Abstract
Wdr68 is a highly conserved scaffolding protein required for craniofacial development and left-right asymmetry. A Ras-Map3k-Wdr68-Dyrk1 signaling relay may mediate these and other diverse signaling events important in development and disease. While the sub-cellular localization of Wdr68 has been shown to be dependent on that of its interaction partners, it is not clear where Wdr68 activity is required during development. Here we show that while a GFP-Wdr68 fusion functionally substituted for craniofacial development in the zebrafish, that a Nuclear Export Signal (NES) fusion protein (GFPNESWdr68) failed to support craniofacial development. As control for NES activity, we show that while GFP-Wdr68 exhibited a pan-cellular distribution in C2C12 cells, the GFPNESWdr68 fusion predominantly localized to the cell cytoplasm, as expected. Interestingly, while GFP-Wdr68 and RFP-Dyrk1a co-localized to the cell nucleus as expected based on the known sub-cellular localization for Dyrk1a, we found that the GFPNESWdr68 fusion redistributed RFP-Dyrk1a to the cell cytoplasm potentially disconnecting the Ras/Dyrk1 signal relay from further downstream targets. Consistent with a nuclear role in gene regulation, we also found that while a transcriptional activation domain fusion, CebpFlagWdr68, functionally substituted for endogenous Wdr68 for craniofacial development, that a transcriptional repression domain fusion, MadFlagWdr68, failed to support craniofacial development. Dyrk1b is required for myogenin (myog) expression in differentiating mouse C2C12 cells and here we report that wdr68 is also important for myog expression in differentiating C2C12 cells. Using a C2C12 cell myog promoter-reporter system, we found that Wdr68 overexpression increased reporter activity while moderate expression levels of MadFlagWdr68 interfered with reporter activity. Taken together, these findings support a nuclear role for Wdr68-containing complexes.
Highlights
IntroductionWdr68/Dcaf (hereafter referred to as Wdr68) is a WD40 repeat domain-containing protein that is highly conserved from animals to plants and is involved in diverse cellular processes
Wdr68/Dcaf7 is a WD40 repeat domain-containing protein that is highly conserved from animals to plants and is involved in diverse cellular processes
18% of animals co-injected with wdr68-MO and negative control GFP mRNA displayed normal M and PQ cartilages indicating a failure of RNA rescue (Figure 1C, D)
Summary
Wdr68/Dcaf (hereafter referred to as Wdr68) is a WD40 repeat domain-containing protein that is highly conserved from animals to plants and is involved in diverse cellular processes. The Petunia ortholog AN11 is important for regulation of anthocyanin biosynthesis [4]. The Arabidopsis ortholog LWD1, which is 58% identical to Wdr, is important for expression of clock genes and localizes to their promoters in vivo [5]. While parallels between such diverse processes are unclear, the ability of the drosophila ortholog CG14614 to substitute for wdr in zebrafish craniofacial development suggests the molecular function of wdr is highly conserved [1]
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