Characterization of the heterodimeric association between Zhangfei and activating transcription factor-4

Zhangfei (ZF), a human cellular protein, was originally identified through its interaction with Host Cell Factor-1 (HCF-1). ZF is a member of the CREB/ATF bZIP family of transcription factors. Unique from most CREB/ATF bZIP factors, ZF lacks a critical asparagine residue in its basic domain, which is believed to hinder ZF from homodimerically binding DNA. The cellular role of ZF is unclear, thus identification of associating proteins and downstream transcriptional targets is essential. In this thesis, both in vitro and in vivo data was presented to support that a bona fide interaction exists between ZF and the cellular factor activating transcription factor-4 (ATF4). Mutational analysis showed that the bZIP region of ZF was required by ZF to interact with the C-terminal bZIP domain of ATF4. An in vivo immunoprecipitation assay suggested that ZF interacted with both the phosphorylated and unphosphorylated form of ATF4. An in vitro CRE-binding assay provided evidence that ZF may enhance ATF4 binding to the cAMP response element (CRE), localized in the promoters of cellular and viral genes. In addition, in vivo CRE-reporter assays showed that ZF-ATF4 heterodimers were able to transactivate CRE enhancers. This transactivation was found to depend on the MEK/MAPK signaling pathway. Additionally, the ability of ZF to affect ATF4 transactivation of the amino acid response element (AARE) of CHOP, and the unfolded protein response element (UPRE) of EDEM was also explored.