Limits...
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.

Show MeSH

Related in: MedlinePlus

NSD3 dimethylates H3K36 at the Sox10 locus. (A) Primer locations in β-actin, Sox10, Snail2, Sox9, and FoxD3 genes used for quantitative PCR after chromatin immunoprecipitation, named by their distance in kilobases from the transcription start site (arrow). (B) Average percentage input (mean ± SD) recovered in three independent chromatin immunoprecipitation experiments performed with 30 pooled NSD3 mmMO1- or MO1-electroporated neural tubes, assaying H3K36me2 occupancy at 12 genomic loci (Chr1nc [chromosome 1 negative control], Chr2nc [chromosome 2 negative control], β-actin, four regions within Sox10, three regions within Snail2, Sox9, and FoxD3). Input recovered is in the normal range for a methylated histone (Cell Signaling Technology, www.cellsignal.com/support/faq_chip.html#a11) and consistent with levels of H3K36me2 occupancy in other systems (e.g., Blackledge et al., 2010; Asangani et al., 2013; fold enrichment in Figure 5 ranges from 5 to 300). Normal rabbit IgG was an immunoprecipitation control (Ab control). Student's t test was used for statistical analysis; *significant; p < 0.05.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4263458&req=5

Figure 5: NSD3 dimethylates H3K36 at the Sox10 locus. (A) Primer locations in β-actin, Sox10, Snail2, Sox9, and FoxD3 genes used for quantitative PCR after chromatin immunoprecipitation, named by their distance in kilobases from the transcription start site (arrow). (B) Average percentage input (mean ± SD) recovered in three independent chromatin immunoprecipitation experiments performed with 30 pooled NSD3 mmMO1- or MO1-electroporated neural tubes, assaying H3K36me2 occupancy at 12 genomic loci (Chr1nc [chromosome 1 negative control], Chr2nc [chromosome 2 negative control], β-actin, four regions within Sox10, three regions within Snail2, Sox9, and FoxD3). Input recovered is in the normal range for a methylated histone (Cell Signaling Technology, www.cellsignal.com/support/faq_chip.html#a11) and consistent with levels of H3K36me2 occupancy in other systems (e.g., Blackledge et al., 2010; Asangani et al., 2013; fold enrichment in Figure 5 ranges from 5 to 300). Normal rabbit IgG was an immunoprecipitation control (Ab control). Student's t test was used for statistical analysis; *significant; p < 0.05.

Mentions: NSD3 binds target gene promoters and coding regions and is believed to affect transcriptional initiation and elongation because gene silencing in NSD3-deficient cells is associated with loss of gene body H3K36 methylation (Fang et al., 2010; Rahman et al., 2011; Wagner and Carpenter, 2012). Thus NSD3 could affect neural crest gene expression (Figures 3 and 4) by H3K36 dimethylating these genes. To investigate this hypothesis, we bilaterally electroporated embryos with NSD3 MO1 or mmMO1 and dissected cranial neural tubes at four to eight somites, when neural crest gene expression was NSD3 dependent (Figures 3 and 4). Because chromatin immunoprecipitation (ChIP)–rated NSD3 antibodies are not available, we then performed ChIP using a validated H3K36me2 antibody (Egelhofer et al., 2011). Because H3K36me2 is highest immediately upstream and downstream of transcription start sites and also marks intergenic regions and constitutive heterochromatin (Bell et al., 2007; Rechsteiner et al., 2010; Chantalat et al., 2011; Kuo et al., 2011), we evaluated the effects of NSD3 knockdown on H3K36me2 occupancy at several locations. Compared to mmMO1-electroporated tissue, NSD3 knockdown did not affect H3K36me2 occupancy at open reading frame–free, intergenic regions of chromosome 1 (Figure 5B; Chr1nc, control for Sox10, p = 0.32) and chromosome 2 (Chr2nc, control for Snail 2, p = 0.12). Similarly, H3K36me2 at the β-actin gene was NSD3 independent (Figure 5B; p = 0.49), indicating that NSD3 knockdown does not affect gene body H3K36 dimethylation generally. In contrast, NSD3 knockdown reduced H3K36me2 occupancy at the Sox10 gene 0.5 and 1.0 kb from the transcription start site (Figure 5, A and B; p = 0.026 and 0.047, respectively). On the other hand, NSD3 knockdown did not diminish H3K36me2 occupancy in the Sox10 promoter or 3′ end, at any location in Snail2, or in the Sox9 or FoxD3 gene bodies (Figure 5, A and B; Sox10, p = 0.38 at −1.0 kb, p = 0.29 at 8.0 kb; Snail2: p = 0.22 at −1.0 kb, p = 0.47 at 0.5 kb, p = 0.35 at 1.0 kb; Sox9: 0.072 at 1.0 kb; FoxD3: p = 0.47 at 0.5 kb). Therefore NSD3 is required for H3K36 dimethylation of Sox10 but not other neural crest transcription factors, despite being required for the expression of all four genes (Figures 3 and 4). This disconnect indicates that the regulation of neural crest specification by NSD3 is more complex than direct H3K36 dimethylation of neural crest genes.


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

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

NSD3 dimethylates H3K36 at the Sox10 locus. (A) Primer locations in β-actin, Sox10, Snail2, Sox9, and FoxD3 genes used for quantitative PCR after chromatin immunoprecipitation, named by their distance in kilobases from the transcription start site (arrow). (B) Average percentage input (mean ± SD) recovered in three independent chromatin immunoprecipitation experiments performed with 30 pooled NSD3 mmMO1- or MO1-electroporated neural tubes, assaying H3K36me2 occupancy at 12 genomic loci (Chr1nc [chromosome 1 negative control], Chr2nc [chromosome 2 negative control], β-actin, four regions within Sox10, three regions within Snail2, Sox9, and FoxD3). Input recovered is in the normal range for a methylated histone (Cell Signaling Technology, www.cellsignal.com/support/faq_chip.html#a11) and consistent with levels of H3K36me2 occupancy in other systems (e.g., Blackledge et al., 2010; Asangani et al., 2013; fold enrichment in Figure 5 ranges from 5 to 300). Normal rabbit IgG was an immunoprecipitation control (Ab control). Student's t test was used for statistical analysis; *significant; p < 0.05.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4263458&req=5

Figure 5: NSD3 dimethylates H3K36 at the Sox10 locus. (A) Primer locations in β-actin, Sox10, Snail2, Sox9, and FoxD3 genes used for quantitative PCR after chromatin immunoprecipitation, named by their distance in kilobases from the transcription start site (arrow). (B) Average percentage input (mean ± SD) recovered in three independent chromatin immunoprecipitation experiments performed with 30 pooled NSD3 mmMO1- or MO1-electroporated neural tubes, assaying H3K36me2 occupancy at 12 genomic loci (Chr1nc [chromosome 1 negative control], Chr2nc [chromosome 2 negative control], β-actin, four regions within Sox10, three regions within Snail2, Sox9, and FoxD3). Input recovered is in the normal range for a methylated histone (Cell Signaling Technology, www.cellsignal.com/support/faq_chip.html#a11) and consistent with levels of H3K36me2 occupancy in other systems (e.g., Blackledge et al., 2010; Asangani et al., 2013; fold enrichment in Figure 5 ranges from 5 to 300). Normal rabbit IgG was an immunoprecipitation control (Ab control). Student's t test was used for statistical analysis; *significant; p < 0.05.
Mentions: NSD3 binds target gene promoters and coding regions and is believed to affect transcriptional initiation and elongation because gene silencing in NSD3-deficient cells is associated with loss of gene body H3K36 methylation (Fang et al., 2010; Rahman et al., 2011; Wagner and Carpenter, 2012). Thus NSD3 could affect neural crest gene expression (Figures 3 and 4) by H3K36 dimethylating these genes. To investigate this hypothesis, we bilaterally electroporated embryos with NSD3 MO1 or mmMO1 and dissected cranial neural tubes at four to eight somites, when neural crest gene expression was NSD3 dependent (Figures 3 and 4). Because chromatin immunoprecipitation (ChIP)–rated NSD3 antibodies are not available, we then performed ChIP using a validated H3K36me2 antibody (Egelhofer et al., 2011). Because H3K36me2 is highest immediately upstream and downstream of transcription start sites and also marks intergenic regions and constitutive heterochromatin (Bell et al., 2007; Rechsteiner et al., 2010; Chantalat et al., 2011; Kuo et al., 2011), we evaluated the effects of NSD3 knockdown on H3K36me2 occupancy at several locations. Compared to mmMO1-electroporated tissue, NSD3 knockdown did not affect H3K36me2 occupancy at open reading frame–free, intergenic regions of chromosome 1 (Figure 5B; Chr1nc, control for Sox10, p = 0.32) and chromosome 2 (Chr2nc, control for Snail 2, p = 0.12). Similarly, H3K36me2 at the β-actin gene was NSD3 independent (Figure 5B; p = 0.49), indicating that NSD3 knockdown does not affect gene body H3K36 dimethylation generally. In contrast, NSD3 knockdown reduced H3K36me2 occupancy at the Sox10 gene 0.5 and 1.0 kb from the transcription start site (Figure 5, A and B; p = 0.026 and 0.047, respectively). On the other hand, NSD3 knockdown did not diminish H3K36me2 occupancy in the Sox10 promoter or 3′ end, at any location in Snail2, or in the Sox9 or FoxD3 gene bodies (Figure 5, A and B; Sox10, p = 0.38 at −1.0 kb, p = 0.29 at 8.0 kb; Snail2: p = 0.22 at −1.0 kb, p = 0.47 at 0.5 kb, p = 0.35 at 1.0 kb; Sox9: 0.072 at 1.0 kb; FoxD3: p = 0.47 at 0.5 kb). Therefore NSD3 is required for H3K36 dimethylation of Sox10 but not other neural crest transcription factors, despite being required for the expression of all four genes (Figures 3 and 4). This disconnect indicates that the regulation of neural crest specification by NSD3 is more complex than direct H3K36 dimethylation of neural crest genes.

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