Prion protein region 23–32 interacts with tubulin and inhibits microtubule assembly

In previous studies we have demonstrated that prion protein (PrP) binds directly to tubulin and this interaction leads to the inhibition of microtubule formation by inducement of tubulin oligomerization. This report is aimed at mapping the regions of PrP and tubulin involved in the interaction and identification of PrP domains responsible for tubulin oligomerization. Preliminary studies focused our attention to the N‐terminal flexible part of PrP encompassing residues 23–110. Using a panel of deletion mutants of PrP, we identified two microtubule‐binding motifs at both ends of this part of the molecule. We found that residues 23–32 constitute a major site of interaction, whereas residues 101–110 represent a weak binding site. The crucial role of the 23–32 sequence in the interaction with tubulin was confirmed employing chymotryptic fragments of PrP. Surprisingly, the octarepeat region linking the above motifs plays only a supporting role in the interaction. The binding of Cu²⁺ to PrP did not affect the interaction. We also demonstrate that PrP deletion mutants lacking residues 23–32 exhibit very low efficiency in the inducement of tubulin oligomerization. Moreover, a synthetic peptide corresponding to this sequence, but not that identical with fragment 101–110, mimics the effects of the full‐length protein on tubulin oligomerization and microtubule assembly. At the cellular level, peptide composed of the PrP motive 23–30 and signal sequence (1–22) disrupted the microtubular cytoskeleton. Using tryptic and chymotryptic fragments of α‐ and β‐tubulin, we mapped the docking sites for PrP within the C‐terminal domains constituting the outer surface of microtubule. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Accumulation and post‐translational modifications of plant tubulins

The microtubular cytoskeleton of plant cells provides support for several functions (including the anchoring of proteins, assembly of the mitotic spindle, cytoplasmic streaming and construction of cell walls). Both α‐ and β‐tubulins are encoded through multigene families that are differentially expressed in different organs and tissues. To increase the variability of expression, both protein subunits are subjected to post‐translational modifications, which could contribute to the assembly of specific microtubule structures. This review aims to highlight the role of specific post‐translational modifications of tubulin in plant cells. We initially describe the expression and accumulation of α‐ and β‐tubulin isoforms in different plants and at different stages of plant development. Second, we discuss the different types of post‐translational modifications that, by adding or removing specific functional groups, increase the isoform heterogeneity and functional variability of tubulin. Modifications are proposed to form a ‘code’ that can be read by proteins interacting with microtubules. Therefore, the subpopulations of microtubules may bind to different associated proteins (motor and non‐motor), thus creating the physical support for various microtubule functions.     

Distinct roles of α‐ and β‐tubulin polyglutamylation in controlling axonal transport and in neurodegeneration

Tubulin polyglutamylation is a post‐translational modification of the microtubule cytoskeleton, which is generated by a variety of enzymes with different specificities. The “tubulin code” hypothesis predicts that modifications generated by specific enzymes selectively control microtubule functions. Our recent finding that excessive accumulation of polyglutamylation in neurons causes their degeneration and perturbs axonal transport provides an opportunity for testing this hypothesis. By developing novel mouse models and a new glutamylation‐specific antibody, we demonstrate here that the glutamylases TTLL1 and TTLL7 generate unique and distinct glutamylation patterns on neuronal microtubules. We find that under physiological conditions, TTLL1 polyglutamylates α‐tubulin, while TTLL7 modifies β‐tubulin. TTLL1, but not TTLL7, catalyses the excessive hyperglutamylation found in mice lacking the deglutamylase CCP1. Consequently, deletion of TTLL1, but not of TTLL7, prevents degeneration of Purkinje cells and of myelinated axons in peripheral nerves in these mice. Moreover, loss of TTLL1 leads to increased mitochondria motility in neurons, while loss of TTLL7 has no such effect. By revealing how specific patterns of tubulin glutamylation, generated by distinct enzymes, translate into specific physiological and pathological readouts, we demonstrate the relevance of the tubulin code for homeostasis.     

Katanin Severing and Binding Microtubules Are Inhibited by Tubulin Carboxy Tails

Microtubule dynamics in cells are regulated by associated proteins that can be either stabilizers or destabilizers. A class of destabilizers that is important in a large number of cellular activities is the microtubule-severing enzymes, yet little is known about how they function. Katanin p60 was the first ATPase associated with microtubule severing. Here, we investigate the activity of katanin severing using a GFP-labeled human version. We quantify the effect of katanin concentration on katanin binding and severing activity. We find that free tubulin can inhibit severing activity by interfering with katanin binding to microtubules. The inhibition is mediated by the sequence of the tubulin and specifically depends on the carboxy-terminal tails. We directly investigate the inhibition effect of tubulin carboxy-terminal tails using peptide sequences of α-, β-, or detyrosinated α-tubulin tails that have been covalently linked to bovine serum albumin. Our results show that β-tubulin tails are the most effective at inhibiting severing, and that detyrosinated α-tubulin tails are the least effective. These results are distinct from those for other severing enzymes and suggest a scheme for regulation of katanin activity in cells dependent on free tubulin concentration and the modification state of the tubulin.     

Polyglutamylation: a fine‐regulator of protein function?

Polyglutamylation is a post-translational modification in which glutamate side chains of variable lengths are formed on the modified protein. It is evolutionarily conserved from protists to mammals and its most prominent substrate is tubulin, the microtubule (MT) building block. Various polyglutamylation states of MTs can be distinguished within a single cell and they are also characteristic of specific cell types or organelles. Polyglutamylation has been proposed to be involved in the functional adaptation of MTs, as it occurs within the carboxy-terminal tubulin tails that participate directly in the binding of many structural and motor MT-associated proteins. The discovery of a new family of enzymes that catalyse this modification has brought new insight into the mechanism of polyglutamylation and now allows for direct functional studies of the role of tubulin polyglutamylation. Moreover, the recent identification of new substrates of polyglutamylation indicates that this post-translational modification could be a potential regulator of diverse cellular processes.     

Neuroligin‐1 performs neurexin‐dependent and neurexin‐independent functions in synapse validation

Postsynaptic neuroligins are thought to perform essential functions in synapse validation and synaptic transmission by binding to, and dimerizing, presynaptic α‐ and β‐neurexins. To test this hypothesis, we examined the functional effects of neuroligin‐1 mutations that impair only α‐neurexin binding, block both α‐ and β‐neurexin binding, or abolish neuroligin‐1 dimerization. Abolishing α‐neurexin binding abrogated neuroligin‐induced generation of neuronal synapses onto transfected non‐neuronal cells in the so‐called artificial synapse‐formation assay, even though β‐neurexin binding was retained. Thus, in this assay, neuroligin‐1 induces apparent synapse formation by binding to presynaptic α‐neurexins. In transfected neurons, however, neither α‐ nor β‐neurexin binding was essential for the ability of postsynaptic neuroligin‐1 to dramatically increase synapse density, suggesting a neurexin‐independent mechanism of synapse formation. Moreover, neuroligin‐1 dimerization was not required for either the non‐neuronal or the neuronal synapse‐formation assay. Nevertheless, both α‐neurexin binding and neuroligin‐1 dimerization were essential for the increase in apparent synapse size that is induced by neuroligin‐1 in transfected neurons. Thus, neuroligin‐1 performs diverse synaptic functions by mechanisms that include as essential components of α‐neurexin binding and neuroligin dimerization, but extend beyond these activities.     

A Single Amino-Acid Substitution at Lysine 40 of an Arabidopsis thaliana α-tubulin Causes Extensive Cell Proliferation and Expansion Defects

Microtubules are highly dynamic cytoskeletal polymers of α/β‐tubulin heterodimers that undergo multiple post‐translational modifications essential for various cellular functions in eukaryotes. The lysine 40 (K40) is largely conserved in α‐tubulins in many eukaryote species, and the post‐translational modification by acetylation at K40 is critical for neuronal development in vertebrates. However, the biological function of K40 of α‐tubulins in plants remains unexplored. In this study, we show in Arabidopsis thaliana that constitutive expression of mutated forms of α‐tubulin6 (TUA6) at K40 (TUA6^K40A or TUA6^K40Q), in which K40 is replaced by alanine or glutamine, result in severely reduced plant size. Phenotypic characterization of the 35S:TUA6^K40A transgenic plants revealed that both cell proliferation and cell expansion were affected. Cytological and biochemical analyses showed that the accumulation of α‐ and β‐tubulin proteins was significantly reduced in the transgenic plants, and the cortical microtubule arrays were severely disrupted, indicating that K40 of the plant α‐tubulin is critical in maintaining microtubule stability. We also constructed 35S:TUA6^K40R transgenic plants in which K40 of the engineered TUA6 protein is replaced by an arginine, and found that the 35S:TUA6^K40R plants were phenotypically indistinguishable from the wild‐type. Since lysine and arginine are similar in biochemical nature but arginine cannot be acetylated, these results suggest a structural importance for K40 of α‐tubulins in cell division and expansion.     

Tyrosination of α‐tubulin controls the initiation of processive dynein–dynactin motility

Post‐translational modifications (PTMs) of α/β‐tubulin are believed to regulate interactions with microtubule‐binding proteins. A well‐characterized PTM involves in the removal and re‐ligation of the C‐terminal tyrosine on α‐tubulin, but the purpose of this tyrosination–detyrosination cycle remains elusive. Here, we examined the processive motility of mammalian dynein complexed with dynactin and BicD2 (DDB) on tyrosinated versus detyrosinated microtubules. Motility was decreased ~fourfold on detyrosinated microtubules, constituting the largest effect of a tubulin PTM on motor function observed to date. This preference is mediated by dynactin's microtubule‐binding p150 subunit rather than dynein itself. Interestingly, on a bipartite microtubule consisting of tyrosinated and detyrosinated segments, DDB molecules that initiated movement on tyrosinated tubulin continued moving into the segment composed of detyrosinated tubulin. This result indicates that the α‐tubulin tyrosine facilitates initial motor–tubulin encounters, but is not needed for subsequent motility. Our results reveal a strong effect of the C‐terminal α‐tubulin tyrosine on dynein–dynactin motility and suggest that the tubulin tyrosination cycle could modulate the initiation of dynein‐driven motility in cells.     

The cytosolic carboxypeptidases CCP2 and CCP3 catalyze posttranslational removal of acidic amino acids

The posttranslational modification of carboxy-terminal tails of tubulin plays an important role in the regulation of the microtubule cytoskeleton. Enzymes responsible for deglutamylating tubulin have been discovered within a novel family of mammalian cytosolic carboxypeptidases. The discovery of these enzymes also revealed the existence of a range of other substrates that are enzymatically deglutamylated. Only four of six mammalian cytosolic carboxypeptidases had been enzymatically characterized. Here we complete the functional characterization of this protein family by demonstrating that CCP2 and CCP3 are deglutamylases, with CCP3 being able to hydrolyze aspartic acids with similar efficiency. Deaspartylation is a novel posttranslational modification that could, in conjunction with deglutamylation, broaden the range of potential substrates that undergo carboxy-terminal processing. In addition, we show that CCP2 and CCP3 are highly regulated proteins confined to ciliated tissues. The characterization of two novel enzymes for carboxy-terminal protein modification provides novel insights into the broadness of this barely studied process.     

Microtubule C‐Terminal Tails Can Change Characteristics of Motor Force Production

Control of intracellular transport is poorly understood, and functional ramifications of tubulin isoform differences between cell types are mostly unexplored. Motors' force production and detachment kinetics are critical for their group function, but how microtubule (MT) details affect these properties – if at all – is unknown. We investigated these questions using both a vesicular transport human kinesin, kinesin‐1, and also a mitotic kinesin likely optimized for group function, kinesin‐5, moving along either bovine brain or MCF7(breast cancer) MTs. We found that kinesin‐1 functioned similarly on the two sets of MTs – in particular, its mean force production was approximately the same, though due to its previously reported decreased processivity, the mean duration of kinesin‐1 force production was slightly decreased on MCF7 MTs. In contrast, kinesin‐5's function changed dramatically on MCF7 MTs: its average detachment force was reduced and its force–velocity curve was different. In spite of the reduced detachment force, the force–velocity alteration surprisingly improved high‐load group function for kinesin‐5 on the cancer‐cell MTs, potentially contributing to functions such as spindle‐mediated chromosome separation. Significant differences were previously reported for C‐terminal tubulin tails in MCF7 versus bovine brain tubulin. Consistent with this difference being functionally important, elimination of the tails made transport along the two sets of MTs similar.     

Perturbation of microtubule polymerization by quercetin through tubulin binding: a novel mechanism of its antiproliferative activity

The dietary flavonoid quercetin has a broad range of biological activities, including potent antitumor activity against several types of tumors. Recently, it has been shown that quercetin inhibits cancer cells proliferation by depleting cellular microtubules and perturbing cellular microtubule functions. However, the direct interactions of quercetin with tubulin and microtubules have not been examined so far. Here, we found that quercetin inhibited polymerization of microtubules and depolymerized microtubules made from purified tubulin in vitro. The binding of quercetin with tubulin was studied using quercetin fluorescence and intrinsic tryptophan fluorescence of tubulin. Quercetin bound to tubulin at a single site with a dissociation constant of 5-7 microM, and it specifically inhibited colchicine binding to tubulin but did not bind at the vinblastine site. In addition, quercetin perturbed the secondary structure of tubulin, and the binding of quercetin stimulated the intrinsic GTPase activity of soluble tubulin. Further, quercetin stabilized tubulin against decay and protected two cysteine residues of tubulin toward chemical modification by 5,5'-dithiobis-2-nitrobenzoic acid. Our data demonstrated that the binding of quercetin to tubulin induces conformational changes in tubulin and a mechanism through which quercetin could perturb microtubule polymerization dynamics has been proposed. The data suggest that quercetin inhibits cancer cells proliferation at least in part by perturbing microtubule functions through tubulin binding.     

Kinesin tail domains are intrinsically disordered

Kinesin motor proteins transport a wide variety of molecular cargoes in a spatially and temporally regulated manner. Kinesin motor domains, which hydrolyze ATP to produce a directed mechanical force along a microtubule, are well conserved throughout the entire superfamily. Outside of the motor domains, kinesin sequences diverge along with their transport functions. The nonmotor regions, particularly the tails, respond to a wide variety of structural and molecular cues that enable kinesins to carry specific cargoes in response to particular cellular signals. Here, we demonstrate that intrinsic disorder is a common structural feature of kinesins. A bioinformatics survey of the full‐length sequences of all 43 human kinesins predicts that significant regions of intrinsically disordered residues are present in all kinesins. These regions are concentrated in the nonmotor domains, particularly in the tails and near sites for ligand binding or post‐translational modifications. In order to experimentally verify these predictions, we expressed and purified the tail domains of kinesins representing three different families (Kif5B, Kif10, and KifC3). Circular dichroism and NMR spectroscopy experiments demonstrate that the isolated tails are disordered in vitro, yet they retain their functional microtubule‐binding activity. On the basis of these results, we propose that intrinsic disorder is a common structural feature that confers functional specificity to kinesins. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

Insights into allosteric control of microtubule dynamics from a buried β‐tubulin mutation that causes faster growth and slower shrinkage

αβ‐tubulin subunits cycle through a series of different conformations in the polymer lattice during microtubule growing and shrinking. How these allosteric responses to different tubulin:tubulin contacts contribute to microtubule dynamics, and whether the contributions are evolutionarily conserved, remains poorly understood. Here, we sought to determine whether the microtubule‐stabilizing effects (slower shrinking) of the β:T238A mutation we previously observed using yeast αβ‐tubulin would generalize to mammalian microtubules. Using recombinant human microtubules as a model, we found that the mutation caused slow microtubule shrinking, indicating that this effect of the mutation is indeed conserved. However, unlike in yeast, β:T238A human microtubules grew faster than wild‐type and the mutation did not appear to attenuate the conformational change associated with guanosine 5′‐triphosphate (GTP) hydrolysis in the lattice. We conclude that the assembly‐dependent conformational change in αβ‐tubulin can contribute to determine the rates of microtubule growing as well as shrinking. Our results also suggest that an allosteric perturbation like the β:T238A mutation can alter the behavior of terminal subunits without accompanying changes in the conformation of fully surrounded subunits in the body of the microtubule.     

Investigation of the Secretory Pathway in Trichomonas vaginalis Argues against a Moonlighting Function of Hydrogenosomal Enzymes

The enzymes pyruvate ferredoxin oxidoreductase (PFO), malic enzyme (ME), and the α‐ and β‐subunits of succinyl‐CoA synthetase (SCS) catalyze key steps of energy metabolism in Trichomonas vaginalis hydrogenosomes. These proteins have also been characterized as the adhesins AP120 (PFO), AP65 (ME), AP33, and AP51 (α‐ and β‐SCS), which are localized on the cell surface and mediate the T. vaginalis cytoadherence. However, the mechanisms that facilitate the targeting of these proteins to the cell surface via the secretory pathway and/or to hydrogenosomes are not known. Here we adapted an in vivo biotinylation system to perform highly sensitive tracing of protein trafficking in T. vaginalis. We showed that α‐ and β‐SCS are biotinylated in the cytosol and imported exclusively into the hydrogenosomes. Neither α‐ nor β‐SCS is biotinylated in the endoplasmic reticulum and delivered to the cell surface via the secretory pathway. In contrast, two surface proteins, tetratricopeptide domain‐containing membrane‐associated protein and tetraspanin family surface protein, as well as soluble‐secreted β‐amylase‐1 are biotinylated in the endoplasmic reticulum and delivered through the secretory pathway to their final destinations. Taken together, these results demonstrate that the α‐ and β‐SCS subunits are targeted only to the hydrogenosomes, which argues against their putative moonlighting function.     

Versatile substrate protein recognition mechanism of the eukaryotic chaperonin CCT

Group II chaperonins, found in eukaryotic and archaeal organisms, recognize substrate proteins through diverse mechanisms that involve either hydrophobic‐ or electrostatic‐dominated interactions. This action is distinct from the universal substrate recognition mechanism of group I chaperonins, which bind a wide spectrum of non‐native proteins primarily through hydrophobic interactions. We use computational approaches to pinpoint the substrate protein binding sites of the γ‐subunit of the eukaryotic chaperonin CCT and to identify its interactions with the stringent substrate β‐tubulin. Protein–protein docking methods reveal intrinsic binding sites of CCT comprising a helical (HL) region, homologous to the GroEL‐binding site, and the helical protrusion (HP) region. We performed molecular dynamics simulations of the solvated CCTγ apical domain, β‐tubulin peptide‐CCTγ complexes, and isolated β‐tubulin peptides. We find that tubulin binds to CCTγ through an extensive interface that spans both the HL region and the HP region. HL interactions involve both hydrophobic and electrostatic contacts, while binding to the HP region is stabilized almost exclusively by a salt bridge network. On the basis of additional simulations of a β‐tubulin‐CCTγ complex that involves a reduced interface, centered onto the HP region, we conclude that this salt bridge network is the minimal stabilizing interaction required. Strong conservation of the charged amino acids that participate in the salt bridge network, Arg306 and Glu271, indicates a general mechanism across the nonidentical CCT subunits and group II chaperonins. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Tubulin tyrosine ligase-like genes ttll3 and ttll6 maintain zebrafish cilia structure and motility

Tubulin post-translational modifications generate microtubule heterogeneity and modulate microtubule function, and are catalyzed by tubulin tyrosine ligase-like (TTLL) proteins. Using antibodies specific to monoglycylated, polyglycylated, and glutamylated tubulin in whole mount immunostaining of zebrafish embryos, we observed distinct, tissue-specific patterns of tubulin modifications. Tubulin modification patterns in cilia correlated with the expression of ttll3 and ttll6 in ciliated cells. Expression screening of all zebrafish tubulin tyrosine ligase-like genes revealed additional tissue-specific expression of ttll1 in brain neurons, ttll4 in muscle, and ttll7 in otic placodes. Knockdown of ttll3 eliminated cilia tubulin glycylation but had surprisingly mild effects on cilia structure and motility. Similarly, knockdown of ttll6 strongly reduced cilia tubulin glutamylation but only partially affected cilia structure and motility. Combined loss of function of ttll3 and ttll6 caused near complete loss of cilia motility and induced a variety of axonemal ultrastructural defects similar to defects previously observed in zebrafish fleer mutants, which were shown to lack tubulin glutamylation. Consistently, we find that fleer mutants also lack tubulin glycylation. These results indicate that tubulin glycylation and glutamylation have overlapping functions in maintaining cilia structure and motility and that the fleer/dyf-1 TPR protein is required for both types of tubulin post-translational modification.     

The C‐terminal tails of heterotrimeric kinesin‐2 motor subunits directly bind to α‐tubulin1: Possible implications for cilia‐specific tubulin entry

The assembly of microtubule‐based cytoskeleton propels the cilia and flagella growth. Previous studies have indicated that the kinesin‐2 family motors transport tubulin into the cilia through intraflagellar transport. Here, we report a direct interaction between the C‐terminal tail fragments of heterotrimeric kinesin‐2 and α‐tubulin1 isoforms in vitro. Blot overlay screen, affinity purification from tissue extracts, cosedimentation with subtilisin‐treated microtubule and LC‐ESI‐MS/MS characterization of the tail‐fragment‐associated tubulin identified an association between the tail domains and α‐tubulin1A/D isotype. The interaction was confirmed by Forster's resonance energy transfer assay in tissue‐cultured cells. The overexpression of the recombinant tails in NIH3T3 cells affected the primary cilia growth, which was rescued by coexpression of a α‐tubulin1 transgene. Furthermore, fluorescent recovery after photobleach analysis in the olfactory cilia of Drosophila indicated that tubulin is transported in a non‐particulate form requiring kinesin‐2. These results provide additional new insight into the mechanisms underlying selective tubulin isoform enrichment in the cilia.      

β‐Tubulin mutations that cause severe neuropathies disrupt axonal transport

Microtubules are fundamental to neuronal morphogenesis and function. Mutations in tubulin, the major constituent of microtubules, result in neuronal diseases. Here, we have analysed β‐tubulin mutations that cause neuronal diseases and we have identified mutations that strongly inhibit axonal transport of vesicles and mitochondria. These mutations are in the H12 helix of β‐tubulin and change the negative charge on the surface of the microtubule. This surface is the interface between microtubules and kinesin superfamily motor proteins (KIF). The binding of axonal transport KIFs to microtubules is dominant negatively disrupted by these mutations, which alters the localization of KIFs in neurons and inhibits axon elongation in vivo. In humans, these mutations induce broad neurological symptoms, such as loss of axons in the central nervous system and peripheral neuropathy. Thus, our data identified the critical region of β‐tubulin required for axonal transport and suggest a molecular mechanism for human neuronal diseases caused by tubulin mutations.     

Sequence divergence of Entamoeba histolytica tubulin is responsible for its altered tertiary structure

Atypical microtubular structures of the protozoan parasite Entamoeba histolytica (Eh) have been attributed to amino acid sequence divergence of Eh tubulin. To investigate if this sequence divergence leads to significant differences in the tertiary structure of the Eh alphabeta-tubulin heterodimer, we have modeled alphabeta-tubulin heterodimer of Eh based on the crystal structure of mammalian tubulin. The predicted 3D homology model exhibits an overall resemblance with the known crystal structure of mammalian tubulin except for the 16 residue long carboxy terminal region of Eh beta-tubulin. We propose that this C-terminal region may provide steric hindrance in the polymerization of Eh alphabeta-tubulin for microtubule formation. Using docking studies, we have identified the binding sites for different microtubule specific drugs on Eh beta-tubulin. Our model provides a rational framework, both for understanding the contribution of Eh beta-tubulin C-terminal region to alphabeta-tubulin polymerization and design of new anti-protozoan drugs in order to control amoebiasis.     

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.