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Neural crest specification and migration independently require NSD3-related lysine methyltransferase activity.

Jacques-Fricke BT, Gammill LS - Mol. Biol. Cell (2014)

Bottom Line: Here we show that the lysine methyltransferase NSD3 is abundantly and specifically expressed in premigratory and migratory neural crest cells.Nevertheless, only Sox10 histone H3 lysine 36 dimethylation requires NSD3, revealing unexpected complexity in NSD3-dependent neural crest gene regulation.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455.

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NSD3 is required for Sox10 and Snail2 expression in premigratory neural crest cells. (A–N) After unilateral electroporation at gastrula stages 4–5 with 3 μg/μl GFP bicistronic expression plasmid pMES (A, H), 3 μg/μl pMES driving NSD3Δ1707 (B, I), 0.75 mM 5– base pair mismatch MO (NSD3 mmMO1; C, J), 0.75 mM NSD3 MO1 (D, K), 0.92 mM NSD3 MO2 (E,L), 1.0 mM NSD3 translation blocking MO (NSD3 trans MO; F, M), or 1.0 mM FITC control MO (FITC con MO; G, N), embryos were incubated to four to seven somites, and Sox10 (A G) or Snail2 (H N) was visualized by in situ hybridization. (A′–N′) GFP- or FITC-tagged MO targeting. Dorsal view, anterior up. Corresponding cross sections are shown beneath dorsal views of embryos. White arrowhead, targeted side; black arrowhead, untargeted side; s, somite. (O) Embryos were categorized by the severity of decrease in Sox10 and Snail2 expression, comparing the targeted side of the embryo to the untargeted control side.
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Figure 3: NSD3 is required for Sox10 and Snail2 expression in premigratory neural crest cells. (A–N) After unilateral electroporation at gastrula stages 4–5 with 3 μg/μl GFP bicistronic expression plasmid pMES (A, H), 3 μg/μl pMES driving NSD3Δ1707 (B, I), 0.75 mM 5– base pair mismatch MO (NSD3 mmMO1; C, J), 0.75 mM NSD3 MO1 (D, K), 0.92 mM NSD3 MO2 (E,L), 1.0 mM NSD3 translation blocking MO (NSD3 trans MO; F, M), or 1.0 mM FITC control MO (FITC con MO; G, N), embryos were incubated to four to seven somites, and Sox10 (A G) or Snail2 (H N) was visualized by in situ hybridization. (A′–N′) GFP- or FITC-tagged MO targeting. Dorsal view, anterior up. Corresponding cross sections are shown beneath dorsal views of embryos. White arrowhead, targeted side; black arrowhead, untargeted side; s, somite. (O) Embryos were categorized by the severity of decrease in Sox10 and Snail2 expression, comparing the targeted side of the embryo to the untargeted control side.

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.


Neural crest specification and migration independently require NSD3-related lysine methyltransferase activity.

Jacques-Fricke BT, Gammill LS - Mol. Biol. Cell (2014)

NSD3 is required for Sox10 and Snail2 expression in premigratory neural crest cells. (A–N) After unilateral electroporation at gastrula stages 4–5 with 3 μg/μl GFP bicistronic expression plasmid pMES (A, H), 3 μg/μl pMES driving NSD3Δ1707 (B, I), 0.75 mM 5– base pair mismatch MO (NSD3 mmMO1; C, J), 0.75 mM NSD3 MO1 (D, K), 0.92 mM NSD3 MO2 (E,L), 1.0 mM NSD3 translation blocking MO (NSD3 trans MO; F, M), or 1.0 mM FITC control MO (FITC con MO; G, N), embryos were incubated to four to seven somites, and Sox10 (A G) or Snail2 (H N) was visualized by in situ hybridization. (A′–N′) GFP- or FITC-tagged MO targeting. Dorsal view, anterior up. Corresponding cross sections are shown beneath dorsal views of embryos. White arrowhead, targeted side; black arrowhead, untargeted side; s, somite. (O) Embryos were categorized by the severity of decrease in Sox10 and Snail2 expression, comparing the targeted side of the embryo to the untargeted control side.
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Related In: Results  -  Collection

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Figure 3: NSD3 is required for Sox10 and Snail2 expression in premigratory neural crest cells. (A–N) After unilateral electroporation at gastrula stages 4–5 with 3 μg/μl GFP bicistronic expression plasmid pMES (A, H), 3 μg/μl pMES driving NSD3Δ1707 (B, I), 0.75 mM 5– base pair mismatch MO (NSD3 mmMO1; C, J), 0.75 mM NSD3 MO1 (D, K), 0.92 mM NSD3 MO2 (E,L), 1.0 mM NSD3 translation blocking MO (NSD3 trans MO; F, M), or 1.0 mM FITC control MO (FITC con MO; G, N), embryos were incubated to four to seven somites, and Sox10 (A G) or Snail2 (H N) was visualized by in situ hybridization. (A′–N′) GFP- or FITC-tagged MO targeting. Dorsal view, anterior up. Corresponding cross sections are shown beneath dorsal views of embryos. White arrowhead, targeted side; black arrowhead, untargeted side; s, somite. (O) Embryos were categorized by the severity of decrease in Sox10 and Snail2 expression, comparing the targeted side of the embryo to the untargeted control side.
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.

Bottom Line: Here we show that the lysine methyltransferase NSD3 is abundantly and specifically expressed in premigratory and migratory neural crest cells.Nevertheless, only Sox10 histone H3 lysine 36 dimethylation requires NSD3, revealing unexpected complexity in NSD3-dependent neural crest gene regulation.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455.

Show MeSH
Related in: MedlinePlus