The long-term objective of our work is to develop new approaches to control the harmful effects of inflammatory processes (such as those observed in periodontitis), while maintaining the beneficial effects (e.g. tissue repair, resolution of infections). Accurate discrimination between normal and dysregulated inflammatory processes requires a deeper understanding of the regulatory mechanisms affecting gene transcription of pro-inflammatory cytokines such as TNF. The studies we propose here build upon our recent results, in which we identified, cloned, and partially characterized the novel transcription factor LITAF. We have defined a role for LITAF as a positive regulator of TNF expression, and have found that binding of the LITAF regulatory element to the TNF promoter occurs within a region from nucleotides -550 to -487.
The Specific Aims of this proposal are designed to test the twin hypotheses that (1) LITAF regulates TNF gene expression in vivo and (2) that LITAF plays an important role in inflammatory disease. The proposed identification of the minimal and specific DNA sequence responsible for protein binding to LITAF Aim (1a) will allow identification of other promoters that may be targeted by this protein, and permit evaluation of the role of LITAF in the regulation of their respective genes Aim (1d): To accomplish this, we will engineer selected mutations within the human and murine TNF promoters, to reveal the exact DNA binding sequence controlled by LITAF Aim (1b). Coupling of the mutant promoters to reporter genes will allow us to assess whether these mutant promoters fail to be activated by LITAF Aim (1c). Mutant LITAF proteins (muteins) will be designed to identify both the DNA- binding and trans-activation domains (Aim 2), and this information will serve as a powerful tool to elucidate LITAF- promoter interactions in other genes. The creation of LITAF knockout mice will build upon the results of Aims 1 and 2 to evaluate the hypothesis that the LITAF gene product, through its capacity to regulate TNF activity, plays an important role in the development of inflammatory diseases, including periodontitis (Aims 3a and 3b).
Aim 3 c will build upon our recent finding linking LITAF to p53, and will investigate how LITAF could be induced through TLR/TNFR and p53-mediated pathways to help mediate apoptosis. Our discovery of the novel protein LITAF, a factor that helps regulates TNF transcription and possibly apoptosis, will serve as a new tool to dissect the complex mechanisms which mediate TNF expression in various inflammatory conditions, including periodontitis.
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