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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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Smyd3 catalytic activity is required for anchorage-independent growth of cancer cells. (A-B) Complementation of Smyd3-depleted cells with wild-type Smyd3 but not catalytically dead Smyd3 restores anchorage-independent growth. (A) Left panel: Quantification of colony formation in methylcellulose after 10 d of Hep3b cells treated with either control shRNA or 3′UTR shSmyd3 reconstituted with GFP, Smyd3-WT or catalytically inactive Smyd3N205A. Right panel: western blot analysis of Hep3B whole cell extracts. (B) Left panel: Quantification of colony formation in soft agar after 14 d of MCF7 cells treated with either control shRNA or 3′UTR shSmyd3 reconstituted with Flag-control vector, Flag-Smyd3-WT, or catalytically inactive Flag-Smyd3F183A. Right panel: western blot analysis of MCF7 whole cell extracts. Bar graphs indicate the number of colonies per field. Error bars indicate the standard deviation (s.d.) from three independent experiments. The p values indicate the statistical significance as determined by t-test between the different conditions marked with * or **.
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Figure 3: Smyd3 catalytic activity is required for anchorage-independent growth of cancer cells. (A-B) Complementation of Smyd3-depleted cells with wild-type Smyd3 but not catalytically dead Smyd3 restores anchorage-independent growth. (A) Left panel: Quantification of colony formation in methylcellulose after 10 d of Hep3b cells treated with either control shRNA or 3′UTR shSmyd3 reconstituted with GFP, Smyd3-WT or catalytically inactive Smyd3N205A. Right panel: western blot analysis of Hep3B whole cell extracts. (B) Left panel: Quantification of colony formation in soft agar after 14 d of MCF7 cells treated with either control shRNA or 3′UTR shSmyd3 reconstituted with Flag-control vector, Flag-Smyd3-WT, or catalytically inactive Flag-Smyd3F183A. Right panel: western blot analysis of MCF7 whole cell extracts. Bar graphs indicate the number of colonies per field. Error bars indicate the standard deviation (s.d.) from three independent experiments. The p values indicate the statistical significance as determined by t-test between the different conditions marked with * or **.

Mentions: Consistent with previous reports, Smyd3 depletion attenuated proliferation of human carcinoma cell lines (Fig. S5).5 In addition, human breast carcinoma MCF7 cells and hepatoma Hep3B cells both lost the ability to form colonies in an anchorage-independent environment upon stable depletion of Smyd3 using shRNA directed to the 3′ UTR of Smyd3 (Figs. 3A and B). Colony formation was restored in Smyd3 depleted cells by complementation with wild-type Smyd3 (lacking the 3′ UTR and therefore RNAi-resistant) (Figs. 3A and B), whereas complementation with catalytically dead Smyd3 (Smyd3N205A (Fig. 3A) and Smyd3F183A (Fig. 3B) failed to reconstitute this activity. Moreover, global levels of H3K4me3 and H4K20me3 were unchanged upon Smyd3 knockdown in MCF7 cells (Fig. S6). Therefore, we conclude that while anchorage independent growth of MCF7 cells requires Smyd3 activity, maintenance of the global levels of H3K4me3 and H4K20me3 does not. Thus, Smyd3 is required for H4K5 methylation in cells and its enzymatic activity is important for maintaining transformed cell phenotypes associated with high Smyd3 expression.


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 catalytic activity is required for anchorage-independent growth of cancer cells. (A-B) Complementation of Smyd3-depleted cells with wild-type Smyd3 but not catalytically dead Smyd3 restores anchorage-independent growth. (A) Left panel: Quantification of colony formation in methylcellulose after 10 d of Hep3b cells treated with either control shRNA or 3′UTR shSmyd3 reconstituted with GFP, Smyd3-WT or catalytically inactive Smyd3N205A. Right panel: western blot analysis of Hep3B whole cell extracts. (B) Left panel: Quantification of colony formation in soft agar after 14 d of MCF7 cells treated with either control shRNA or 3′UTR shSmyd3 reconstituted with Flag-control vector, Flag-Smyd3-WT, or catalytically inactive Flag-Smyd3F183A. Right panel: western blot analysis of MCF7 whole cell extracts. Bar graphs indicate the number of colonies per field. Error bars indicate the standard deviation (s.d.) from three independent experiments. The p values indicate the statistical significance as determined by t-test between the different conditions marked with * or **.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Smyd3 catalytic activity is required for anchorage-independent growth of cancer cells. (A-B) Complementation of Smyd3-depleted cells with wild-type Smyd3 but not catalytically dead Smyd3 restores anchorage-independent growth. (A) Left panel: Quantification of colony formation in methylcellulose after 10 d of Hep3b cells treated with either control shRNA or 3′UTR shSmyd3 reconstituted with GFP, Smyd3-WT or catalytically inactive Smyd3N205A. Right panel: western blot analysis of Hep3B whole cell extracts. (B) Left panel: Quantification of colony formation in soft agar after 14 d of MCF7 cells treated with either control shRNA or 3′UTR shSmyd3 reconstituted with Flag-control vector, Flag-Smyd3-WT, or catalytically inactive Flag-Smyd3F183A. Right panel: western blot analysis of MCF7 whole cell extracts. Bar graphs indicate the number of colonies per field. Error bars indicate the standard deviation (s.d.) from three independent experiments. The p values indicate the statistical significance as determined by t-test between the different conditions marked with * or **.
Mentions: Consistent with previous reports, Smyd3 depletion attenuated proliferation of human carcinoma cell lines (Fig. S5).5 In addition, human breast carcinoma MCF7 cells and hepatoma Hep3B cells both lost the ability to form colonies in an anchorage-independent environment upon stable depletion of Smyd3 using shRNA directed to the 3′ UTR of Smyd3 (Figs. 3A and B). Colony formation was restored in Smyd3 depleted cells by complementation with wild-type Smyd3 (lacking the 3′ UTR and therefore RNAi-resistant) (Figs. 3A and B), whereas complementation with catalytically dead Smyd3 (Smyd3N205A (Fig. 3A) and Smyd3F183A (Fig. 3B) failed to reconstitute this activity. Moreover, global levels of H3K4me3 and H4K20me3 were unchanged upon Smyd3 knockdown in MCF7 cells (Fig. S6). Therefore, we conclude that while anchorage independent growth of MCF7 cells requires Smyd3 activity, maintenance of the global levels of H3K4me3 and H4K20me3 does not. Thus, Smyd3 is required for H4K5 methylation in cells and its enzymatic activity is important for maintaining transformed cell phenotypes associated with high Smyd3 expression.

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