This group works on gene regulation in the immune system. Our focus has been to elucidate the role of two transcription factors, RXRbeta and ICSBP that we have isolated previously. RXRbeta is a member of the nuclear hormone receptor superfamily and by heterodimerizing with retinoic acid receptor(RAR), it regulates retinoic acid (RA) responsive genes. ICSBP is a member of the interferon regulatory factor (IRF) family and is involved in viral/interferon (IFN) mediated gene regulation. To further study the role of ICSBP and other members of the IRF family in the immune system, a dominant negative ICSBP has been constructed which has only the DNA binding domain (DBD) and lacks a large C-terminal domain. This construct was stably transfected into a human monocytic cell line U937 cells, and a number of clones expressing the DBD have been established. The DBD clones exhibited altered infectivity by several viruses: they were refractory to infection by vaccinia virus, ectromelia virus as well as human immunodeficiency virus-1. In contrast, control clones and those expressing the intact ICSBF were strongly infected by these viruses. The DBD clones were, however, infected by vesicular stomatitis virus (VSV) as efficiently as control clones. But, IFN pretreatment known to confer protection against VSV, completely failed to protect DBD clones. As expected control clones and those expressing intact ICSBF were fully protected from VSV infection by IFNs. These results indicate that some viruses depends on the IRF proteins for their infection and growth in host cells. By genomic footprinting analysis we have studied binding of RXR heterodimers to the promoter of an RA responsive gene (RARbeta) in Pl9 EC cells. We show that the RA responsive element (RARE) in the RARbeta gene is not occupied prior to RA treatment, but becomes occupied within 1 h of RA treatment, without requiring new protein synthesis. This in vivo occupancy accompanied factor binding to other elements in the promoter. The occupancy at all sites was rapidly reversed when RA was withdrawn from culture medium. On the other hand, Pl9 cells expressing a dominant negative RXRbeta that blocks binding of endogenous RAR/RXR heterodimers to the RARE, did not exhibit occupancy at all elements in the promoter. Our observations underscore the significance of ligand for receptor DNA interaction in vivo and demonstrate the presence of a hierarchy in factor binding processes.
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