Abstract
Cytosine methylation of genomic DNA controls gene expression and maintains genome stability. How a specific DNA sequence is targeted for methylation by a methyltransferase is largely unknown. Here, we show that histone H3 tails lacking lysine 4 (K4) methylation function as an allosteric activator for methyltransferase Dnmt3a by binding to its plant homeodomain (PHD). In vitro, histone H3 peptides stimulated the methylation activity of Dnmt3a up to 8-fold, in a manner reversely correlated with the level of K4 methylation. The biological significance of allosteric regulation was manifested by molecular modeling and identification of key residues in both the PHD and the catalytic domain of Dnmt3a whose mutations impaired the stimulation of methylation activity by H3 peptides but not the binding of H3 peptides. Significantly, these mutant Dnmt3a proteins were almost inactive in DNA methylation when expressed in mouse embryonic stem cells while their recruitment to genomic targets was unaltered. We therefore propose a two-step mechanism for de novo DNA methylation – first recruitment of the methyltransferase probably assisted by a chromatin- or DNA-binding factor, and then allosteric activation depending on the interaction between Dnmt3a and the histone tails – the latter might serve as a checkpoint for the methylation activity.
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Acknowledgements
We thank Gerd Pfeifer (Beckman Research Institute of City of Hope) and Taiping Chen (Novartis Institutes for Biomedical Research) for helpful discussions and Jiemin Wong (East China Normal University) for critical reading of the manuscript. We are grateful to Masaki Okano (RIKEN Center for Developmental Biology) for providing Dnmt triple knockout ES cells. This work was supported by grants from the Ministry of Science and Technology of China (2005CB522400 and 2006CB943900), the National Natural Science Foundation of China (30730059) and the Shanghai Municipal Government (08dj1400501, 0852S11223) to GLX.
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( Supplementary information is linked to the online version of the paper on the Cell Research website.)
Supplementary information
Supplementary information, Figure S1
The Dnmt3a PHD domain interacts with the catalytic domain. (PDF 83 kb)
Supplementary information, Figure S2
Measurement of binding affinity of Flag-Dnmt3a toward DNA. (PDF 98 kb)
Supplementary information, Figure S3
Coomassie staining of Flag-tagged full-length Dnmt3a proteins purified from transiently transfected HEK293T cells and GST-fusion proteins of 3aPHD purified from E. coli BL21 (DE3). (PDF 169 kb)
Supplementary information, Figure S4
Functional analysis of Dnmt3a D308A and M325W mutants. (PDF 146 kb)
Supplementary information, Figure S5
Southern hybridization analysis of the methylation activity of transiently transfected Dnmt3a interface mutants. (PDF 121 kb)
Supplementary information, Figure S6
Expression and distribution of Dnmt3a Q304A and R580A mutants in mouse ES cells. (PDF 160 kb)
Supplementary information, Figure S7
Methylation analysis at the Oct4 and Sox30 promoters in ES cells ectopically expressing wild-type and mutant Dnmt3a. (PDF 146 kb)
Supplementary Table 1.
Mutational analysis of residues on the interfaces of the PHD and catalytic domains of Dnmt3a. (PDF 90 kb)
Supplementary information, Data S1
Materials and Methods (PDF 61 kb)
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Li, BZ., Huang, Z., Cui, QY. et al. Histone tails regulate DNA methylation by allosterically activating de novo methyltransferase. Cell Res 21, 1172–1181 (2011). https://doi.org/10.1038/cr.2011.92
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DOI: https://doi.org/10.1038/cr.2011.92
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