Wnt responsive SUMOylation regulates ZIC protein activity to promote murine neural crest specification

Abstract

Neural crest cells are a transient population of multipotent cells which arise at the border of the neural and non-neural ectoderm (known as the neural plate border) during neurulation. These cells migrate through the embryo and differentiate into a range of cell types so diverse that the neural crest is sometimes termed the fourth germ layer. Neural crest cell formation and differentiation is driven by a cascade of transcription factor activity at the neural plate border in response to the combinatorial action of several major signal transduction pathways, including canonical Wnt signalling. Canonical Wnt signalling culminates in the conversion of TCF transcription factors from repressors to activators and TCF consensus DNA binding sites are known as Wnt responsive elements (WREs). The zinc finger of the cerebellum (ZIC) proteins are a family of transcriptional regulators that promote neural crest cell specification in a variety of vertebrate organisms. The ZIC proteins, however, are known to bind TCF proteins and repress Wnt mediated transcription at WREs, implying a role in preventing neural crest cell differentiation. The work presented in this thesis resolves the apparent paradox of ZIC behaviour. Cell-based reporter assays demonstrate a bi-phasic response of ZIC proteins to Wnt signalling. In a low Wnt environment ZIC proteins repress transcription at WREs but this repression is lost in a high Wnt environment. Wnt signalling promotes post-translational modification of ZIC proteins, via SUMOylation at the highly conserved but hitherto functionally uncharacterised Zinc Finger N-terminally Conserved (ZF-NC) domain. SUMOylated ZIC proteins exhibit decreased TCF interaction and decreased inhibition at WREs. Simultaneously, SUMOylated ZIC proteins exhibit increased transactivation activity of an enhancer element known to drive expression of the neural crest specifier gene Foxd3 in the specified trunk neural crest cells. These in vitro findings have a direct in vivo correlate as a mouse strain in which ZIC5 protein is refractory to SUMOylation exhibits decreased Foxd3 expression in trunk neural crest and decreased numbers of differentiated neural crest cells. Canonical Wnt signalling controls the activation of the ZIC proteins and impacts gene expression not only at classical WREs but also at ZIC responsive elements. Neural crest cell specification can therefore be restricted to the neural plate border despite a broad neurectodermal domain of ZIC expression. High concentration Wnt signals at the neural plate border will simultaneously convert ZIC and TCF proteins into transactivators, whereas in the future lateral neurectoderm repression at WREs will persist.