The effect of the C. elegans transcription factor CES-2 on mammalian apoptosis

The basic region leucine zipper (bZIP) transcription factor CES-2 (cell death specification) controls the apoptotic fate of a subset of neurones in the pharynx of the nematode C.elegans. Conserved components of the cell death pathway have previously been shown to function across species. The aim of the work described in this thesis was to use ces-2 to identify critical genes involved in apoptotic control in mammalian cells. Preliminary experiments were performed to ascertain whether mammalian cells contained endogenous CES-2 like proteins, and whether the C.elegans protein, when expressed in mammalian cells, is correctly localised. The former was confirmed in HeLa nuclear extracts utilising the specific DNA binding sequence of CES-2 in an electrophoretic mobility shift assay; and the latter by immunofluorescence studies established that expression of CES-2 in several mammalian cell types was confined to the nucleus, consistent with its role as a nuclear transcription factor. The bZIP family of proteins function as specific homo- and hetero-dimers. The strategy used, therefore, to identify CES-2-like or CES-2-interacting proteins was by two-hybrid screening of a HeLa cDNA library. Two mammalian bZIP transcription factors were identified which interacted specifically with CES-2, CHOP (C/EBP homologous protein) and ATF4/CREB-2 (activating transcription factor 4 / cAMP response element binding protein-2). The bZIP domain of CES-2 was sufficient for these interactions and this was confirmed by co-immunoprecipitation studies. Since CHOP is upregulated in response to ER-stress and apoptosis induced by the N-linked glycosylation inhibitor tunicamycin, the effect of expressing CES-2 on the response of HeLa cells to tunicamycin was investigated. In contrast to its pro-apoptotic role in the C.elegans neurones, overexpression of CES-2 in HeLa cells, in both transient and stable transfectants, increased the resistance of the cells to apoptosis induced by tunicamycin. Experiments using thapsigargin, calcium ionophore and taxol suggested that CES-2 protection depended on the upregulation of CHOP. Results obtained from expressing the CES-2 bZIP domain alone point to protection being by a dominant negative mechanism to down-regulate CHOP activity. Analysis of the second CES-2 interacting protein showed that ATF4 is also upregulated by agents that induce ER-stress, with a time course similar to CHOP. In the light of recent published data that ATF4 activates CHOP by binding its promoter element, CES-2 binding to ATF4 may also affect the regulation of ATF4-dependent chop transcription. Thus CES-2 is able to bind proteins positioned at different levels of the ER-stress pathway to influence apoptosis. The results presented in this thesis demonstrate that CES-2 can function in mammalian cells and suggest that CES-2-like proteins exist as conserved elements of the apoptotic program