Nucleosome occupancy is an important determinant of global DNA methylation patterns in vivo
(Chodavarapu et al., 2010
). The association of CMT3 with nucleosomes suggests that CMT3 may methylate nucleosome-bound DNA, which may explain our previous observations from whole genome shotgun bisulfite sequencing that CHG methylation was enhanced on nucleosomal DNA relative to linker DNA and that CHG sites facing on the outside of nucleosomes were methylated at a higher level than those facing on the inside (Chodavarapu et al., 2010
). This model also explains the 167-nt periodicity pattern of CHG methylation previously observed in bulk analysis of whole genome methylation data (Cokus et al., 2008
The observation that CMT3 is predominately expressed in cells undergoing active replication implies that methylation by CMT3 takes place during DNA replication, when H3.1 is incorporated onto newly synthesized chromatin and modified by the histone H3K9 methyltransferase, KYP. One plausible model is that CMT3 is displaced from nucleosomes during replication. Once replication is completed, it is recruited back to the newly assembled nucleosomes that have been methylated by KYP (or other SUVHs) on H3K9. Alternatively, CMT3 may be recruited to chromatin during DNA replication by pre-modified H3K9me2 marks from parental histones. In turn, CHG methylation would recruit KYP to deposit methylation marks on newly synthesized histones.
We attempted to test whether the H3K9-specific histone methyltransferase KYP (SUVH4) or possibly SUVH5 and SUVH6 (two other H3K9 methyltransferases) would form a complex with CMT3. However, the CMT3 IP-MS analysis did not detect any KYP, SUVH5, or SUVH6 peptides. Reciprocal IP experiments with protein extracts from Nicotiana benthamiana leaves co-expressing MYC-tagged CMT3 and FLAG-tagged KYP also failed to detect interaction between CMT3 and KYP (data not shown). These findings suggest either that KYP is not stably associated with nucleosomes, or that KYP and CMT3 cannot simultaneously remain bound to the same nucleosomes.
Our in vitro
activity assays show that CMT3 has some activity on unmethylated DNA. We speculate that this “de novo
” activity is in fact part of the “maintenance” loop of KYP and CMT3. Unlike the mammalian maintenance DNA methyltransferase DNMT1, which has much greater activity on hemi-methylated DNA than on unmethylated DNA (Bestor and Ingram, 1983
), CMT3 only has slightly higher activity on hemimethylated substrates than unmethylated substrates. In addition, our data suggested that CMT3 showed only a small tendency to fully methylate CHG sites in vivo
rather than leaving them hemimethylated, which is in contrast to the strong tendency of DNMT1 to fully methylate CG sites in vivo
. This suggests that CMT3 is not as good as DNMT1 at restoring hemimethylated sites to fully methylated sites and consequently, methylation at many sites will be completely lost during multiple cycles of replication. The “de novo”
activity of CMT3, however, could persistently target methylation to regions marked with H3K9 methylation. CMT3’s inefficiency at maintaining methylation is also reflected by the fact that the overall global levels of CHG methylation (6.7%) are much lower than those of CG methylation (24%) (Cokus et al., 2008
). In addition to the preference for hemimethylated DNA as substrate, DNMT1 and MET1 work with the UHRF1/VIM cofactor that specifically recognizes hemimethylated DNA through its SRA domain (Bostick et al., 2007
). However, the SRA domain of KYP and SUVH5 bind equally well to hemimethylated and fully methylated DNAs (Johnson et al., 2007
; Rajakumara et al., 2011a
). Therefore, CMT3’s “maintenance” activity is likely to be driven mainly by the feedback loop between KYP and CMT3, rather than by the inherent preference of CMT3 for hemimethylated DNA.
H3K27me1 is enriched in heterochromatin and was proposed to be involved in CMT3 function based on in vitro
data showing that the chromodomain of CMT3 bound to histone H3 peptides only when both H3K9 and H3K27 were methylated (Lindroth et al., 2004
). However, these doubly methylated peptides were longer than the singly methylated control peptides, which might be an alternative explanation for this result. More critically, a recent study showed that depletion of H3K27me1 in vivo
did not reduce CHG methylation (Jacob et al., 2009
). In addition, our peptide array and structural analyses showed that H3K9me2 is necessary and sufficient to bind CMT3. Therefore, H3K27me is unlikely to play a role in CMT3 targeting.
The BAH domain functions as a mediator of protein-protein interactions. Yeast Sir3 BAH domain in complex with mononucleosomes revealed that the BAH domain makes contacts through unmodified H4K16 and H3K79 (Armache et al., 2011
), which are important for BAH-nucleosome binding (Onishi et al., 2007
). Recent studies of the mouse ORC1 BAH domain bound to H4K20me2 peptide have established that methylated-lysine is recognized through positioning within an aromatic cage on the surface of the BAH domain, and the methyllysine recognition mode is similar as the ZMET2 BAH domain recognizing H3K9me2 peptide reported here (Figure S4E
) (Kuo et al., 2012
). In the current study, we also observed that the methylated-lysine of H3K9me2 peptide was positioned within an aromatic cage of the BAH domain of ZMET2. The aromatic cage residues in the BAH domain of ZMET2, that are conserved amongst the BAH domains of other plant chromomethylases, are also observed within the BAH1 domain of human DNMT1. These results imply that the BAH1 domain of DNMT1 may also be a reader of methylated-lysine marks using aromatic cage capture. By contrast, no aromatic cage was identified following alignment of the BAH2 domain of DNMT1.
How is CHG methylation precisely controlled and faithfully maintained? We propose a dual recognition mechanism, in which the CMT3 BAH and chromo domains simultaneously read the H3K9me2 on the two tails emanating from a single nucleosome to ensure a higher fidelity of CHG DNA methylation (). In the crystal, we are restricted to one or the other complex most likely due to packing interactions, but there should be no such constraints for simultaneous recognition of H3K9me2 marks by both BAH and chromo domains in solution. Indeed, ITC binding studies yield a stoichiometry of 1.8 for binding of H3(1-15)K9me2 peptide to ZMET2 (Figure S6A
). Modeling efforts indicate that it is possible that two H3K9m2 modified tails could extend from the single nucleosome and bind to the chromo and BAH domains simultaneously. The nucleosomal DNA between the two H3 tails could be clamped by the TRD subdomain and the catalytic loop and subsequently methylated (Figures S6B-C
). The H3 tail has sufficient length so as to likely allow the enzyme enough flexibility to catalyze on a range of nucleosomal DNA. The two-domain binding mode could potentially increase CMT3’s binding affinity and specificity towards H3K9me2 marks and thus help to recruit CMT3 to H3K9me2-containing nucleosomes. It is also conceivable that the dual binding modules could help CMT3 walk from nucleosomes to adjacent nucleosomes, characteristic of a spreading mechanism. Interestingly, the mammalian de novo
DNA methyltransferase DNMT3A/3L complex also binds stably to nucleosomes (Jeong et al., 2009
), and DNMT3A and DNMT3L each have an unmodified H3K4-recognizing ADD domain, suggesting that the DNMT3A/3L complex may also have the potential for dual recognition of two unmodified H3K4-containing tails of a nucleosome. By contrast, DNMT1 only contains one BAH domain that has a potential methyl-lysine binding cage and is not tightly associated with nucleosomes (Jeong et al., 2009
). Thus a dual histone tail recognition mode may be a common feature of DNA methyltransferase that are stably bound to chromatin.