Here we report an association between the DNA methyltransferases Dnmt1 and Dnmt3a and the SUV39H1–HP1 histone methylation system. We show that Dnmt3a associates primarily with histone H3 Lys9 methyltransferase activity but also to a lesser extent with H3 Lys4 activity. The H3 Lys9 enzymatic activity is likely provided by the H3-K9 HMTase SUV39H1 since we observed that it binds both in vitro and in vivo to Dnmt3a. We find that SUV39H1 also binds to Dnmt1 and consistent with these associations, SUV39H1 purifies DNA methyltransferase activity. In addition, we show that HP1β, a known SUV39H1-interacting protein, can bind directly to Dnmt1 as well as Dnmt3a and that endogenous HP1β purifies DNA methyltransferase activity. Taken together, the present study reveals a direct link between the enzymatic activities responsible for two distinct epigenetic layers.
Recently, a connection between DNA and histone methylation has been uncovered in N.crassa
. It was found that the genes dim-5
encode H3-K9-specific HMTase. Interestingly, mutations in dim-5
that abolish their enzymatic activity lead to a decrease in DNA methylation (11
). The mechanisms by which histone methylation influences DNA methylation are still unclear. Given that in A.thaliana
the CMT3 DNA methyltransferase associates with the Arabidopsis
homologue of HP1, LHP1, it was proposed that Kryptonite would methylate H3-K9, creating a binding platform for LHP1. CMT3 would then be recruited by the adaptor LHP1 to methylate DNA and thereby DNA methylation would be dependent on prior histone methylation (12
). Our data suggest that a similar scenario could also be operational in mammals. We found that endogenous HP1β purifies DNA methyltransferase activity and, in agreement with our observation, Dnmt3a was found to co-localise with HP1β (6
). Thus, similarly to what was found in Neurospora
, mammalian Dnmts could be recruited by the adaptor molecule HP1 to chromatin that contains methylated histones. In addition, our finding that the DNA methyltransferases themselves associate with the H3-K9 SUV39H1 HMTase extend this initial model and imply a much more direct connection between DNA methylation and histone methylation. Indeed, the present data suggest that the Dnmts could be directly recruited to H3-K9 methylated chromatin through their interaction with the SUV39H1 histone methyltransferase. This possibility is interesting because it would mean that the histone methyltransferase activity associated with the DNA methyltransferases may be required for the Dnmts to methylate DNA. In other words, the Dnmts could only access and methylate DNA that is first wrapped around histones modified at Lys9 of H3. The notion that chromatin remodelling is required for DNA methylation to occur is supported by recent studies on the SNF2-like ATPase family. In Arabidopsis
, mutation of the putative SNF2-like ATPase DDM1 leads to a severe reduction in DNA methylation (24
) and targeted disruption of its mammalian homologue LSH causes loss of DNA methylation and lethality in mice (25
). These data suggest that the putative nucleosome remodelling activity of these ATPases is required so that DNA becomes suitable to serve as a substrate for methylation.
A model of epigenetic transmission in the opposite direction should also be considered, namely that DNA methylation can influence the methylation of histones. This reverse scenario is supported by our recent finding that the methyl-CpG-binding protein MeCP2 associates with H3-K9 methyltransferase activity and is involved in targeting methylation of histone H3 Lys9 to a DNA methylated gene that it regulates (13
Taken together, the recent findings in Neurospora and Arabidopsis as well as our data suggest the following order of events that connect DNA to histone methylation. In the initial phase, the Dnmts would add methyl groups to DNA only on chromatin that is methylated at Lys9 and bind HP1. The direct physical link identified here between the Dnmts and the SUV39H1–HP1 system would make sure that the H3-K9 methylation status directly influences DNA methylation patterns. In a second step, the generation of methylated DNA by the Dnmts would allow DNA binding of MeCP2, which in turn associates and favours histone methylation at Lys9 of H3. This sequential process of coupling DNA with histone methylation is particularly attractive because it would suggest that DNA methylation may also feed back to facilitate histone methylation, thereby reinforcing the two modes of epigenetic silencing and creating a self-propagating epigenetic cycle for long-term transcriptional repression.
Beside contacting H3-K9 HMTase activity, we found that Dnmt3a also associates, albeit weakly, with histone methyltransferase activity at Lys4 of H3. This finding was somewhat unexpected as DNA methylation is associated with gene silencing and H3-K4 methylation has been described to correlate with transcriptional activation (19
). However, recent work in Saccharomyces cerevisiae
indicates that the Set1 H3-K4 HMTase is required for gene silencing of rDNA (26
). The mechanisms by which H3-K4 methylation is linked to transcriptional silencing are currently unknown. One explanation could be related to the histone code hypothesis (28
), namely that the effect of H3-K4 methylation on transcription may well depend on its combination with other histone modifications. Our finding that a DNA methyltransferase can contact H3-K4 activity opens up an alternative possibility whereby methylation at Lys4 of H3 might function in concert with DNA methylation to repress transcription. By analogy to what we propose here for the connection between the Dnmts and H3-K9 methylation, it is possible that H4-K4 HMTase recruits the Dnmts to the DNA that has to be modified or conversely DNA methyltransferases may influence H3-K4 methylation. Future work will be needed to test this attractive possibility.
In conclusion, our data further emphasise the intimate relationship that exists between DNA methylation and histone methylation. The physical interaction identified here between the enzymes responsible for these distinct methylation layers further substantiates the concept of a self-reinforcing cycle for the propagation of a stable state of inactive chromatin.