Neural crest specification and migration independently require NSD3-related lysine methyltransferase activity.
Bottom Line: Nevertheless, only Sox10 histone H3 lysine 36 dimethylation requires NSD3, revealing unexpected complexity in NSD3-dependent neural crest gene regulation.In addition, by temporally limiting expression of a dominant negative to migratory stages, we identify a novel, direct requirement for NSD3-related methyltransferase activity in neural crest migration.These results identify NSD3 as the first protein methyltransferase essential for neural crest gene expression during specification and show that NSD3-related methyltransferase activity independently regulates migration.
Affiliation: Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455.Show MeSH
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Mentions: The genomic loci of neural crest transcription factors Sox10 and Snail2 exhibit H3K36 trimethylation in neural crest cells (Strobl-Mazzulla et al., 2010), consistent with H3K36me3 being a mark of active chromatin that represses spurious initiation of actively transcribed genes (reviewed in Wagner and Carpenter, 2012). To investigate the necessity of this methylation for Sox10 and Snail2 expression, we used NSD3Δ1707 to disrupt H3K36me2 and me3 during neural crest specification. We electroporated HH stage 4–6 embryos with NSD3Δ1707 or pMES, incubated to four to seven somites, and analyzed Sox10 and Snail2 expression by in situ hybridization. NSD3Δ1707 significantly reduced Sox10 and Snail2 expression in >90% of embryos (Sox10: Figure 3, B and O, 92.3% with reduced expression, p = 0.038; Snail2: Figure 3, I and O, 92.3% with reduced expression, p = 0.0042; see Supplemental Figure S3, A–C, for representative embryos in each scoring class), compared with a minority of embryos exhibiting mild defects after pMES electroporation (Sox10: Figure 3, A and O, 46.2% with reduced expression; Snail2: Figure 3, H and O, 36.4% with reduced expression). This outcome was not due to indirect effects on cell death or proliferation, as the frequency of cleaved caspase 3 and phospho-histone H3–immunopositive cells was not significantly changed in the targeted versus untargeted dorsal neural tube or in GFP-positive cells of embryos electroporated with NSD3Δ1707 or pMES (Supplemental Figure S4). The ability of SET domain–deleted NSD3 to interfere with neural crest specification suggests that the methyltransferase activity of NSD3 and its relatives is essential in the neural crest.
Affiliation: Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455.