Hughes, R.; (2010) An investigation into the mechanisms by which bim gene expression is regulated in sympathetic neurons. Doctoral thesis , UCL (University College London).

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Abstract

Neuronal apoptosis plays a critical role in development and disease. Developing sympathetic neurons require nerve growth factor (NGF) for their survival and die by apoptosis in its absence. Studies with sympathetic neurons have provided important insights into the molecular mechanisms of neuronal apoptosis and the signalling pathways that regulate the cell death programme in neurons. Bim is a BH3-only member of the Bcl-2 family that increases in level after NGF withdrawal and which is required for NGF withdrawal-induced death. Regulation of bim expression is complex and remains incompletely understood. By analysing the DNA sequence of the rat bim promoter I identified a conserved inverted CCAAT box (ICB) that is bound by the heterotrimeric transcription factor NF-Y in vitro and in chromatin. Interestingly, mutational analysis revealed that the ICB is critical for the induction of a bim-LUC reporter construct following NGF withdrawal. Use of a well-characterised dominant negative NF-YA mutant (YA13 m29) showed that NF-Y is required for bim promoter activity and its induction following NGF withdrawal. Overexpression of YA13 m29 also demonstrated that NF-Y activity is essential for the expression of the endogenous Bim protein and that NF-Y is important for apoptosis following NGF withdrawal. Furthermore, I found that the transcriptional coactivators CBP/p300 are required for the activation of bim-LUC following NGF withdrawal and that CBP/p300 may interact with NF-Y to enhance bim transcription. In addition to this, the prosurvival MEK/ERK pathway has been found to inhibit bim expression independently of the PI3-K/Akt pathway. 3' RACE and experiments in sympathetic neurons with a new bim-LUC+3'UTR reporter construct revealed that this negative regulation is mediated through the bim 3' UTR. Mutational analysis and RNA stability experiments have been employed to further investigate this mechanism.

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