Approaching the catalytic mechanism of protein lysine methyltransferases by biochemical and simulation techniques.

Protein lysine methyltransferases (PKMTs) transfer up to three methyl groups to the side chains of lysine residues in proteins and fulfill important regulatory functions by controlling protein stability, localization and protein/protein interactions. The methylation reactions are highly regulated, and aberrant methylation of proteins is associated with several types of diseases including neurologic disorders, cardiovascular diseases, and various types of cancer. This review describes novel insights into the catalytic machinery of various PKMTs achieved by the combined application of biochemical experiments and simulation approaches during the last years, focusing on clinically relevant and well-studied enzymes of this group like DOT1L, SMYD1-3, SET7/9, G9a/GLP, SETD2, SUV420H2, NSD1/2, different MLLs and EZH2. Biochemical experiments have unraveled many mechanistic features of PKMTs concerning their substrate and product specificity, processivity and the effects of somatic mutations observed in PKMTs in cancer cells. Structural data additionally provided information about the substrate recognition, enzyme-substrate complex formation, and allowed for simulations of the substrate peptide interaction and mechanism of PKMTs with atomistic resolution by molecular dynamics and hybrid quantum mechanics/molecular mechanics methods. These simulation technologies uncovered important mechanistic details of the PKMT reaction mechanism including the processes responsible for the deprotonation of the target lysine residue, essential conformational changes of the PKMT upon substrate binding, but also rationalized regulatory principles like PKMT autoinhibition. Further developments are discussed that could bring us closer to a mechanistic understanding of catalysis of this important class of enzymes in the near future. The results described here illustrate the power of the investigation of enzyme mechanisms by the combined application of biochemical experiments and simulation technologies.

Methylation of DNA Ligase 1 by G9a/GLP Recruits UHRF1 to Replicating DNA and Regulates DNA Methylation.

DNA methylation is an essential epigenetic mark in mammals that has to be re-established after each round of DNA replication. The protein UHRF1 is essential for this process; it has been proposed that the protein targets newly replicated DNA by cooperatively binding hemi-methylated DNA and H3K9me2/3, but this model leaves a number of questions unanswered. Here, we present evidence for a direct recruitment of UHRF1 by the replication machinery via DNA ligase 1 (LIG1). A histone H3K9-like mimic within LIG1 is methylated by G9a and GLP and, compared with H3K9me2/3, more avidly binds UHRF1. Interaction with methylated LIG1 promotes the recruitment of UHRF1 to DNA replication sites and is required for DNA methylation maintenance. These results further elucidate the function of UHRF1, identify a non-histone target of G9a and GLP, and provide an example of a histone mimic that coordinates DNA replication and DNA methylation maintenance.

On the accuracy of the epigenetic copy machine: comprehensive specificity analysis of the DNMT1 DNA methyltransferase.

The specificity of DNMT1 for hemimethylated DNA is a central feature for the inheritance of DNA methylation. We investigated this property in competitive methylation kinetics using hemimethylated (HM), hemihydroxymethylated (OH) and unmethylated (UM) substrates with single CpG sites in a randomized sequence context. DNMT1 shows a strong flanking sequence dependent HM/UM specificity of 80-fold on average, which is slightly enhanced on long hemimethylated DNA substrates. To explain this strong effect of a single methyl group, we propose a novel model in which the presence of the 5mC methyl group changes the conformation of the DNMT1-DNA complex into an active conformation by steric repulsion. The HM/OH preference is flanking sequence dependent and on average only 13-fold, indicating that passive DNA demethylation by 5hmC generation is not efficient in many flanking contexts. The CXXC domain of DNMT1 has a moderate flanking sequence dependent contribution to HM/UM specificity during DNA association to DNMT1, but not if DNMT1 methylates long DNA molecules in processive methylation mode. Comparison of genomic methylation patterns from mouse ES cell lines with various deletions of DNMTs and TETs with our data revealed that the UM specificity profile is most related to cellular methylation patterns, indicating that de novo methylation activity of DNMT1 shapes the DNA methylome in these cells.

The T1150A cancer mutant of the protein lysine dimethyltransferase NSD2 can introduce H3K36 trimethylation.

Protein lysine methyltransferases (PKMTs) play essential roles in gene expression regulation and cancer development. Somatic mutations in PKMTs are frequently observed in cancer cells. In biochemical experiments, we show here that the NSD1 mutations Y1971C, R2017Q, and R2017L observed mostly in solid cancers are catalytically inactive suggesting that NSD1 acts as a tumor suppressor gene in these tumors. In contrast, the frequently observed T1150A in NSD2 and its T2029A counterpart in NSD1, both observed in leukemia, are hyperactive and introduce up to three methyl groups in H3K36 in biochemical and cellular assays, while wildtype NSD2 and NSD1 only introduce up to two methyl groups. In Molecular Dynamics simulations, we determined key mechanistic and structural features controlling the product specificity of this class of enzymes. Simulations with NSD2 revealed that H3K36me3 formation is possible due to an enlarged active site pocket of T1150A and loss of direct contacts of T1150 to critical residues which regulate the product specificity of NSD2. Bioinformatic analyses of published data suggested that the generation of H3K36me3 by NSD2 T1150A could alter gene regulation by antagonizing H3K27me3 finally leading to the upregulation of oncogenes.

Methylation of the transcription factor E2F1 by SETD6 regulates SETD6 expression via a positive feedback mechanism.

The protein lysine methyltransferase SET domain-containing protein 6 (SETD6) has been shown to influence different cellular activities and to be critically involved in the regulation of diverse developmental and pathological processes. However, the upstream signals that regulate the mRNA expression of SETD6 are not known. Bioinformatic analysis revealed that the SETD6 promoter has a binding site for the transcription factor E2F1. Using various experimental approaches, we show that E2F1 binds to the SETD6 promoter and regulates SETD6 mRNA expression. Our further observation that this phenomenon is SETD6 dependent suggested that SETD6 and E2F1 are linked. We next demonstrate that SETD6 monomethylates E2F1 specifically at K117 in vitro and in cells. Finally, we show that E2F1 methylation at K117 positively regulates the expression level of SETD6 mRNA. Depletion of SETD6 or overexpression of E2F1 K117R mutant, which cannot be methylated by SETD6, reverses the effect. Taken together, our data provide evidence for a positive feedback mechanism, which regulates the expression of SETD6 by E2F1 in a SETD6 methylation-dependent manner, and highlight the importance of protein lysine methyltransferases and lysine methylation signaling in the regulation of gene transcription.

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.

Genome-wide investigation of the dynamic changes of epigenome modifications after global DNA methylation editing.

Chromatin properties are regulated by complex networks of epigenome modifications. Currently, it is unclear how these modifications interact and if they control downstream effects such as gene expression. We employed promiscuous chromatin binding of a zinc finger fused catalytic domain of DNMT3A to introduce DNA methylation in HEK293 cells at many CpG islands (CGIs) and systematically investigated the dynamics of the introduced DNA methylation and the consequent changes of the epigenome network. We observed efficient methylation at thousands of CGIs, but it was unstable at about 90% of them, highlighting the power of genome-wide molecular processes that protect CGIs against DNA methylation. Partially stable methylation was observed at about 1000 CGIs, which showed enrichment in H3K27me3. Globally, the introduced DNA methylation strongly correlated with a decrease in gene expression indicating a direct effect. Similarly, global but transient reductions in H3K4me3 and H3K27ac were observed after DNA methylation but no changes were found for H3K9me3 and H3K36me3. Our data provide a global and time-resolved view on the network of epigenome modifications, their connections with DNA methylation and the responses triggered by artificial DNA methylation revealing a direct repressive effect of DNA methylation in CGIs on H3K4me3, histone acetylation, and gene expression.

Structural and biochemical insight into the mechanism of dual CpG site binding and methylation by the DNMT3A DNA methyltransferase.

DNMT3A/3L heterotetramers contain two active centers binding CpG sites at 12 bp distance, however their interaction with DNA not containing this feature is unclear. Using randomized substrates, we observed preferential co-methylation of CpG sites with 6, 9 and 12 bp spacing by DNMT3A and DNMT3A/3L. Co-methylation was favored by AT bases between the 12 bp spaced CpG sites consistent with their increased bending flexibility. SFM analyses of DNMT3A/3L complexes bound to CpG sites with 12 bp spacing revealed either single heterotetramers inducing 40° DNA bending as observed in the X-ray structure, or two heterotetramers bound side-by-side to the DNA yielding 80° bending. SFM data of DNMT3A/3L bound to CpG sites spaced by 6 and 9 bp revealed binding of two heterotetramers and 100° DNA bending. Modeling showed that for 6 bp distance between CpG sites, two DNMT3A/3L heterotetramers could bind side-by-side on the DNA similarly as for 12 bp distance, but with each CpG bound by a different heterotetramer. For 9 bp spacing our model invokes a tetramer swap of the bound DNA. These additional DNA interaction modes explain how DNMT3A and DNMT3A/3L overcome their structural preference for CpG sites with 12 bp spacing during the methylation of natural DNA.

A functional LSD1 coregulator screen reveals a novel transcriptional regulatory cascade connecting R-loop homeostasis with epigenetic regulation.

The lysine specific demethylase 1 (LSD1) plays a pivotal role in cellular differentiation by regulating the expression of key developmental genes in concert with different coregulatory proteins. This process is impaired in different cancer types and incompletely understood. To comprehensively identify functional coregulators of LSD1, we established a novel tractable fluorescent reporter system to monitor LSD1 activity in living cells. Combining this reporter system with a state-of-the-art multiplexed RNAi screen, we identify the DEAD-box helicase 19A (DDX19A) as a novel coregulator and demonstrate that suppression of Ddx19a results in an increase of R-loops and reduced LSD1-mediated gene silencing. We further show that DDX19A binds to tri-methylated lysine 27 of histone 3 (H3K27me3) and it regulates gene expression through the removal of transcription promoting R-loops. Our results uncover a novel transcriptional regulatory cascade where the downregulation of genes is dependent on the LSD1 mediated demethylation of histone H3 lysine 4 (H3K4). This allows the polycomb repressive complex 2 (PRC2) to methylate H3K27, which serves as a binding site for DDX19A. Finally, the binding of DDX19A leads to the efficient removal of R-loops at active promoters, which further de-represses LSD1 and PRC2, establishing a positive feedback loop leading to a robust repression of the target gene.

Recognition of nonproline N-terminal residues by the Pro/N-degron pathway.

Eukaryotic N-degron pathways are proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal (Nt) degradation signals called N-degrons, and to target these proteins for degradation by the 26S proteasome or autophagy. GID4, a subunit of the GID ubiquitin ligase, is the main recognition component of the proline (Pro)/N-degron pathway. GID4 targets proteins through their Nt-Pro residue or a Pro at position 2, in the presence of specific downstream sequence motifs. Here we show that human GID4 can also recognize hydrophobic Nt-residues other than Pro. One example is the sequence Nt-IGLW, bearing Nt-Ile. Nt-IGLW binds to wild-type human GID4 with a Kd of 16 µM, whereas the otherwise identical Nt-Pro-bearing sequence PGLW binds to GID4 more tightly, with a Kd of 1.9 µM. Despite this difference in affinities of GID4 for Nt-IGLW vs. Nt-PGLW, we found that the GID4-mediated Pro/N-degron pathway of the yeast Saccharomyces cerevisiae can target an Nt-IGLW-bearing protein for rapid degradation. We solved crystal structures of human GID4 bound to a peptide bearing Nt-Ile or Nt-Val. We also altered specific residues of human GID4 and measured the affinities of resulting mutant GID4s for Nt-IGLW and Nt-PGLW, thereby determining relative contributions of specific GID4 residues to the GID4-mediated recognition of Nt-Pro vs. Nt-residues other than Pro. These and related results advance the understanding of targeting by the Pro/N-degron pathway and greatly expand the substrate recognition range of the GID ubiquitin ligase in both human and yeast cells.

A loss-of-function variant in SUV39H2 identified in autism-spectrum disorder causes altered H3K9 trimethylation and dysregulation of protocadherin ß-cluster genes in the developing brain.

Recent evidence has documented the potential roles of histone-modifying enzymes in autism-spectrum disorder (ASD). Aberrant histone H3 lysine 9 (H3K9) dimethylation resulting from genetic variants in histone methyltransferases is known for neurodevelopmental and behavioral anomalies. However, a systematic examination of H3K9 methylation dynamics in ASD is lacking. Here we resequenced nine genes for histone methyltransferases and demethylases involved in H3K9 methylation in individuals with ASD and healthy controls using targeted next-generation sequencing. We identified a novel rare variant (A211S) in the SUV39H2, which was predicted to be deleterious. The variant showed strongly reduced histone methyltransferase activity in vitro. In silico analysis showed that the variant destabilizes the hydrophobic core and allosterically affects the enzyme activity. The Suv39h2-KO mice displayed hyperactivity and reduced behavioral flexibility in learning the tasks that required complex behavioral adaptation, which is relevant for ASD. The Suv39h2 deficit evoked an elevated expression of a subset of protocadherin ß (Pcdhb) cluster genes in the embryonic brain, which is attributable to the loss of H3K9 trimethylation (me3) at the gene promoters. Reduced H3K9me3 persisted in the cerebellum of Suv39h2-deficient mice to an adult stage. Congruently, reduced expression of SUV39H1 and SUV39H2 in the postmortem brain samples of ASD individuals was observed, underscoring the role of H3K9me3 deficiency in ASD etiology. The present study provides direct evidence for the role of SUV39H2 in ASD and suggests a molecular cascade of SUV39H2 dysfunction leading to H3K9me3 deficiency followed by an untimely, elevated expression of Pcdhb cluster genes during early neurodevelopment.

Complex DNA sequence readout mechanisms of the DNMT3B DNA methyltransferase.

DNA methyltransferases interact with their CpG target sites in the context of variable flanking sequences. We investigated DNA methylation by the human DNMT3B catalytic domain using substrate pools containing CpX target sites in randomized flanking context and identified combined effects of CpG recognition and flanking sequence interaction together with complex contact networks involved in balancing the interaction with different flanking sites. DNA methylation rates were more affected by flanking sequences at non-CpG than at CpG sites. We show that T775 has an essential dynamic role in the catalytic mechanism of DNMT3B. Moreover, we identify six amino acid residues in the DNA-binding interface of DNMT3B (N652, N656, N658, K777, N779, and R823), which are involved in the equalization of methylation rates of CpG sites in favored and disfavored sequence contexts by forming compensatory interactions to the flanking residues including a CpG specific contact to an A at the +1 flanking site. Non-CpG flanking preferences of DNMT3B are highly correlated with non-CpG methylation patterns in human cells. Comparison of the flanking sequence preferences of human and mouse DNMT3B revealed subtle differences suggesting a co-evolution of flanking sequence preferences and cellular DNMT targets.

Mechanisms and Biological Roles of DNA Methyltransferases and DNA Methylation: From Past Achievements to Future Challenges.

DNA methylation and DNA methyltransferases (MTases)-the enzymes that introduce the methylation mark into the DNA-have been studied for almost 70 years. In this chapter, we review the key developments in the DNA methylation field that have led to our current understanding of the structures and mechanisms of DNA MTases. We discuss the essential biological roles of DNA methylation, including the discovery of DNA methylation, cloning and sequence analysis of the bacterial and eukaryotic MTases, and the elucidation of their structure, mechanism, regulation, and molecular evolution. We describe genetic studies that contributed greatly to the evolving views on the role of DNA methylation in development and diseases, the invention of methods for the genome-wide analysis of DNA methylation, and the biochemical identification of DNA MTases and the TET enzyme family, which is involved in DNA demethylation. We summarize the roles of MTases in bacterial epigenetics and the application of MTases in synthetic biology to generate artificial signaling systems. We finish by highlighting some open questions for the next years of research in the field.

Enzymology of Mammalian DNA Methyltransferases.

DNA methylation is a hot topic in basic and biomedical research. Despite tremendous progress in understanding the structures and biochemical properties of the mammalian DNA methyltransferases (DNMTs), principles of their targeting and 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 containing a catalytic C-terminal domain and a complex N-terminal part with diverse targeting and regulatory functions. The sub-nuclear localization of DNMTs plays an important role in their biological function: DNMT1 is localized to replicating DNA and heterochromatin via interactions with PCNA and UHRF1 and direct binding to the heterochromatic histone modifications H3K9me3 and H4K20me3. DNMT3 enzymes bind to heterochromatin via protein multimerization and are targeted to chromatin by their ADD, PWWP, and UDR domains, binding to unmodified H3K4, H3K36me2/3, and H2AK119ub1, respectively. In recent years, a novel regulatory principle has been discovered in DNMTs, as structural and functional data demonstrated that the catalytic activities of DNMT enzymes are under a tight allosteric control by their different N-terminal domains with autoinhibitory functions. This mechanism provides numerous possibilities for the precise regulation of the methyltransferases via controlling the binding and release of the autoinhibitory domains by protein partners, chromatin interactions, non-coding RNAs, or posttranslational modifications of the DNMTs. In this chapter, we summarize key enzymatic properties of DNMTs, viz. their specificity and processivity, and afterwards focus on the regulation of their activity and targeting via allosteric processes, protein interactions, and posttranslational modifications.

PWWP-DOMAIN INTERACTOR OF POLYCOMBS1 Interacts with Polycomb-Group Proteins and Histones and Regulates Arabidopsis Flowering and Development.

Polycomb-group (PcG) proteins mediate epigenetic gene regulation by setting H3K27me3 via Polycomb Repressive Complex 2 (PRC2). In plants, it is largely unclear how PcG proteins are recruited to their target genes. Here, we identified the PWWP-DOMAIN INTERACTOR OF POLYCOMBS1 (PWO1) protein, which interacts with all three Arabidopsis thaliana PRC2 histone methyltransferases and is required for maintaining full H3 occupancy at several Arabidopsis genes. PWO1 localizes and recruits CURLY LEAF to nuclear speckles in Nicotiana benthamiana nuclei, suggesting a role in spatial organization of PcG regulation. PWO1 belongs to a gene family with three members having overlapping activities: pwo1 pwo2 pwo3 triple mutants are seedling lethal and show shoot and root meristem arrest, while pwo1 single mutants are early flowering. Interestingly, the PWWP domain of PWO1 confers binding to histones, which is reduced by a point mutation in a highly conserved residue of this domain and blocked by phosphorylation of H3S28. PWO1 carrying this mutation is not able to fully complement the pwo1 pwo2 pwo3 triple mutant, indicating the requirement of this domain for PWO1 in vivo activity. Thus, the PWO family may present a novel class of histone readers that are involved in recruiting PcG proteins to subnuclear domains and in promoting Arabidopsis development.

Mutations of R882 change flanking sequence preferences of the DNA methyltransferase DNMT3A and cellular methylation patterns.

Somatic DNMT3A mutations at R882 are frequently observed in AML patients including the very abundant R882H, but also R882C, R882P and R882S. Using deep enzymology, we show here that DNMT3A-R882H has more than 70-fold altered flanking sequence preferences when compared with wildtype DNMT3A. The R882H flanking sequence preferences mainly differ on the 3' side of the CpG site, where they resemble DNMT3B, while 5' flanking sequence preferences resemble wildtype DNMT3A, indicating that R882H behaves like a DNMT3A/DNMT3B chimera. Investigation of the activity and flanking sequence preferences of other mutations of R882 revealed that they cause similar effects. Bioinformatic analyses of genomic methylation patterns focusing on flanking sequence effects after expression of wildtype DNMT3A and R882H in human cells revealed that genomic methylation patterns reflect the details of the altered flanking sequence preferences of R882H. Concordantly, R882H specific hypermethylation in AML patients was strongly correlated with the R882H flanking sequence preferences. R882H specific DNA hypermethylation events in AML patients were accompanied by R882H specific mis-regulation of several genes with strong cancer connection, which are potential downstream targets of R882H. In conclusion, our data provide novel and detailed mechanistic understanding of the pathogenic mechanism of the DNMT3A R882H somatic cancer mutation.

Analysis of the Substrate Specificity of the SMYD2 Protein Lysine Methyltransferase and Discovery of Novel Non-Histone Substrates.

The SMYD2 protein lysine methyltransferase methylates various histone and non-histone proteins and is overexpressed in several cancers. Using peptide arrays, we investigated the substrate specificity of the enzyme, revealing a recognition of leucine (or weaker phenylalanine) at the -1 peptide site and disfavor of acidic residues at the +1 to +3 sites. Using this motif, novel SMYD2 peptide substrates were identified, leading to the discovery of 32 novel peptide substrates with a validated target site. Among them, 19 were previously reported to be methylated at the target lysine in human cells, strongly suggesting that SMYD2 is the protein lysine methyltransferase responsible for this activity. Methylation of some of the novel peptide substrates was tested at the protein level, leading to the identification of 14 novel protein substrates of SMYD2, six of which were more strongly methylated than p53, the best SMYD2 substrate described so far. The novel SMYD2 substrate proteins are involved in diverse biological processes such as chromatin regulation, transcription, and intracellular signaling. The results of our study provide a fundament for future investigations into the role of this important enzyme in normal development and cancer.

The DNMT3A R882H mutant displays altered flanking sequence preferences.

The DNMT3A R882H mutation is frequently observed in acute myeloid leukemia (AML). It is located in the subunit and DNA binding interface of DNMT3A and has been reported to cause a reduction in activity and dominant negative effects. We investigated the mechanistic consequences of the R882H mutation on DNMT3A showing a roughly 40% reduction in overall DNA methylation activity. Biochemical assays demonstrated that R882H does not change DNA binding affinity, protein stability or subnuclear distribution of DNMT3A. Strikingly, DNA methylation experiments revealed pronounced changes in the flanking sequence preference of the DNMT3A-R882H mutant. Based on these results, different DNA substrates with selected flanking sequences were designed to be favored or disfavored by R882H. Kinetic analyses showed that the R882H favored substrate was methylated by R882H with 45% increased rate when compared with wildtype DNMT3A, while methylation of the disfavored substrate was reduced 7-fold. Our data expand the model of the potential carcinogenic effect of the R882H mutation by showing CpG site specific activity changes. This result suggests that R882 is involved in the indirect readout of flanking sequence preferences of DNMT3A and it may explain the particular enrichment of the R882H mutation in cancer patients by revealing mutation specific effects.

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 tail modifications 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.

Mechanistic Insights into the Allosteric Regulation of the Clr4 Protein Lysine Methyltransferase by Autoinhibition and Automethylation.

Clr4 is a histone H3 lysine 9 methyltransferase in Schizosaccharomyces pombe that is essential for heterochromatin formation. Previous biochemical and structural studies have shown that Clr4 is in an autoinhibited state in which an autoregulatory loop (ARL) blocks the active site. Automethylation of lysine residues in the ARL relieves autoinhibition. To investigate the mechanism of Clr4 regulation by autoinhibition and automethylation, we exchanged residues in the ARL by site-directed mutagenesis leading to stimulation or inhibition of automethylation and corresponding changes in Clr4 catalytic activity. Furthermore, we demonstrate that Clr4 prefers monomethylated (H3K9me1) over unmodified (H3K9me0) histone peptide substrates, similar to related human enzymes and, accordingly, H3K9me1 is more efficient in overcoming autoinhibition. Due to enzyme activation by automethylation, we observed a sigmoidal dependence of Clr4 activity on the AdoMet concentration, with stimulation at high AdoMet levels. In contrast, an automethylation-deficient mutant showed a hyperbolic Michaelis-Menten type relationship. These data suggest that automethylation of the ARL could act as a sensor for AdoMet levels in cells and regulate the generation and maintenance of heterochromatin accordingly. This process could connect epigenome modifications with the metabolic state of cells. As other human protein lysine methyltransferases (for example, PRC2) also use automethylation/autoinhibition mechanisms, our results may provide a model to describe their regulation as well.