Investigating the effects of CIITA,-TAF7 interaction on programmed cell death

It has been proposed that TAF7 could be a eukaryotic analogue of HIV Tat. HIV-Tat functions can be summarized as: 1) transcriptional transactivation of the provirus 2) transcriptional inhibition of host genes and 3) induction of apoptosis in host cells. TAF7 has been shown to both support transcriptional elongation and to inhibit transcription by mechanisms very similar to HIV Tat; and we have also previously reported that TAF7 over-expression induces cell death. An important feature of the Tat protein is its interaction with CIITA, the transcriptional activator for MHC class II genes. While Tat can suppress MHC class II expression through its interaction with CIITA; CIITA over-expression can suppress Tat and thereby inhibit HIV transcription and host cell death. Given the functional similarities between HIV-Tat and TAF7 we aimed to investigate the effects of CIITA-TAF7 interactions on programmed cell death. Another important feature of the Tat protein is its ability to bind tubulin proteins during apoptosis. We also aimed to investigate whether TAF7 also binds to tubulin proteins.TAF7 gene was cloned in to expression vectors which were transfected in to HeLa and Kelly cells. In addition to transient transfection, a tetracycline regulated expression system (TREX) was used for TAF7 over-expression. TAF7 was cloned to TREX system?s plasmid and was stable transfected into a Tet-repressor expressing Hela cell line.TAF7 ? Beta Tubulin interaction was shown by immune precipitation experiments performed using both pure isolated proteins and cell lysates. Functional outcome of the interaction was analyzed by invitro tubulin polymerization assays. We?ve shown that TAF7 over-expression induced cell death and CIITA expression suppressed TAF7's apoptotic effect . Our data supports a model in which TAF7 causes stabilization of tubulin polymerization. I the presence of an apoptotic signal, TAF7 remains bound to tubulin proteins and the cell cycle halts. In the absence of an apoptotic signal, the chromosomes attach to the mitotic spindle properly and cell cycle continues with the termination of mitosis. We also showed that the phosphorylation mutant of TAF7 did not induce apoprosis as its wild type form. In addition by using CIITA mutants we were able to show that the inhibition of apoptosis is specific to the presence of a functional CIITA molecule When taking the role TAF7 plays in apoptosis under consideration, its interactions with CIITA during this process has very important implication fort he field of immunology. Futhermore when one considers CIITA and Tat interactions during HIV infection; the importance to elucidating molecular interactions between CIITA and TAF7 is evident.