Product specificity of the HMT activity of MDU

HMTs can mono-, di- and tri-methylate target lysine residues (Zhang et al., 2003b). The methylation status of lysine residues plays an important role in the histone code hypothesis, because the methylation status of the lysine residue determines the biological function of histone methylation (Ruthenburg et al., 2007). For example, tri-methylated H3K4 has been associated with transcriptional activation, whereas di-methylated H3K4 has been detected in eu- and heterochromatin (Santos-Rosa et al., 2002).

Previous studies revealed that the structure of the lysine access channel in the SET domain determines the product specificity of HMTs (Xiao et al., 2003b, Trievel et al., 2003, Zhang et al., 2003b) (Figure 37).

Figure 37. The geometry of the lysine access channel determines the number of methyl groups that can be transferred. (a–c) Surface representations of the lysine access channels of the three ternary structures viewed from the peptide-binding side: (a) Set7/9, (b) Rubisco LSMT and (c) DIM-5. Key residues are shown in stick formation, and the substrate lysine side chain is shown in yellow. (d,e) Diagrams illustrating how the channel geometry either prevents rotation around the C--N bond after the addition of a methyl group, (d) as in the case of SET7/9, or

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allows rotation and therefore the addition of further methyl groups, (e) as in the case of DIM-5 and LSMT (Xiao et al., 2003a).

The lysine access channel is a narrow channel that connects the cofactor-binding site on one surface with the substrate-binding site on the opposite surface of the domain (Xiao et al., 2003a). The geometry of the lysine access channel determines the number of methyl groups that can be transferred.

In Figure 37, the lysine access channels of the three HMTase (SET7/9, DIM-5 and Rubisco LSMT) ternary complex structures are shown from the viewpoint of the peptide-binding side. DIM-5 and Rubisco LSMT catalyze tri-methylation of their target lysine residues, whereas SET7/9 adds just a single methyl group to its lysine substrate (Xiao et al., 2003b; Trievel et al., 2003; Zhang et al., 2003b). The authors of these studies propose that the geometry and shape of the bottom of the access channel are responsible for determining how many methyl groups the SET domain can methylate. This endpoint is achieved either by preventing rotation around the C-N bond of the lysine side chain after the addition of a methyl group or permitting the rotation (Figure 37).

Our results indicate that MDU predominantly tri-methylates H3K9 in vitro and mediates tri-methylation of H3K9 at target genes in vivo.

A recent study demonstrated that MDU mono-, di- and tri-methylates H3K9 in a sequential manner (Tzeng et al., 2007). In vitro HMTase assay was performed with immunopurified MDU from transfected S2 cells. At different times, the reaction products were analysed by Western blotting with antibodies against different forms of methylated H3K9. The results revealed that MDU catalyzed H3 methylation switches gradually from predominantly mono-methylation to tri-methylation (Tzeng et al., 2007). We cannot rule out that MDU methylates H3K9 in a sequential fashion. However the presence of only tri-methylated H3K9 at MDU target genes in vivo provides strong evidence that MDU establishes tri-methylated H3K9 at target genes rather than mono-, di- and tri-methylated H3K9. The HMTs DIM-5 and LSMT mediate tri-methylation of H3K9 in a sequential fashion. Thus the sequential methylation of H3K9 by MDU in vitro reflects on the mechanism by which MDU methylates H3K9 of HMTs rather than revealing the product specificity of the HMT-activity of MDU. Collectively, the results support a model in which MDU tri-methylates H3K9 in vitro.

This model is challenged and confirmed by contradicting in vivo studies of the HMT-activity of MDU.

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Tzeng et al. analyzed methylation of histone 3 in third-instar larvae of Drosophila by Western blotting (Tzeng et al., 2007). Mono-, di- and tri-methylated H3K9 were detected in wild type larvae, whereas mono-, di- and tri-methylation of H3K9 were decreased in MDU mutant larvae. All three methylated H3K9 species could be rescued by overexpression of MDU. The authors concluded that MDU is one of the major histone H3K9 methyltransferases in the fly and produces mono-, di- and tri-methylated H3K9 in the third-instar larvae (Tzeng et al., 2007).

Another study investigated the role of MDU in Drosophila ovaries. In MDU mutant flies, H3K9 tri-methylation was absent throughout the germarium. The H3K9 di-methylation pattern did not change in the MDU mutant ovaries (Clough et al., 2007). It was concluded that MDU is required for tri-methylation, but not dimethylation, of histone H3 at the K9 residue. MDU mediates H3K9 tri-methylation in both the germ and somatic cells of the germarium and is the major HMT conferring H3K9 tri-methylation in these cells (Clough et al., 2007).

A second study dissecting the role of MDU in ovaries showed that MDU is responsible for the synthesis of H3K9 tri-methylation signals in the inner germarium, where GSCs (germline stem cells) and their early descendants are found. When these cells move to region-3 germarium, the H3K9 tri-methylation task is transferred to a combination of MDU and SU(VAR)3–9, because both enzymes act cooperatively in all other somatic-type cells of the germarium. After the egg chamber buds off from the germarium, the tri-methylation is regulated only by SU(VAR)3–9 (Yoon et al., 2008).

All three studies focused on the pattern of MDU-mediated H3K9 methylation at the chromatin level, but did not investigate histone methylation of MDU at specific target genes.

Our results suggest that MDU tri-methylates H3K9 at artificial and endogenous target genes in vivo. Tri-methylation of H3K9 by MDU triggers de novo DNA methylation. The different isoforms of MDU detected throughout development could establish different H3K9 methylation patterns. Our results support a model in which gene-specific tri-methylation of H3K9 plays an important role in gene silencing and de novo DNA methylation.

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