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Biomedical subjects

L Dorman

Publications and source records attributed to L Dorman.

6 recordsLinked to original sources
PubMed1997

Interaction of NF-kappaB and NFAT with the interferon-gamma promoter.

Interferon-gamma (IFN-gamma) is a pleiotropic lymphokine whose production is restricted to activated T cells and NK cells. Along with other cytokines, IFN-gamma gene expression is inhibited by the immunosuppressant cyclosporin A. We have previously identified an intronic enhancer region (C3) of the IFN-gamma gene that binds the NF-kappaB protein c-Rel and that shows partial DNA sequence homology with the cyclosporin A-sensitive NFAT binding site and the 3'-half of the NF-kappaB consensus site. Sequence analysis of the IFN-gamma promoter revealed the presence of two additional C3-related elements (C3-1P and C3-3P). In addition, an NF-kappaB site (IFN-gamma kappaB) was identified within the promoter region. Based on this observation, we have analyzed the potential role of NF-kappaB and NFAT family members in regulating IFN-gamma transcription. Electrophoretic mobility shift assay analysis demonstrated that after T cell activation, the p50 and p65 NF-kappaB subunits bind specifically to the newly identified IFN-gamma kappaB and C3-related sites. In addition, we identified the NFAT proteins as a component of the inducible complexes that bind to the C3-3P site. Site-directed mutagenesis and transfection studies demonstrate that calcineurin-inducible transcriptional factors enhance the transcriptional activity of the IFN-gamma promoter through the cyclosporin-sensitive C3-3P site, whereas NF-kappaB proteins functionally interact with the C3-related sites. In addition, when located downstream to the beta-galactosidase gene driven by the IFN-gamma promoter, the intronic C3 site worked in concert with both the IFN-gamma kappaB and the C3-3P site to enhance gene transcription. These results demonstrate that the coordinate activities of NFAT and NF-kappaB proteins are involved in the molecular mechanisms controlling IFN-gamma gene transcription.

Base Sequence↗
PubMed1996

The nuclear factor YY1 suppresses the human gamma interferon promoter through two mechanisms: inhibition of AP1 binding and activation of a silencer element.

Our group has previously reported that the nuclear factor Yin-Yang 1 (YY1), a ubiquitous DNA-binding protein, is able to interact with a silencer element (BE) in the gamma interferon (IFN-gamma) promoter region. In this study, we demonstrated that YY1 can directly inhibit the activity of the IFN-gamma promoter by interacting with multiple sites in the promoter. In cotransfection assays, a YY1 expression vector significantly inhibited IFN-gamma promoter activity. Mutation of the YY1 binding site in the native IFN-gamma promoter was associated with an increase in the IFN-gamma promoter activity. Analysis of the DNA sequences of the IFN-gamma promoter revealed a second functional YY1 binding site (BED) that overlaps with an AP1 binding site. In this element, AP1 enhancer activity was suppressed by YY1. Since the nuclear level of YY1 does not change upon cell activation, our data support a model that the nuclear factor YY1 acts to suppress basal IFN-gamma transcription by interacting with the promoter at multiple DNA binding sites. This repression can occur through two mechanisms: (i) cooperation with an as-yet-unidentified AP2-like repressor protein and (ii) competition for DNA binding with the transactivating factor AP1.

Base Sequence↗
PubMed1994

Induction of multiple cytokine gene expression and IRF-1 mRNA by flavone acetic acid in a murine macrophage cell line.

Flavone-8-acetic (FAA) acid is a potential chemotherapeutic agent that has demonstrated strong immunomodulatory activity in murine model systems. The immunomodulatory activity of this drug in murine systems has been linked to its ability to rapidly induce cytokine gene expression in vivo and in mouse splenocytes ex vivo. We have now developed a tissue culture model for studying the molecular basis of induction of cytokine expression by FAA. Using the mouse macrophage cell line, ANA-1, we can demonstrate the direct induction of interferon beta (IFN beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF alpha), and interferon response factor-1 (IRF-1) mRNA expression following treatment with FAA. Furthermore, the induction of the IFN beta mRNA can occur in the absence of new protein synthesis. Nuclear run-on experiments indicate that at least part of the induction of IFN beta, IL-6, and TNF alpha mRNA occurs at the transcriptional level while the increase in IRF-1 mRNA appears largely post-transcriptional or due to the production of IFN beta protein. Additionally, experiments using agents that interfere with second messengers demonstrate that activation of the protein kinase C pathway is possibly involved in FAA gene induction. The use of this tissue culture model system should lead to a more complete understanding of the mechanisms involved in FAA-induced gene expression and help determine why this drug is inactive on human cells.

Adjuvants, Immunologic↗
PubMed1988

Characterization of interleukin 2 and phorbol myristate acetate augmentation of expression of transfected human interferon-gamma genomic DNA.

We have reported that human interferon-gamma (IFN-gamma) genomic DNA is expressed after transfection into a mouse T-lymphoblastoid cell line and expression can be enhanced by both interleukin 2 (IL2) and phorbol myristate acetate (PMA). It can now be shown that PMA rapidly induces new transcription of the IFN-gamma gene and that increased human (Hu) IFN-gamma can be detected by 2 h after the addition of PMA to the mouse cells. Enhancement of IFN-gamma production by IL2 takes place with similar kinetics with increased IFN-gamma production observed at 4-6 h after IL2 addition, although the maximum amount of IFN-gamma produced in response to IL2 was significantly lower than that produced in response to PMA. Furthermore, along with increased levels of cytoplasmic IFN-gamma RNA, we were able to demonstrate increased nuclear transcription at 4 h after IL2 treatment. Stimulation of IFN-gamma mRNA by both PMA and IL2 could occur in the presence of cycloheximide, indicating that protein synthesis was not required for the initial stimulation to occur. However, the functional half-life of IFN-gamma mRNA after actinomycin D treatment was higher in cells that had been treated with PMA when compared with untreated or IL2-treated cells. This data indicates that there are quantitative differences in the ability of PMA or IL2 to augment IFN-gamma production, and that PMA may increase IFN-gamma production in the transfected cell by additional mechanisms, such as increasing mRNA stability.

Cell Division↗