Structural Basis And Biological Effects Of Histone Modification Recognition On DNMT3A Targeting

Histone modifications and DNA methylation represent two distinct modes of varying epigenetic “landscapes”, but whose exact molecular relationship remains unclear. Previous studies have shown that histone H3 lysine 4 trimethylation(H3K4me3) inhibits the binding of de novo DNA methyltransferase DNMT3 A through the ATRX-DNMT3-DNMT3L(ADD) domain, thus protects CpG islands(CGI) from DNA methylation. Key questions remain including in vivo links between H3K4me3 and unmethylated CGI and a potential role of other H3 modification on de novo Dnmts.Here, we solved a 2.4 ? crystal structure of the DNMT3 A ADD domain bound to the unmodified N-terminus of histone H3. Using biochemical approaches such as peptides array assay and isothermal titration calorimetry, we found a novel antagonistic relationship between H3T3/H3T6 phosphorylation and ADD binding to the unmodified H3 N-terminus. Based on solved ADD-H3 complex structure, we elucidated the molecular mechanism by which H3K4me3 and H3T3ph/H3T6 ph modifications disrupt the binding of DNMT3 A to histone H3. To assess the physiological relevance of these restrictions, with careful structure comparation and analysis, we had engineered the ADD domain of DNMT3 A to permit additional binding to H3K4me3 or H3T3 ph respectively. Isothermal titration calorimetry was used to measure the comparable binding affinity of mutant and wild-type ADD with correspondent modified peptides and crystal structures were solved to confirm the effectivity of mutant ADD bound to H3K4me3 and H3T3 ph respectively. Cooperated with David Allis? lab, we investigate the correlation of these mutant Dnmt3a2 with distribution of DNA methylation and its biological function in murine Dnmt1-Dnmt3a-Dnmt3 b triple knockout embryonic stem celllines. Engineered Dnmt3a2 tolerable to H3K4me3 binds to a subset of CGI marked with H3K4me3,induces modest “ecotopic” DNA methylation thus leads to a failure of cell lineage specification. Dnmt3a2 tolerable to H3T3 ph was found seldom to localize at centromeres during metaphase therefore results in chromosome instability.These results demonstrate a fundamental role for histone H3 modifications in guiding DNA methylation in vivo, and highlight the biological importance of the site-specific, methyl-lysine histone recognition domain within Dnmt3 a in bringing about physiologically-relevant epigenetic “landscapes”.