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 is detected in both the cytoplasm and nucleus (Fig. S2). Thus, while methylation of H4K5 in human cells has not been described to date, Smyd3 nuclear localization suggests that it could generate this mark in vivo. In this context, an unbiased MS/MS based approach was used to quantify the methylation states of all lysine residues present on histones H3 and H4 in two model cells systems, Smyd3 depleted HeLa cells and Smyd3 knockout mouse embryonic fibroblasts (MEFs) (Fig. 2C and D). H4K5me1 was detected in both cell types and the levels of this mark were significantly reduced upon Smyd3 knockdown or knockout (Fig. 2D and Table S2). Notably, in this analysis, no significant changes were observed in the methylation states of any of the other lysine residues including the previously implicated Smyd3 substrate sites of H3K4 and H4K20 (Table S2 and Fig. S3). Furthermore, global levels of H3K4 and H4K20 methylation in HeLa cells did not increase upon Smyd3 overexpression. (Fig. S4).

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.

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