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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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NSD3Δ1707 prevents H3K36me2 and me3. (A) Diagram of the protein domain organization of NSD3 (top) and NSD3Δ1707 (bottom). PHD, plant homeodomain fingers; PWWP, Pro-Trp-Trp-Pro domain; SET, suppressor of variegation3-9, Enhancer of Zeste and Trithorax domain. (B–K) Chick fibroblast DF-1 cells were transfected with the GFP (green) bicistronic expression plasmid pMES (B, D, F, H, J) or pMES driving NSD3Δ1707 (C, E, G, I, K). Twenty-four hours after transfection, cells were fixed and stained with anti-methyl (CH3; red) antibodies against dimethylated (me2) lysines H3K9me2 (B, C), H3K27me2 (D, E), H3K4me2 (F, G), or H3K36me2 (H, I) or trimethylated (me3) lysine H3K36me3 (J, K) and DAPI (blue). (L) Mean ± SEM of the ratios of the mean integrated density of fluorescence between cells transfected with pMES or NSD3Δ1707 and neighboring untransfected cells are shown for H3K9me2, H3K27me2, H3K4me2, H3K36me2, and H3K36me3. A ratio of 1 would indicate equal staining between transfected and untransfected cells. White arrowhead, pMES- transfected cell; white arrow, NSD3Δ1707-transfected cell. n ≥ 20 fields of cells/condition. Student's t test was used for statistical analysis; *significant, p < 0.05; **highly significant, p < 0.001.
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Figure 2: NSD3Δ1707 prevents H3K36me2 and me3. (A) Diagram of the protein domain organization of NSD3 (top) and NSD3Δ1707 (bottom). PHD, plant homeodomain fingers; PWWP, Pro-Trp-Trp-Pro domain; SET, suppressor of variegation3-9, Enhancer of Zeste and Trithorax domain. (B–K) Chick fibroblast DF-1 cells were transfected with the GFP (green) bicistronic expression plasmid pMES (B, D, F, H, J) or pMES driving NSD3Δ1707 (C, E, G, I, K). Twenty-four hours after transfection, cells were fixed and stained with anti-methyl (CH3; red) antibodies against dimethylated (me2) lysines H3K9me2 (B, C), H3K27me2 (D, E), H3K4me2 (F, G), or H3K36me2 (H, I) or trimethylated (me3) lysine H3K36me3 (J, K) and DAPI (blue). (L) Mean ± SEM of the ratios of the mean integrated density of fluorescence between cells transfected with pMES or NSD3Δ1707 and neighboring untransfected cells are shown for H3K9me2, H3K27me2, H3K4me2, H3K36me2, and H3K36me3. A ratio of 1 would indicate equal staining between transfected and untransfected cells. White arrowhead, pMES- transfected cell; white arrow, NSD3Δ1707-transfected cell. n ≥ 20 fields of cells/condition. Student's t test was used for statistical analysis; *significant, p < 0.05; **highly significant, p < 0.001.

Mentions: NSD3 is a SET-domain lysine methyltransferase (Angrand et al., 2001; Kim et al., 2006; Li et al., 2009). In other lysine methyltransferases, mutation or deletion of the SET domain results in dominant-negative activity (Roopra et al., 2004; Lee et al., 2005; Houston et al., 2008; Huang, 2008; Joshi et al., 2008; Tanaka et al., 2008; Fujiki et al., 2009). To create a dominant negative and investigate a role for NSD3 in neural crest development, we truncated NSD3 at the start of the SET domain (NSD3Δ1707, Figure 2A) and drove expression with the green fluorescent protein (GFP) bicistronic expression plasmid pMES (Swartz et al., 2001).


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

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

NSD3Δ1707 prevents H3K36me2 and me3. (A) Diagram of the protein domain organization of NSD3 (top) and NSD3Δ1707 (bottom). PHD, plant homeodomain fingers; PWWP, Pro-Trp-Trp-Pro domain; SET, suppressor of variegation3-9, Enhancer of Zeste and Trithorax domain. (B–K) Chick fibroblast DF-1 cells were transfected with the GFP (green) bicistronic expression plasmid pMES (B, D, F, H, J) or pMES driving NSD3Δ1707 (C, E, G, I, K). Twenty-four hours after transfection, cells were fixed and stained with anti-methyl (CH3; red) antibodies against dimethylated (me2) lysines H3K9me2 (B, C), H3K27me2 (D, E), H3K4me2 (F, G), or H3K36me2 (H, I) or trimethylated (me3) lysine H3K36me3 (J, K) and DAPI (blue). (L) Mean ± SEM of the ratios of the mean integrated density of fluorescence between cells transfected with pMES or NSD3Δ1707 and neighboring untransfected cells are shown for H3K9me2, H3K27me2, H3K4me2, H3K36me2, and H3K36me3. A ratio of 1 would indicate equal staining between transfected and untransfected cells. White arrowhead, pMES- transfected cell; white arrow, NSD3Δ1707-transfected cell. n ≥ 20 fields of cells/condition. Student's t test was used for statistical analysis; *significant, p < 0.05; **highly significant, p < 0.001.
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Figure 2: NSD3Δ1707 prevents H3K36me2 and me3. (A) Diagram of the protein domain organization of NSD3 (top) and NSD3Δ1707 (bottom). PHD, plant homeodomain fingers; PWWP, Pro-Trp-Trp-Pro domain; SET, suppressor of variegation3-9, Enhancer of Zeste and Trithorax domain. (B–K) Chick fibroblast DF-1 cells were transfected with the GFP (green) bicistronic expression plasmid pMES (B, D, F, H, J) or pMES driving NSD3Δ1707 (C, E, G, I, K). Twenty-four hours after transfection, cells were fixed and stained with anti-methyl (CH3; red) antibodies against dimethylated (me2) lysines H3K9me2 (B, C), H3K27me2 (D, E), H3K4me2 (F, G), or H3K36me2 (H, I) or trimethylated (me3) lysine H3K36me3 (J, K) and DAPI (blue). (L) Mean ± SEM of the ratios of the mean integrated density of fluorescence between cells transfected with pMES or NSD3Δ1707 and neighboring untransfected cells are shown for H3K9me2, H3K27me2, H3K4me2, H3K36me2, and H3K36me3. A ratio of 1 would indicate equal staining between transfected and untransfected cells. White arrowhead, pMES- transfected cell; white arrow, NSD3Δ1707-transfected cell. n ≥ 20 fields of cells/condition. Student's t test was used for statistical analysis; *significant, p < 0.05; **highly significant, p < 0.001.
Mentions: NSD3 is a SET-domain lysine methyltransferase (Angrand et al., 2001; Kim et al., 2006; Li et al., 2009). In other lysine methyltransferases, mutation or deletion of the SET domain results in dominant-negative activity (Roopra et al., 2004; Lee et al., 2005; Houston et al., 2008; Huang, 2008; Joshi et al., 2008; Tanaka et al., 2008; Fujiki et al., 2009). To create a dominant negative and investigate a role for NSD3 in neural crest development, we truncated NSD3 at the start of the SET domain (NSD3Δ1707, Figure 2A) and drove expression with the green fluorescent protein (GFP) bicistronic expression plasmid pMES (Swartz et al., 2001).

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