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The jumonji domain demethylases catalyze demethylation of histones, thus affecting modifications that are associated with transcription status. Structural and functional analyses of two PHF8 jumonji orthologs now indicates that the PHD domain, conserved in this family, binds H3K4me3, a modification associated with transcriptional activation, thus directing and augmenting the target of jumonji-mediated demethylation. Combinatorial readout of multiple covalent histone modifications is poorly understood. We provide insights into how an activating histone mark, in combination with linked repressive marks, is differentially 'read' by two related human demethylases, PHF8 and KIAA1718 (also known as JHDM1D). Both enzymes harbor a plant homeodomain (PHD) that binds Lys4-trimethylated histone 3 (H3K4me3) and a jumonji domain that demethylates either H3K9me2 or H3K27me2. The presence of H3K4me3 on the same peptide as H3K9me2 makes the doubly methylated peptide a markedly better substrate of PHF8, whereas the presence of H3K4me3 has the opposite effect, diminishing the H3K9me2 demethylase activity of KIAA1718 without adversely affecting its H3K27me2 activity. The difference in substrate specificity between the two is explained by PHF8 adopting a bent conformation, allowing each of its domains to engage its respective target, whereas KIAA1718 adopts an extended conformation, which prevents its access to H3K9me2 by its jumonji domain when its PHD engages H3K4me3.

Clostridioides difficile infections are an urgent medical problem. The newly discovered C. difficile a denine m ethyltransferase A (CamA) is specified by all C. difficile genomes sequenced to date (>300), but is rare among other bacteria. CamA is an orphan methyltransferase, unassociated with a restriction endonuclease. CamA-mediated methylation at CAAAA A is required for normal sporulation, biofilm formation, and intestinal colonization by C. difficile . We characterized CamA kinetic parameters, and determined its structure bound to DNA containing the recognition sequence. CamA contains an N-terminal domain for catalyzing methyl transfer, and a C-terminal DNA recognition domain. Major and minor groove DNA contacts in the recognition site involve base-specific hydrogen bonds, van der Waals contacts and the Watson-Crick pairing of a rearranged A:T base pair. These provide sufficient sequence discrimination to ensure high specificity. Finally, the surprisingly weak binding of the methyl donor S -adenosyl- l -methionine (SAM) might provide avenues for inhibiting CamA activity using SAM analogs. Clostridioides difficile adenine methyltransferase A (CamA) is required for the sporulation and colonization of the pathogen that causes gastrointestinal infections. Here, the authors characterise CamA kinetically and present its crystal structure bound to the DNA recognition sequence, which reveals DNA distortions including bending and the flipping of the target adenine out of the DNA helix, as well as protein conformational changes upon cofactor binding.

BCL11A, a transcription factor, is vital for hematopoiesis, including B and T cell maturation and the fetal-to-adult hemoglobin switch. Mutations in BCL11A are linked to neurodevelopmental disorders. BCL11A contains two DNA-binding zinc-finger arrays, low-affinity ZF2-3 and high-affinity ZF4-6, separated by a 300-amino-acid linker. ZF2-3 and ZF4-5 share 73% identity, including five out of six DNA base-interacting residues. These arrays bind similar short sequence motifs in clusters, with the linker enabling a broader binding span. Crystallographic structures of ZF4-6, in complex with oligonucleotides from the β-globin locus region, reveal DNA sequence recognition by residues Asn756 (ZF4), Lys784 and Arg787 (ZF5). A Lys784-to-Thr mutation, linked to a neurodevelopmental disorder with persistent fetal globin expression, reduces DNA binding over 10-fold but gains interaction with a variable base pair. BCL11A isoforms may form oligomers, enhancing chromatin occupancy and repressor functions by allowing multiple copies of both low- and high-affinity ZF arrays to bind DNA. These distinctive properties, apparently conserved among vertebrates, provide essential functional flexibility to this crucial regulator. BCL11A, key to hematopoiesis and the hemoglobin switch, binds DNA as an oligomer. Here, the authors show its two zinc-finger arrays, separated by 300 amino acids, enhance chromatin occupancy and repression by targeting clustered short sequence motifs.

Keeping DNA methylation on track DNA methylation is a key epigenetic process and the faithful maintenance of DNA methylation patterns is essential to the wellbeing of mammalian cells. This means that cells need a mechanism to identify the partially methylated version of CpG once a new DNA strand has been replicated or repaired, so that it can be further methylated by the DNA methyltransferase, DNMT1. As part of this process the protein UHRF1 (or Np95/ICBP90) facilitates the loading of DNMT1 onto the hemimethylated CpG sequences during DNA replication. Three papers in this issue describe crystal structures of the SRA domain of UHRF1 bound to DNA containing a hemi-methylated CpG site. The structures show that methyl-cytosine is flipped out of the DNA helix and inserted into a binding pocket on the SRA domain. Maintenance methylation of hemimethylated CpG dinucleotides at DNA replication forks is the key to faithful mitotic inheritance of genomic methylation patterns. UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is required for maintenance methylation by interacting with DNA nucleotide methyltransferase 1 (DNMT1), the maintenance methyltransferase, and with hemimethylated CpG, the substrate for DNMT1 (refs 1 and 2 ). Here we present the crystal structure of the SET and RING-associated (SRA) domain of mouse UHRF1 in complex with DNA containing a hemimethylated CpG site. The DNA is contacted in both the major and minor grooves by two loops that penetrate into the middle of the DNA helix. The 5-methylcytosine has flipped completely out of the DNA helix and is positioned in a binding pocket with planar stacking contacts, Watson–Crick polar hydrogen bonds and van der Waals interactions specific for 5-methylcytosine. Hence, UHRF1 contains a previously unknown DNA-binding module and is the first example of a non-enzymatic, sequence-specific DNA-binding protein domain to use the base flipping mechanism to interact with DNA.

SETD3 is an actin histidine-N 3 methyltransferase, whereas other characterized SET-domain enzymes are protein lysine methyltransferases. We report that in a pre-reactive complex SETD3 binds the N 3 -protonated form (N 3 -H) of actin His73, and in a post-reactive product complex, SETD3 generates the methylated histidine in an N 1 -protonated (N 1 -H) and N 3 -methylated form. During the reaction, the imidazole ring of His73 rotates ~105°, which shifts the proton from N 3 to N 1 , thus ensuring that the target atom N 3 is deprotonated prior to the methyl transfer. Under the conditions optimized for lysine deprotonation, SETD3 has weak lysine methylation activity on an actin peptide in which the target His73 is substituted by a lysine. The structure of SETD3 with Lys73-containing peptide reveals a bent conformation of Lys73, with its side chain aliphatic carbons tracing along the edge of imidazole ring and the terminal ε-amino group occupying a position nearly identical to the N 3 atom of unmethylated histidine. SETD3 is the first known metazoan protein histidine methyltransferase but the molecular basis for its target specificity is unclear. Here, the authors elucidate the structural and molecular determinants for the histidine specificity of SETD3.

The Caulobacter crescentus cell cycle-regulated DNA methyltransferase (CcrM) methylates the adenine of hemimethylated GANTC after replication. Here we present the structure of CcrM in complex with double-stranded DNA containing the recognition sequence. CcrM contains an N-terminal methyltransferase domain and a C-terminal nonspecific DNA-binding domain. CcrM is a dimer, with each monomer contacting primarily one DNA strand: the methyltransferase domain of one molecule binds the target strand, recognizes the target sequence, and catalyzes methyl transfer, while the C-terminal domain of the second molecule binds the non-target strand. The DNA contacts at the 5-base pair recognition site results in dramatic DNA distortions including bending, unwinding and base flipping. The two DNA strands are pulled apart, creating a bubble comprising four recognized base pairs. The five bases of the target strand are recognized meticulously by stacking contacts, van der Waals interactions and specific Watson–Crick polar hydrogen bonds to ensure high enzymatic specificity. CcrM is a cell cycle-regulated DNA methyltransferase that methylates an adenine within a specific sequence following replication in the gram negative bacterium Caulobacter crescentus . Here the authors present a crystal structure of DNA-bound CcrM that reveals the molecular mechanism leading to sequence-specific methylation.

DNA methyltransferase-1 (DNMT1) is involved in CpG methylation, and its stability is known to be regulated by lysine methylation. Now it has been shown that methylation of DNMT1 and AKT1 phosphorylation of DNMT1 are mutually exclusive and that antagonism between these modifications affects DNMT1 turnover. The protein lysine methyltransferase SET7 regulates DNA methyltransferase-1 (DNMT1) activity in mammalian cells by promoting degradation of DNMT1 and thus allows epigenetic changes via DNA demethylation. Here we reveal an interplay between monomethylation of DNMT1 Lys142 by SET7 and phosphorylation of DNMT1 Ser143 by AKT1 kinase. These two modifications are mutually exclusive, and structural analysis suggests that Ser143 phosphorylation interferes with Lys142 monomethylation. AKT1 kinase colocalizes and directly interacts with DNMT1 and phosphorylates Ser143. Phosphorylated DNMT1 peaks during DNA synthesis, before DNMT1 methylation. Depletion of AKT1 or overexpression of dominant-negative AKT1 increases methylated DNMT1, resulting in a decrease in DNMT1 abundance. In mammalian cells, phosphorylated DNMT1 is more stable than methylated DNMT1. These results reveal cross-talk on DNMT1, through modifications mediated by AKT1 and SET7, that affects cellular DNMT1 levels.

BHC80 is a component of the LSD1 co-repressor complex that demethylates histone H3 at lysine 4. The PHD domain of BHC80 interacts with the histone H3 tail only when lysine 4 is unmethylated, and BHC80 function is coupled to that of LSD1 in gene repression. Histone methylation is crucial for regulating chromatin structure, gene transcription and the epigenetic state of the cell. LSD1 is a lysine-specific histone demethylase that represses transcription by demethylating histone H3 on lysine 4 (ref. 1 ). The LSD1 complex contains a number of proteins, all of which have been assigned roles in events upstream of LSD1-mediated demethylation 2 , 3 , 4 apart from BHC80 (also known as PHF21A), a plant homeodomain (PHD) finger-containing protein. Here we report that, in contrast to the PHD fingers of the bromodomain PHD finger transcription factor (BPTF) and inhibitor of growth family 2 (ING2), which bind methylated H3K4 (H3K4me3) 5 , 6 , the PHD finger of BHC80 binds unmethylated H3K4 (H3K4me0), and this interaction is specifically abrogated by methylation of H3K4. The crystal structure of the PHD finger of BHC80 bound to an unmodified H3 peptide has revealed the structural basis of the recognition of H3K4me0. Knockdown of BHC80 by RNA inhibition results in the de-repression of LSD1 target genes, and this repression is restored by the reintroduction of wild-type BHC80 but not by a PHD-finger mutant that cannot bind H3. Chromatin immunoprecipitation showed that BHC80 and LSD1 depend reciprocally on one another to associate with chromatin. These findings couple the function of BHC80 to that of LSD1, and indicate that unmodified H3K4 is part of the ‘histone code’ 7 . They further raise the possibility that the generation and recognition of the unmodified state on histone tails in general might be just as crucial as post-translational modifications of histone for chromatin and transcriptional regulation.

The G9a-like lysine methyltransferases can be inhibited by the small molecule BIX-01294, recently identified through a chemical screen and shown to be capable of replacing Oct3/4. The structure of GLP in complex with BIX-01294 indicates an overlap with the known position of histone peptide binding, and further work indicates that the drug inhibits methylation of DNMT1, indicating that it is enzyme specific but non specific with regard to substrate. Histone lysine methylation is an important epigenetic mark that regulates gene expression and chromatin organization. G9a and G9a-like protein (GLP) are euchromatin-associated methyltransferases that repress transcription by methylating histone H3 Lys9. BIX-01294 was originally identified as a G9a inhibitor during a chemical library screen of small molecules and has previously been used in the generation of induced pluripotent stem cells. Here we present the crystal structure of the catalytic SET domain of GLP in complex with BIX-01294 and S -adenosyl- L -homocysteine. The inhibitor is bound in the substrate peptide groove at the location where the histone H3 residues N-terminal to the target lysine lie in the previously solved structure of the complex with histone peptide. The inhibitor resembles the bound conformation of histone H3 Lys4 to Arg8, and is positioned in place by residues specific for G9a and GLP through specific interactions.

DNA CpG methylation and histone H3 lysine 9 (H3K9) methylation are two major repressive epigenetic modifications, and these methylations are positively correlated with one another in chromatin. Here we show that G9a or G9a-like protein (GLP) dimethylate the amino-terminal lysine 44 (K44) of mouse Dnmt3a (equivalent to K47 of human DNMT3A) in vitro and in cells overexpressing G9a or GLP. The chromodomain of MPP8 recognizes the dimethylated Dnmt3aK44me2. MPP8 also interacts with self-methylated GLP in a methylation-dependent manner. The MPP8 chromodomain forms a dimer in solution and in crystals, suggesting that a dimeric MPP8 molecule could bridge the methylated Dnmt3a and GLP, resulting in a silencing complex of Dnmt3a–MPP8–GLP/G9a on chromatin templates. Together, these findings provide a molecular explanation, at least in part, for the co-occurrence of DNA methylation and H3K9 methylation in chromatin. The methylation of DNA and histone H3 lysine 9 in chromatin are positively correlated. This study shows that the DNA methyl transferase Dnmt3a is methylated, and a crystal structure of Dnmt3a bound to the chromodomain protein MPP8 suggests a molecular mechanism.