Modular fluorescence complementation sensors for live cell detection of epigenetic signals at endogenous genomic sites

Investigation of the fundamental role of epigenetic processes requires methods for the locus-specific detection of epigenetic modifications in living cells. Here, we address this urgent demand by developing four modular fluorescence complementation-based epigenetic biosensors for live cell microscopy applications. These tools combine engineered DNA-binding proteins with domains recognizing target epigenetic marks, both fused to non-fluorescent fragments of a fluorescent protein. The presence of the epigenetic mark at the target DNA sequence leads to the reconstitution of a functional fluorophore. With this approach, we could for the first time directly detect DNA methylation and histone 3 lysine 9 trimethylation at endogenous genomic sites in live cells and follow dynamic changes in these marks upon drug treatment, induction of epigenetic enzymes and during the cell cycle. We anticipate that this versatile technology will improve our understanding of how specific epigenetic signatures are set, erased and maintained during embryonic development or disease onset

Chromatin-dependent allosteric regulation of DNMT3A activity by MeCP2

Despite their central importance in mammalian development, the mechanisms that regulate the DNA methylation machinery and thereby the generation of genomic methylation patterns are still poorly understood. Here, we identify the 5mC-binding protein MeCP2 as a direct and strong interactor of DNA methyltransferase 3( DNMT3) proteins. We mapped the interaction interface to the transcriptional repression domain of MeCP2 and the ADD domain of DNMT3A and find that binding of MeCP2 strongly inhibits the activity of DNMT3A in vitro. This effect was reinforced by cellular studies where a global reduction of DNA methylation levels was observed after overexpression of MeCP2 in human cells. By engineering conformationally locked DNMT3A variants as novel tools to study the allosteric regulation of this enzyme, we show that MeCP2 stabilizes the closed, autoinhibitory conformation of DNMT3A. Interestingly, the interaction with MeCP2 and its resulting inhibition were relieved by the binding of K4 unmodified histone H3 N-terminal tail to the DNMT3A-ADD domain. Taken together, our data indicate that the localization and activity of DNMT3A are under the combined control of MeCP2 and H3 tailmodifications where, depending on the modification status of the H3 tail at the binding sites, MeCP2 can act as either a repressor or activator of DNA methylation

Chromatin methylation activity of Dnmt3a and Dnmt3a/3L is guided by interaction of the ADD domain with the histone H3 tail

Using peptide arrays and binding to native histone proteins, we show that the ADD domain of Dnmt3a specifically interacts with the H3 histone 1–19 tail. Binding is disrupted by di- and trimethylation of K4, phosphorylation of T3, S10 or T11 and acetylation of K4. We did not observe binding to the H4 1–19 tail. The ADD domain of Dnmt3b shows the same binding specificity, suggesting that the distinct biological functions of both enzymes are not related to their ADD domains. To establish a functional role of the ADD domain binding to unmodified H3 tails, we analyzed the DNA methylation of in vitro reconstituted chromatin with Dnmt3a2, the Dnmt3a2/Dnmt3L complex, and the catalytic domain of Dnmt3a. All Dnmt3a complexes preferentially methylated linker DNA regions. Chromatin substrates with unmodified H3 tail or with H3K9me3 modification were methylated more efficiently by full-length Dnmt3a and full-length Dnmt3a/3L complexes than chromatin trimethylated at H3K4. In contrast, the catalytic domain of Dnmt3a was not affected by the H3K4me3 modification. These results demonstrate that the binding of the ADD domain to H3 tails unmethylated at K4 leads to the preferential methylation of DNA bound to chromatin with this modification state. Our in vitro results recapitulate DNA methylation patterns observed in genome-wide DNA methylation studies

Analysis of the Substrate Specificity of the Dim-5 Histone Lysine Methyltransferase Using Peptide Arrays

Histone methylation is an epigenetic mark essential for gene regulation and development. We introduce peptide SPOT synthesis to study sequence specificity of the Dim-5 histone-3 lysine-9 methyltransferase. Dim-5 recognizes R8-G12 of the H3 tail with T11 and G12 being the most important specificity determinants. Exchange of H3 tail residue S10 and T11 by E strongly reduced methylation by Dim-5, suggesting that phosphorylation of S10 or T11 may regulate the activity of Dim-5. In the Dim-5/peptide structure, E227 interacts with H3R8 and D209 with H3-S10. Mutations of E227 or D209 caused predictable changes in the substrate preference, illustrating that peptide recognition of histone methyltransferases can be altered by protein design. Comparative analyses of peptide arrays with wild-type and mutant enzymes, therefore, are well suited to investigate the target specificity of protein methyltransferases and study epigenetic crosstalk

Application of DNA methyltransferases in targeted DNA methylation

DNA methylation is an essential epigenetic modification. In bacteria, it is involved in gene regulation, DNA repair, and control of cell cycle. In eukaryotes, it acts in concert with other epigenetic modifications to regulate gene expression and chromatin structure. In addition to these biological roles, DNA methyltransferases have several interesting applications in biotechnology, which are the main focus of this review, namely, (1) in vivo footprinting: as several bacterial DNA methyltransferases cannot methylate DNA bound to histone proteins, the pattern of DNA methylation after expression of DNA methyltransferases in the cell allows determining nucleosome positioning; (2) mapping the binding specificity of DNA binding proteins: after fusion of a DNA methyltransferase to a DNA-binding protein and expression of the fusion protein in a cell, the DNA methylation pattern reflects the DNA-binding specificity of the DNA-binding protein; and (3) targeted gene silencing: after fusion of a DNA methyltransferase to a suitable DNA-binding domain, DNA methylation can be directed to promoter regions of target genes. Thereby, gene expression can be switched off specifically, efficiently, and stably, which has a number of potential medical applications