DNA methyltransferase DNMT3A forms interaction networks with the CpG site and flanking sequence elements for efficient methylation.

Specific DNA methylation at CpG and non-CpG sites is essential for chromatin regulation. The DNA methyltransferase DNMT3A interacts with target sites surrounded by variable DNA sequences with its TRD and RD loops, but the functional necessity of these interactions is unclear. We investigated CpG and non-CpG methylation in a randomized sequence context using WT DNMT3A and several DNMT3A variants containing mutations at DNA-interacting residues. Our data revealed that the flanking sequence of target sites between the -2 and up to the +8 position modulates methylation rates >100-fold. Non-CpG methylation flanking preferences were even stronger and favor C(+1). R836 and N838 in concert mediate recognition of the CpG guanine. R836 changes its conformation in a flanking sequence-dependent manner and either contacts the CpG guanine or the +1/+2 flank, thereby coupling the interaction with both sequence elements. R836 suppresses activity at CNT sites but supports methylation of CAC substrates, the preferred target for non-CpG methylation of DNMT3A in cells. N838 helps to balance this effect and prevent the preference for C(+1) from becoming too strong. Surprisingly, we found L883 reduces DNMT3A activity despite being highly conserved in evolution. However, mutations at L883 disrupt the DNMT3A-specific DNA interactions of the RD loop, leading to altered flanking sequence preferences. Similar effects occur after the R882H mutation in cancer cells. Our data reveal that DNMT3A forms flexible and interdependent interaction networks with the CpG guanine and flanking residues that ensure recognition of the CpG and efficient methylation of the cytosine in contexts of variable flanking sequences

H3K14ac is linked to methylation of H3K9 by the triple Tudor domain of SETDB1

SETDB1 is an essential H3K9 methyltransferase involved in silencing of retroviruses and gene regulation. We show here that its triple Tudor domain (3TD) specifically binds to doubly modified histone H3 containing K14 acetylation and K9 methylation. Crystal structures of 3TD in complex with H3K14ac/K9me peptides reveal that peptide binding and K14ac recognition occurs at the interface between Tudor domains (TD) TD2 and TD3. Structural and biochemical data demonstrate a pocket switch mechanism in histone code reading, because K9me1 or K9me2 is preferentially recognized by the aromatic cage of TD3, while K9me3 selectively binds to TD2. Mutations in the K14ac/K9me binding sites change the subnuclear localization of 3TD. ChIP-seq analyses show that SETDB1 is enriched at H3K9me3 regions and K9me3/K14ac is enriched at SETDB1 binding sites overlapping with LINE elements, suggesting that recruitment of the SETDB1 complex to K14ac/K9me regions has a role in silencing of active genomic regions

Rewriting DNA Methylation Signatures at Will:The Curable Genome Within Reach?

DNA methyltransferases are important enzymes in a broad range of organisms. Dysfunction of DNA methyltransferases in humans leads to many severe diseases, including cancer. This book focuses on the biochemical properties of these enzymes, describing their structures and mechanisms in bacteria, humans and other species, including plants, and also explains the biological processes of reading of DNA methylation and DNA demethylation. It covers many emerging aspects of the biological roles of DNA methylation functioning as an essential epigenetic mark and describes the role of DNA methylation in diseases. Moreover, the book explains modern technologies, like targeted rewriting of DNA methylation by designed DNA methyltransferases, as well as technological applications of DNA methyltransferases in DNA labelling. Finally, the book summarizes recent methods for the analysis of DNA methylation in human DNA. Overall, this book represents a comprehensive state-of-the-art- work and is a must-have for advanced researchers in the field of DNA methylation and epigenetics

Enzymology of mammalian DNA methyltransferases

DNA methylation is currently one of the hottest topics in basic and biomedical research. Despite tremendous progress in understanding the structures and biochemical properties of the mammalian DNA nucleotide methyltransferases (DNMTs), principles of their regulation in cells have only begun to be uncovered. In mammals, DNA methylation is introduced by the DNMT1, DNMT3A, and DNMT3B enzymes, which are all large multi-domain proteins. These enzymes contain a catalytic C-terminal domain with a characteristic cytosine-C5 methyltransferase fold and an N-terminal part with different domains that interacts with other proteins and chromatin and is involved in targeting and regulation of the DNMTs. The subnuclear localization of the DNMT enzymes plays an important role in their biological function: DNMT1 is localized to replicating DNA via interaction with PCNA and UHRF1. DNMT3 enzymes bind to heterochromatin via protein multimerization and are targeted to chromatin by their ADD and PWWP domains. Recently, a novel regulatory mechanism has been discovered in DNMTs, as latest structural and functional data demonstrated that the catalytic activities of all three enzymes are under tight allosteric control of their N-terminal domains having autoinhibitory functions. This mechanism provides numerous possibilities for the precise regulation of the methyltransferases via controlling the binding and release of autoinhibitory domains by protein factors, noncoding RNAs, or by posttranslational modifications of the DNMTs. In this chapter, we summarize key enzymatic properties of DNMTs, including their specificity and processivity, and afterward we focus on the regulation of their activity and targeting via allosteric processes, protein interactors, and posttranslational modifications