Transcriptional regulation of the apoptosis regulator Bfl-1 by Rel/NF-kappaB transcription factors

Bfl-1/A1 is a pro-survival member of the Bcl-2 family of cell death regulators. We have previously demonstrated that Bfl-1 is a direct transcriptional target of NF-κB that can suppress apoptosis under conditions where endogenous NF-κB, activity is inhibited. This dissertation will characterize in detail how bfl-1 gene expression is regulated. Sequence analysis of the 5′ regulatory region of the human bfl-1 gene revealed binding sites for transcription factors NF-κB, AP-1, and C/EBPβ, clustered within a 160 base pair region. Gel-shift analysis confirmed the legitimacy of these motifs. DNA-binding studies demonstrated that the κB site was necessary for the concerted binding of NF-κB, AP-1, and C/EBPβ, to the 160bp region and that c-Rel nucleated an enhanceosome-like complex containing these proteins. Functionally, c-Rel, c-Jun, and C/EBPβ, synergistically activated the bfl-1 promoter with one another as well as with the coactivators p300 and TAF_II105. Studies of endogenous activation of the bfl-1 promoter revealed a mutual dependence on all three transcription factors, supporting the enhanceosome model. Gel-shift and competition assays revealed that the stability of this large complex is dependent on the orientation and phasing of the NF-κB, site with the Bfl-1 regulatory region. This is consistent with the orientation and positional dependence of enhanceosome complexes. Chromatin immunoprecipitation assays demonstrated the in vivo recruitment of endogenous NF-κB, AP-1, C/EBPβ, and HMGI-C factors to the bfl-1 regulatory region upon T cell activation. Also recruited were the coactivator p300 along with histone H3 acetylation. To our knowledge, this is the first demonstration of enhanceosome-mediated regulation of a cell death inhibitor. The involvement of NF-κB, C/EBPβ, and AP-1 in the expression of genes important for immune and inflammatory responses suggests that Bfl-1 may be critical for carrying out the protective role of Rel/NF-κB in the immune system and points to the need for a complex and precise regulatory network to control its expression.