Tumor necrosis factor (TNF) is a key mediator of inflammatory and immune responses produced by many cells including T and B lymphocytes, macrophages and dendritic cells. It is a critical cytokine for eradication of intracellular pathogens such as Mycobacterium tuberculosis (MTb), but uncontrolled can lead to severe disorders such as septic shock, cerebral malaria and autoimmunity. This is illustrated by the efficacy of anti-TNF antibody treatment of rheumatoid arthritis, inflammatory bowel disease and other inflammatory pathologies. TNF is a tightly regulated gene at the level of transcription. Our research has shown that a distinct enhanceosome is recruited to the TNF promoter when macrophage cell lines are stimulated with lipopolysaccharide (LPS) or MTb, distinct from the enhanceosomes recruited to the promoter upon stimulation of T cell lines with virus or calcium. Furthermore, genetic approaches and DNAse I hypersensitivity (DH) analysis in cell lines have identified regulatory elements involved in inducer and cell type specific regulation of the gene outside of the promoter in other non-coding sequences. Based on these findings, our hypothesis is that through a dynamic process of enhanceosome recruitment to the TNF promoter, which contains shared binding sites for distinct activators, together with remodeling of chromatin, the TNF gene achieves both the flexibility and specificity required for its regulation. We will test this hypothesis in primary murine T cells and macrophages in Aims 1 and 2 through the characterization of the chromatin environment of the TNF locus in these cells under different conditions, and by determining the transcription factors and signaling molecules involved in TNF gene expression.
In Aim 3, we will examine the chromatin environment and transcription factors involved in the modulation of TNF gene expression by interferon-gamma (IFN-gamma) and interleukin-10 (IL-10). The checkpoints of TNF gene expression will be elucidated using a combination of approaches including: (i) chromatin remodeling by a combination of DH analysis and bioinformatic approaches; (ii) the use of specific inhibitors, RNAi knockdown or mice deficient for candidate transcription factor/signaling molecules; and/or retroviral vectors expressing candidate transcription factors or signalling molecules; (iii) the recruitment of specific TNF enhancer complexes to regulatory elements or the TNF promoter by ChIP analysis of primary cells under different conditions. These experiments will allow us to link the recruitment of specific TNF enhancer complexes with distinct adaptor proteins and subsequent signal transduction pathways. The overarching goals of this proposal are: 1) the identification of specific transcriptional targets allowing for cell and inducer directed therapeutic manipulation of TNF in inflammatory and infectious diseases, and 2) the further elucidation of the mechanisms of control and specificity of eukaryotic gene transcription.
