Direct coupling between DNA methyltransferase enzymes and po
Direct coupling between DNA methyltransferase enzymes and posttranslational modifications on histone proteins has been observed in mammals. For example, DNMT3A recognizes unmodified H3 through an ATRX-DNMT3-DNMT3L (ADD) domain, and its activity is inhibited by methylation of H3 at K4 (Li et al., 2011, Otani et al., 2009, Zhang et al., 2010). Full activity requires the formation of a complex of two DNMT3A molecules with two accessory DNMT3L molecules. The resultant tetramer allows for multivalent histone recognition, in which all four subunits make contact with the unmodified H3 tails on nucleosomes (Jia et al., 2007). This type of multivalent histone recognition likely serves to promote fidelity and specificity for DNA methyltransferase activity, because DNA methylation will depend not only on the successful formation of the tetrameric complex, but also successful recognition of the H3 tail by each subunit. Crystal structures of the tetramer formed by the C-terminal domains of DNMT3A and DNMT3L suggest models in which the complex bridges across nucleosomes to target DNA, but these structures do not include nucleosomes (Jia et al., 2007, Zhang et al., 2018b).
In plants, the first example of positive feedback between DNA methylation and histone methylation came from studies of CMT3 and the histone H3 methyltransferase KRYPTONITE (KYP). Mutation of KYP was found to reduce CHG methylation, whereas Thyroid Hormone Receptor Antagonist (1-850) clinical of CMT3 led to reduced levels of H3K9me (Jackson et al., 2002, Malagnac et al., 2002, Mathieu et al., 2005, Soppe et al., 2002, Tariq et al., 2003). H3K9me and CHG methylation were also shown to be highly correlated throughout the Arabidopsis genome (Bernatavichute et al., 2008). This positive reinforcement was found to be driven in part by the KYP SET and RING finger associated (SRA) domain, which recognizes methylated CHG sites, and a SET (Su(var)3-9, enhancer-of-zeste and Trithorax) domain, which deposits H3K9me (Du et al., 2014). Conversely, CMT3 contains a chromodomain (CD) with a canonical aromatic cage that specifically binds H3K9me. In addition to CMT3’s H3K9me recognition via its CD, it was discovered that CMT3’s bromo adjacent homology (BAH) domain is also competent to bind H3K9me (Du et al., 2012). Crystal structures of an N-terminal truncation of ZMET2 demonstrate recognition of methylated H3 tail peptides by either the CD or the BAH domain (Du et al., 2012). Thus, CMT3 and ZMET2 appear to have bypassed the necessity for adaptor proteins or heterodimerization to engage in multivalent histone tail recognition. How CMT3 and ZMET2 harness this “all-in-one” architecture to methylate target DNA sequences with high fidelity in the context of chromatin remains an open question.
Discussion In this study, we sought to understand the mechanistic roles of ZMET2/H3K9me interactions that were uncovered previously using genetics and structural biology (Du et al., 2012). Our biochemical and structural results suggest that the two H3K9me recognition domains in ZMET2 play binding and allosteric roles allowing ZMET2 to couple maximal DNA methylation to recognition of the appropriate DNA methylation status and chromatin architecture. Below we discuss the mechanistic implications of our findings.
Acknowledgments We thank D. Patel for ZMET2 expression plasmids and initial protein purification advice. We thank K. Armache for initial 601 dinucleosomal DNA plasmids, the Kruglyak lab for use of the Illumina MiSeq sequencer, S. Poepsel for guidance on generating ligated dinucleosomes, and E. Palovcak, A. Lyon, M. Ravalin, and D. Elnatan for technical assistance. We thank R.S. Isaac and P.A. Dumesic for critical reading of the manuscript, and members of the Narlikar, Cheng, and Jacobsen labs for useful discussion. This work was funded by an NSF grant (1517081) to G.J.N., an NSF Graduate Research Fellowship (1144247) awarded to C.I.S., an NSF-CAREER grant (MCB-1552455) and NIH-MIRA grant (R35GM124806) to X.Z., an NIH grant (GM60398) to S.E.J., and NIH grants (S10OD020054, R01GM098672, and R01GM082893) awarded to Y.C. S.E.J. and Y.C. are investigators of the Howard Hughes Medical Institute. We thank B. Carragher and Z. Zhang for preliminary EM experiments conducted at the National Resource for Automated Molecular Microscopy (NRAMM) located at the New York Structural Biology Center, supported by the NIH (grant GM103310) and the Simons Foundation (grant 349247).