of either the putative newly synthesized protein factor or its product involved in IDO gene regulation. It is planned to purify and characterize the IFN-gamma- regulated factor(s), attempt to clone it, and to study its role IFN-gamma- induced expression of IDO (and perhaps other) gene(s). It is of interest that lymphocytes and lymphocyte-derived cell lines do not show induction of IDO when treated with IFN-gamma, which will be investigated. These studies will contribute to an understanding of the biochemistry of action of IFN- gamma and its differential biological activities as against IFN-alpha and - beta.
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