This section has been studying gene regulation in the developing immune system. Our focus has been transcriptional regulation of genes mediated by retinoids/ vitamins and interferons (IFNs). These agents exert regulatory activities partly through transcription factors called RXRbeta and ICSBP that we have previously isolated. RXRbeta is a member of the nuclear hormone receptor superfamily and heterodimerizes with many other members of the superfamily. We have shown that RXRbeta heterodimerizes with retinoic acid (RA) receptor beta, and plays a key role in RA induction of MHC class I genes in human embryonal carcinoma (EC) cells. To further elucidate the role of RXRbeta heterodimers in vivo, several dominant negative mutants of RXRbeta have been constructed and their activities examined in P19 EC cells. Our data show that a mutant lacking the DNA binding domain inhibits RA induction of several genes, by inhibiting the function of RXR/RAR heterodimers formed in vivo. We found that this inhibition also leads to an altered growth regulation in the EC cells. By genomic footprinting analysis, it was found that the RA responsive element of an RA inducible gene is not occupied prior to RA treatment in undifferentiated EC cells, but the occupancy is promptly induced after RA treatment. This is the first demonstration that RXR heterodimers bind to cognate DNA only after RA treatment in vivo, which is in contrast to the RA independent DNA binding in vitro. IFNs are cytokines produced in many types of cells, and are being used for treatment of cancers and viral infections. IFNs' biological activities are realized through the action of the IRF family, to which ICSBP belongs to. We showed that ICSBP represses IFN induction of many IFN regulated genes, which is attributed to the interaction of ICSBP and other members of the family. We found that ICSBP directly binds to IRF-1 and IRF-2, which enhances binding of ICSBP to cognate DNA. This association can be detected in vitro, but occurs also in vivo, and in vitro. In the coming year we will focus on mechanistic analysis of how these factors acts. Interactions of RXRbeta and IRF family members with basal transcription factors will be studied for both physical and functional interactions. The recently developed in vitro transcription assay involving recombinant RXRbeta will be used for functional analysis.
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