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Smyd3 regulates cancer cell phenotypes and catalyzes histone H4 lysine 5 methylation.

Van Aller GS, Reynoird N, Barbash O, Huddleston M, Liu S, Zmoos AF, McDevitt P, Sinnamon R, Le B, Mas G, Annan R, Sage J, Garcia BA, Tummino PJ, Gozani O, Kruger RG - Epigenetics (2012)

Bottom Line: Smyd3 is a lysine methyltransferase implicated in chromatin and cancer regulation.Here we show that Smyd3 catalyzes histone H4 methylation at lysine 5 (H4K5me).This novel histone methylation mark is detected in diverse cell types and its formation is attenuated by depletion of Smyd3 protein.

View Article: PubMed Central - PubMed

Affiliation: GlaxoSmithKline, Collegeville, PA, USA.

ABSTRACT
Smyd3 is a lysine methyltransferase implicated in chromatin and cancer regulation. Here we show that Smyd3 catalyzes histone H4 methylation at lysine 5 (H4K5me). This novel histone methylation mark is detected in diverse cell types and its formation is attenuated by depletion of Smyd3 protein. Further, Smyd3-driven cancer cell phenotypes require its enzymatic activity. Thus, Smyd3, via H4K5 methylation, provides a potential new link between chromatin dynamics and neoplastic disease.

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Related in: MedlinePlus

Smyd3 methylates H4K5 in vitro and in vivo. (A) Methylation assay on H4 peptide residues 1–36, and H4 derived peptides with all lysines mutated to arginine (∆Kall) or single lysine maintained and all other lysines mutated to arginine as indicated (ΔK5* refers to a similar H4 peptide with all lysines mutated except K5). (B) LC-MS/MS analysis of Smyd3 methylated recombinant H4. (C) western blot analysis with the indicated antibodies of whole cell extracts (left panel) from HeLa cells expressing the indicated siRNAs and (right panel) wild-type and Smyd3−/− MEFs. (D) Quantitative mass spectrometry of the relative amounts of the indicated histone methylation marks in HeLa cells for Smyd3 positive/Smyd3 depleted cells (gray bars) and in wild type MEFs/ SMYD3−/− MEFs (black bars).
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Figure 2: Smyd3 methylates H4K5 in vitro and in vivo. (A) Methylation assay on H4 peptide residues 1–36, and H4 derived peptides with all lysines mutated to arginine (∆Kall) or single lysine maintained and all other lysines mutated to arginine as indicated (ΔK5* refers to a similar H4 peptide with all lysines mutated except K5). (B) LC-MS/MS analysis of Smyd3 methylated recombinant H4. (C) western blot analysis with the indicated antibodies of whole cell extracts (left panel) from HeLa cells expressing the indicated siRNAs and (right panel) wild-type and Smyd3−/− MEFs. (D) Quantitative mass spectrometry of the relative amounts of the indicated histone methylation marks in HeLa cells for Smyd3 positive/Smyd3 depleted cells (gray bars) and in wild type MEFs/ SMYD3−/− MEFs (black bars).

Mentions: To determine which H4 lysine residue is modified by Smyd3, a recombinant library was generated in which only a single lysine residue on the H4 tail is present and available to undergo methylation. In this assay, H4K20 did not serve as a substrate, but methylation was observed primarily on K5, with very low activity also observed on K8 and K12 (Fig. 2A). Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis of recombinant H4 methylated by Smyd3 identified H4K5me1 as the most abundant species, and also detected H4K5me2, H4K5me3 and H4K12me1 species, but no other methylation sites were detected including H4K20 (Fig. 2B). Thus, we conclude that in vitro Smyd3 primarily methylates histone H4 at lysine 5.


Smyd3 regulates cancer cell phenotypes and catalyzes histone H4 lysine 5 methylation.

Van Aller GS, Reynoird N, Barbash O, Huddleston M, Liu S, Zmoos AF, McDevitt P, Sinnamon R, Le B, Mas G, Annan R, Sage J, Garcia BA, Tummino PJ, Gozani O, Kruger RG - Epigenetics (2012)

Smyd3 methylates H4K5 in vitro and in vivo. (A) Methylation assay on H4 peptide residues 1–36, and H4 derived peptides with all lysines mutated to arginine (∆Kall) or single lysine maintained and all other lysines mutated to arginine as indicated (ΔK5* refers to a similar H4 peptide with all lysines mutated except K5). (B) LC-MS/MS analysis of Smyd3 methylated recombinant H4. (C) western blot analysis with the indicated antibodies of whole cell extracts (left panel) from HeLa cells expressing the indicated siRNAs and (right panel) wild-type and Smyd3−/− MEFs. (D) Quantitative mass spectrometry of the relative amounts of the indicated histone methylation marks in HeLa cells for Smyd3 positive/Smyd3 depleted cells (gray bars) and in wild type MEFs/ SMYD3−/− MEFs (black bars).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3368817&req=5

Figure 2: Smyd3 methylates H4K5 in vitro and in vivo. (A) Methylation assay on H4 peptide residues 1–36, and H4 derived peptides with all lysines mutated to arginine (∆Kall) or single lysine maintained and all other lysines mutated to arginine as indicated (ΔK5* refers to a similar H4 peptide with all lysines mutated except K5). (B) LC-MS/MS analysis of Smyd3 methylated recombinant H4. (C) western blot analysis with the indicated antibodies of whole cell extracts (left panel) from HeLa cells expressing the indicated siRNAs and (right panel) wild-type and Smyd3−/− MEFs. (D) Quantitative mass spectrometry of the relative amounts of the indicated histone methylation marks in HeLa cells for Smyd3 positive/Smyd3 depleted cells (gray bars) and in wild type MEFs/ SMYD3−/− MEFs (black bars).
Mentions: To determine which H4 lysine residue is modified by Smyd3, a recombinant library was generated in which only a single lysine residue on the H4 tail is present and available to undergo methylation. In this assay, H4K20 did not serve as a substrate, but methylation was observed primarily on K5, with very low activity also observed on K8 and K12 (Fig. 2A). Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis of recombinant H4 methylated by Smyd3 identified H4K5me1 as the most abundant species, and also detected H4K5me2, H4K5me3 and H4K12me1 species, but no other methylation sites were detected including H4K20 (Fig. 2B). Thus, we conclude that in vitro Smyd3 primarily methylates histone H4 at lysine 5.

Bottom Line: Smyd3 is a lysine methyltransferase implicated in chromatin and cancer regulation.Here we show that Smyd3 catalyzes histone H4 methylation at lysine 5 (H4K5me).This novel histone methylation mark is detected in diverse cell types and its formation is attenuated by depletion of Smyd3 protein.

View Article: PubMed Central - PubMed

Affiliation: GlaxoSmithKline, Collegeville, PA, USA.

ABSTRACT
Smyd3 is a lysine methyltransferase implicated in chromatin and cancer regulation. Here we show that Smyd3 catalyzes histone H4 methylation at lysine 5 (H4K5me). This novel histone methylation mark is detected in diverse cell types and its formation is attenuated by depletion of Smyd3 protein. Further, Smyd3-driven cancer cell phenotypes require its enzymatic activity. Thus, Smyd3, via H4K5 methylation, provides a potential new link between chromatin dynamics and neoplastic disease.

Show MeSH
Related in: MedlinePlus