WNT-responsive SUMOylation of ZIC5 promotes murine neural crest cell development, having multiple effects on transcription

Abstract

Zinc finger of the cerebellum (Zic) proteins act as classical transcription factors to promote transcription of the Foxd3 gene during neural crest cell specification. Additionally, they can act as co-factors that bind TCF molecules to repress WNT/β-catenin-dependent transcription without contacting DNA. Here, we show ZIC activity at the neural plate border is influenced by WNT-dependent SUMOylation. In a high WNT environment, a lysine within the highly conserved ZF-NC domain of ZIC5 is SUMOylated, which decreases formation of the TCF/ZIC co-repressor complex and shifts the balance towards transcription factor function. The modification is critical in vivo, as a ZIC5 SUMO-incompetent mouse strain exhibits neural crest specification defects. This work reveals the function of the ZIC ZF-NC domain, provides in vivo validation of target protein SUMOylation, and demonstrates that WNT/β-catenin signaling directs transcription at non-TCF DNA binding sites. Furthermore, it can explain how WNT signals convert a broad domain of Zic ectodermal expression into a restricted domain of neural crest cell specification.

SUMOylation Potentiates ZIC Protein Activity to Influence Murine Neural Crest Cell Specification

“…During murine NCC formation, ZIC5 regulation of Foxd3 expression requires post-translational modification of the ZIC5 protein by SUMOylation at a lysine within the highly conserved ZF-NC domain [ 25 ]. The paralogous ZIC3 lysine is also a SUMO substrate [ 49 ], and a high-throughput proteomic study [ 50 ] and search for high-probability consensus SUMOylation motifs within the ZIC proteins [ 25 , 51 ] suggest all human ZIC proteins can be SUMOylated. To determine if this is the case, the various ZICs were assessed using the UBC9 fusion-directed SUMOylation (UFDS) system.…”

“…SUMOylation generally modifies a lysine within a motif of conserved residues (ΨKXE), enabling the identification of high-probability SUMO sites via sequence analysis. In addition to the highly conserved SUMOylation site in the ZF-NC domain within ZIC1-4, our previous analysis showed that human ZIC1 and ZIC3 also contain a second high-probability canonical SUMOylation site at the boundary of zinc fingers 3 and 4 [ 25 ] ( Figure 1 a). To determine which of the predicted SUMOylation sites within ZIC1-4 are bona fide sites of SUMO attachment in HEK293T cells, each putative, modified lysine (K) was converted to an arginine (R) (which cannot be modified be SUMOylation) in the V5 epitope-tagged mammalian expression construct.…”

“…We have recently shown that another protein associated with neural crest, ZIC5, is also regulated by SUMOylation [ 25 ]. ZIC5 is a member of the ZIC ( zi nc finger of the c erebellum) gene family that encodes multifunctional transcriptional regulators.…”

“…Other members of the murine ZIC family ( Zic2 , Zic3, and Zic5 ) are expressed in the neurectoderm during gastrulation and neurulation, including in the neural plate border region that is the site of neural crest specification [ 30 , 32 , 33 ]. Loss-of-function mutations in either Zic2 or Zic5 result in the depletion of NCCs and lead to neural crest-related phenotypes including ventral spotting and reduced cephalic neural crest tissues [ 25 , 34 , 35 , 36 , 37 , 38 ], suggesting the other Zic genes have acquired the role of neural plate border genes from Zic1 in mammalian species.…”

“…Outside the zinc finger domain, the ZICs are more varied, but all vertebrate ZICs share the zinc finger N-terminally conserved (ZF-NC) domain, a small domain (14–21 amino acids) that is located immediately adjacent to the zinc finger domain [ 48 ]. The function of the ZF-NC is unknown; however, we recently showed it contains a highly conserved SUMOylation consensus sequence that is targeted for SUMOylation in response to canonical WNT signaling in ZIC5 [ 25 ]. Here, we further investigated the connection between WNT signaling, ZIC function, and SUMOylation during mammalian NCC induction and specification.…”