Molecular analysis of the ZIC3 and TCF7L1 interaction and its implication in human Heterotaxy

Abstract

Heterotaxy is a congenital disorder that occurs due to the defective establishment of the left-right (L-R) body axis during early embryo development. The disorder affects 1/10,000 live births, results in the abnormal alignment of internal organs with respect to the L-R axis, and is often accompanied by severe cardiac malformations. The most commonly mutated gene in human cases of Heterotaxy is the X-linked ZIC3, which was first associated with the condition in 1997. ZIC3 belongs to a family of multifunctional transcriptional regulator proteins (ZIC family) all characterised by a C2H2 type zinc finger domain (ZFD). This domain is implicated in the transactivation (DNA binding) and co-factor (protein binding) function of the proteins. However, despite both capabilities, it is still not known when during development the co-factor role of ZIC3 is necessary over the transcription factor role. Consequently, the exact cellular and molecular causes underlying ZIC3-associated Heterotaxy remain unknown.

Mouse models of Zic3 loss-of-function reveal phenotypes that resemble human Heterotaxy and exhibit a defective node (the structure that confers the L-R axis during development). More recently, studies have shown murine Zic3 alleles lead to incompletely penetrant, partial (posterior) axis duplications and anterior truncation, phenotypes characteristic of elevated canonical Wnt signalling. The expression pattern of Zic3 during mammalian gastrulation overlaps with a known repressor of the canonical Wnt pathway, Tcf7l1 and it has also been shown that a related ZIC protein (ZIC2) can interact with TCF proteins to co-repress Wnt/b-catenin mediated transcription when overexpressed in cell lines. Overall, this leads to the novel hypothesis that ZIC3 controls the establishment of the L-R axis by interacting with TCF7L1 to repress Wnt signalling during mammalian gastrulation. In this thesis I examine the molecular role of ZIC3 in the Wnt pathway and investigate the functional consequences of ZIC3 Heterotaxy-associated protein variants.

A physical interaction between ZIC3 and TCF7L1 was demonstrated by several molecular assays. Additionally, 17 Heterotaxy-associated human ZIC3 variants were functionally assessed in well-established cell-based assays. Four of the human protein variants exhibit non-pathogenic properties as they retain wildtype ZIC3 function in these assays. The remaining variants, which affect key residues within the ZFD or contain a premature termination codon, display impaired transcription factor and co-factor functions: they can no longer activate transcription at a ZIC responsive element and have reduced ability to inhibit b-catenin-mediated Wnt transcription. Loss of Wnt inhibition did not always correlate with loss of TCF7L1 interaction, suggesting ZIC3 and/or TCF7L1 must interact with additional co-repressors or repressive chromatin complexes to mediate transcriptional repression in the Wnt pathway. Additionally, in this thesis I identified several evolutionary conserved domains implicated in transactivation, including the newly annotated SANC and SACC domains, enhancing our understanding of the structural domains required for ZIC3 molecular function. Overall, this thesis supports a new model of ZIC3 molecular function: ZIC3 interacts with TCF7L1 to inhibit canonical Wnt signalling during mammalian gastrulation and correctly establish the L-R axis. Ultimately, ZIC3-associated Heterotaxy arises when ZIC3 is unable to function as a Wnt co-repressor. Show more