Mapping Domains for ZIC3 Molecular Function

Abstract

The zinc finger of the cerebellum (Zic) genes encode a family of transcriptional regulators critical for early embryogenesis. All ZIC proteins contain a zinc finger domain (ZFD) and other evolutionary conserved regions. They are pleiotropic in nature since they can influence gene expression directly by acting as transcription factors due to their ability to bind target DNA sequences, or indirectly as co-factors by interacting with protein partners. Little is known, however, about the structural components that allow ZIC proteins to perform these functions. Among ZIC family members the protein structure of ZIC3 is relatively well characterised, yet details regarding its transactivation domain remain unknown. During embryonic development ZIC3 is involved in maintaining pluripotency of embryonic stem cells, formation of the left-right (L-R) axis and arrangement of visceral organs. Mutations in Zic3 in humans and animal models cause congenital L-R axis defects. The work presented in this thesis maps structural domains required for ZIC3 molecular function. Characterisation of a novel allele of murine Zic3 revealed that removal of the ZFD and C-terminus renders the mutant protein functionally null and incapable of dominantly interfering with the function of other ZIC proteins. To further assess the transcription factor function of ZIC3, a new cell-based transactivation assay system using target ZIC3-DNA binding sequences was designed. This assay was used to identify regions within the ZFD and C-terminus vital for transactivation via ZIC3. In addition other evolutionary conserved domains were implicated in transactivation. This study provides a reliable and robust platform to investigate the transcription factor function of ZIC proteins and their variants. Show more