Dynamics of epigenetic control in plants via SET domain containing proteins: Structural and functional insights

Plants control expression of their genes in a way that involves manipulating the chromatin structural dynamics in order to adapt to environmental changes and carry out developmental processes. Histone modifications like histone methylation are significant epigenetic marks which profoundly and globally modify chromatin, potentially affecting the expression of several genes. Methylation of histones is catalyzed by histone lysine methyltransferases (HKMTs), that features an evolutionary conserved domain known as SET [Su(var)3–9, E(Z), Trithorax]. This methylation is directed at particular lysine (K) residues on H3 or H4 histone. Plant SET domain group (SDG) proteins are categorized into different classes that have been conserved through evolution, and each class have specificity that influences how the chromatin structure operates. The domains discovered in plant SET domain proteins have typically been linked to protein-protein interactions, suggesting that majority of the SDGs function in complexes. Additionally, SDG-mediated histone mark deposition also affects alternative splicing events. In present review, we discussed the diversity of SDGs in plants including their structural properties. Additionally, we have provided comprehensive summary of the functions of the SDG-domain containing proteins in plant developmental processes and response to environmental stimuli have also been highlighted.     

Provenance of SET-Domain Histone Methyltransferases Through Duplication of a Simple Structural Unit

SET domains are protein lysine methyltransferases that methylate diverse proteins, such as, histones, Rubisco and cytochrome C. In particular, they play an important role in the dynamics of the eukaryotic chromatin and are present in several chromatin-associated proteins. Recently, structures of several SET domains have been solved, and they contain a conserved fold that is unrelated to previously characterized methyltransferases, which possess either Rossmann fold or SPOUT domains. Phylogenetic and phyletic-profile analysis of the SET domain suggests that it was an evolutionary “invention” of the eukaryotic lineage, with secondary lateral transfers to bacteria. We show that the conserved N- and C- terminal regions, which comprise the core barrel-like module of the SET domain, are symmetric repeats of a simple 3-stranded unit. Furthermore, the two symmetrically arranged repeats contribute to the binding sites for the two substrates of the SET domain. This suggests the SET domain arose from an ancestral dimer of this 3-stranded unit, with each unit probably functioning as generic-ligand binding structure. The divergence between the two repeat units appears to have arisen as a result of their interactions with the central module of the SET domain, which was inserted between the two repeats. One of the repeats appears to have acquired adaptations, which helped it to specialize in AdoMet binding, whereas the second repeat contributed to histone-interaction, and in orienting a crucial active site residue. The central module of the SET domain supplies a critical asparagine to the active site, and its structural features suggest that it may have also arisen from a further duplication of one of the repeats comprising the core barrel. However, it appears to have structurally diverged from the two canonical repeats due to the lack of an obligate dimerization partner. The spatial position of the two repeats in the ancestral dimer appears to have favored the formation of the structural knot typical of the SET domain. A comparable knot is seen in the SPOUT-domain methyltransferases, and this represents a case of convergent evolution of an active-site-associated configuration in two otherwise unrelated classes of methylases. Thus, the SET domain provides a model for the innovation of a complex enzymatic fold through the duplications of a structurally simple non-enzymatic unit.     

Provenance of SET-domain histone methyltransferases through duplication of a simple structural unit

SET domains are protein lysine methyltransferases that methylate diverse proteins, such as, histones, Rubisco and cytochrome C. In particular, they play an important role in the dynamics of the eukaryotic chromatin and are present in several chromatin-associated proteins. Recently, structures of several SET domains have been solved, and they contain a conserved fold that is unrelated to previously characterized methyltransferases, which possess either Rossmann fold or SPOUT domains. Phylogenetic and phyletic-profile analysis of the SET domain suggests that it was an evolutionary "invention" of the eukaryotic lineage, with secondary lateral transfers to bacteria. We show that the conserved N- and C- terminal regions, which comprise the core barrel-like module of the SET domain, are symmetric repeats of a simple 3-stranded unit. Furthermore, the two symmetrically arranged repeats contribute to the binding sites for the two substrates of the SET domain. This suggests the SET domain arose from an ancestral dimer of this 3-stranded unit, with each unit probably functioning as generic-ligand binding structure. The divergence between the two repeat units appears to have arisen as a result of their interactions with the central module of the SET domain, which was inserted between the two repeats. One of the repeats appears to have acquired adaptations, which helped it to specialize in AdoMet binding, whereas the second repeat contributed to histone-interaction, and in orienting a crucial active site residue. The central module of the SET domain supplies a critical asparagine to the active site, and its structural features suggest that it may have also arisen from a further duplication of one of the repeats comprising the core barrel. However, it appears to have structurally diverged from the two canonical repeats due to the lack of an obligate dimerization partner. The spatial position of the two repeats in the ancestral dimer appears to have favored the formation of the structural knot typical of the SET domain. A comparable knot is seen in the SPOUT-domain methyltransferases, and this represents a case of convergent evolution of an active-site-associated configuration in two otherwise unrelated classes of methylases. Thus, the SET domain provides a model for the innovation of a complex enzymatic fold through the duplications of a structurally simple non-enzymatic unit.     

Three-dimensional structures of fibrillar Sm proteins: Hfq and other Sm-like proteins

SET domain lysine methyltransferases are known to catalyze site and state-specific methylation of lysine residues in histones that is fundamental in epigenetic regulation of gene activation and silencing in eukaryotic organisms. Here we report the three-dimensional solution structure of the SET domain histone lysine methyltransferase (vSET) from Paramecium bursaria chlorella virus 1 bound to cofactor S-adenosyl-L-homocysteine and a histone H3 peptide containing mono-methylated lysine 27. The dimeric structure, mimicking an enzyme/cofactor/substrate complex, yields the structural basis of the substrate specificity and methylation multiplicity of the enzyme. Our results from mutagenesis and enzyme kinetics analyses argue that a general base mechanism is less likely for lysine methylation by SET domains; and that the only invariant active site residue tyrosine 105 in vSET facilitates methyl transfer from cofactor to the substrate lysine by aligning intermolecular interactions in the lysine access channel of the enzyme.     

Characterization of the PR domain of RIZ1 histone methyltransferase

RIZ1 (PRDM2) and PRDI-BF1 (PRDM1) are involved in B cell differentiation and the development of B cell lymphomas. These proteins are expressed in two forms that differ by the presence or absence of a PR domain. The protein product that retains the PR domain is anti-tumorigenic while the product that lacks the PR domain is oncogenic and over-expressed in tumor cells. The conserved PR domain is homologous to the SET domain from a family of histone methyltransferases. RIZ1 is also a histone methyltransferase and methylates lysine 9 in histone H3. This activity has been mapped to the PR domain. In the present study, deuterium exchange mass spectrometry was used to define the structural boundaries of the RIZ1 PR domain and to map sites of missense mutations that occur in human cancers and reduce methyltransferase activity. Flexible segments were selectively deleted to produce protein products that crystallize for structural studies. Segments at the carboxyl terminus of the PR domain that are involved in methylation of H3 were shown to be flexible, similar to SET domains, suggesting that the PR and SET methyltransferases may belong to an emerging class of proteins that contain mobile functional regions.     

The function of histone lysine methylation related SET domain group proteins in plants

Histone methylation, which is mediated by the histone lysine (K) methyltransferases (HKMTases), is a mechanism associated with many pathways in eukaryotes. Most HKMTases have a conserved SET (Su(var) 3‐9,E(z),Trithorax) domain, while the HKMTases with SET domains are called the SET domain group (SDG) proteins. In plants, only SDG proteins can work as HKMTases. In this review, we introduced the classification of SDG family proteins in plants and the structural characteristics of each subfamily, surmise the functions of SDG family members in plant growth and development processes, including pollen and female gametophyte development, flowering, plant morphology and the responses to stresses. This review will help researchers better understand the SDG proteins and histone methylation in plants and lay a basic foundation for further studies on SDG proteins.     

Structures of SET domain proteins: protein lysine methyltransferases make their mark

Proteins bearing the widely distributed SET domain have been shown to methylate lysine residues in histones and other proteins. In this issue, three-dimensional structures are reported for three very different SET domain-containing proteins. The structures reveal novel folds for several new domains, including SET, and provide early insights into mechanisms of catalysis and molecular recognition in this family of enzymes.     

Comparative analysis of SET domain proteins in maize and Arabidopsis reveals multiple duplications preceding the divergence of monocots and dicots

Histone proteins play a central role in chromatin packaging, and modification of histones is associated with chromatin accessibility. SET domain [Su(var)3-9, Enhancer-of-zeste, Trithorax] proteins are one class of proteins that have been implicated in regulating gene expression through histone methylation. The relationships of 22 SET domain proteins from maize (Zea mays) and 32 SET domain proteins from Arabidopsis were evaluated by phylogenetic analysis and domain organization. Our analysis reveals five classes of SET domain proteins in plants that can be further divided into 19 orthology groups. In some cases, such as the Enhancer of zeste-like and trithorax-like proteins, plants and animals contain homologous proteins with a similar organization of domains outside of the SET domain. However, a majority of plant SET domain proteins do not have an animal homolog with similar domain organization, suggesting that plants have unique mechanisms to establish and maintain chromatin states. Although the domains present in plant and animal SET domain proteins often differ, the domains found in the plant proteins have been generally implicated in protein-protein interactions, indicating that most SET domain proteins operate in complexes. Combined analysis of the maize and Arabidopsis SET domain proteins reveals that duplication of SET domain proteins in plants is extensive and has occurred via multiple mechanisms that preceded the divergence of monocots and dicots.     

The Saccharomyces cerevisiae Set1 complex includes an Ash2 homologue and methylates histone 3 lysine 4.

The SET domain proteins, SUV39 and G9a have recently been shown to be histone methyltransferases specific for lysines 9 and 27 (G9a only) of histone 3 (H3). The SET domains of the Saccharomyces cerevisiae Set1 and Drosophila trithorax proteins are closely related to each other but distinct from SUV39 and G9a. We characterized the complex associated with Set1 and Set1C and found that it is comprised of eight members, one of which, Bre2, is homologous to the trithorax-group (trxG) protein, Ash2. Set1C requires Set1 for complex integrity and mutation of Set1 and Set1C components shortens telomeres. One Set1C member, Swd2/Cpf10 is also present in cleavage polyadenylation factor (CPF). Set1C methylates lysine 4 of H3, thus adding a new specificity and a new subclass of SET domain proteins known to methyltransferases. Since methylation of H3 lysine 4 is widespread in eukaryotes, we screened the databases and found other Set1 homologues. We propose that eukaryotic Set1Cs are H3 lysine 4 methyltransferases and are related to trxG action through association with Ash2 homologues.     

Biochemical characterization of human SET and MYND domain-containing protein 2 methyltransferase

SET and MYND domain-containing protein 2 (SMYD2) is a protein lysine methyltransferase that catalyzes the transfer of methyl groups from S-adenosylmethionine (AdoMet) to acceptor lysine residues on histones and other proteins. To understand the kinetic mechanism and the function of individual domains, human SMYD2 was overexpressed, purified, and characterized. Substrate specificity and product analysis studies established SMYD2 as a monomethyltransferase that prefers nonmethylated p53 peptide substrate. Steady-state kinetic and product inhibition studies showed that SMYD2 operates via a rapid equilibrium random Bi Bi mechanism at a rate of 0.048 ± 0.001 s(-1), with K(M)s for AdoMet and the p53 peptide of 0.031 ± 0.01 μM and 0.68 ± 0.22 μM, respectively. Metal analyses revealed that SMYD2 contains three tightly bound zinc ions that are important for maintaining the structural integrity and catalytic activity of SMYD2. Catalytic activity was also shown to be dependent on the GxG motif in the S-sequence of the split SET domain, as a G18A/G20A double mutant and a sequence deletion within the conserved motif impaired AdoMet binding and significantly decreased enzymatic activity. The functional importance of other SMYD2 domains including the MYND domain, the cysteine-rich post-SET domain, and the C-terminal domain (CTD), were also investigated. Taken together, these results demonstrated the functional importance of distinct domains in the SMYD family of proteins and further advanced our understanding of the catalytic mechanism of this family.     

Structure of SET domain proteins: a new twist on histone methylation

The methylation of lysine residues on histone tails is catalyzed by proteins containing a conserved SET domain. A recent flurry of structures of SET domain proteins has revealed a new protein fold and a scaffold for understanding catalysis and substrate binding by these enzymes. The prospect that histone methylation might form an epigenetic code and the implicated involvement of SET domain proteins in cancer assures that structure-function studies of these enzymes will continue until their detailed mechanism of action is determined.     

Structural and biochemical studies of human lysine methyltransferase Smyd3 reveal the important functional roles of its post-SET and TPR domains and the regulation of its activity by DNA binding

The SET- and MYND-domain containing (Smyd) proteins constitute a special subfamily of the SET-containing lysine methyltransferases. Here we present the structure of full-length human Smyd3 in complex with S-adenosyl-l-homocysteine at 2.8 Å resolution. Smyd3 affords the first example that other region(s) besides the SET domain and its flanking regions participate in the formation of the active site. Structural analysis shows that the previously uncharacterized C-terminal domain of Smyd3 contains a tetratrico-peptide repeat (TPR) domain which together with the SET and post-SET domains forms a deep, narrow substrate binding pocket. Our data demonstrate the important roles of both TPR and post-SET domains in the histone lysine methyltransferase (HKMT) activity of Smyd3, and show that the hydroxyl group of Tyr239 is critical for the enzymatic activity. The characteristic MYND domain is located nearby to the substrate binding pocket and exhibits a largely positively charged surface. Further biochemical assays show that DNA binding of Smyd3 can stimulate its HKMT activity and the process may be mediated via the MYND domain through direct DNA binding.     

The Human Mixed Lineage Leukemia 5 (MLL5), a Sequentially and Structurally Divergent SET Domain-Containing Protein with No Intrinsic Catalytic Activity

Mixed Lineage Leukemia 5 (MLL5) plays a key role in hematopoiesis, spermatogenesis and cell cycle progression. Chromatin binding is ensured by its plant homeodomain (PHD) through a direct interaction with the N-terminus of histone H3 (H3). In addition, MLL5 contains a Su(var)3-9, Enhancer of zeste, Trithorax (SET) domain, a protein module that usually displays histone lysine methyltransferase activity. We report here the crystal structure of the unliganded SET domain of human MLL5 at 2.1 Å resolution. Although it shows most of the canonical features of other SET domains, both the lack of key residues and the presence in the SET-I subdomain of an unusually large loop preclude the interaction of MLL5 SET with its cofactor and substrate. Accordingly, we show that MLL5 is devoid of any in vitro methyltransferase activity on full-length histones and histone H3 peptides. Hence, the three dimensional structure of MLL5 SET domain unveils the structural basis for its lack of methyltransferase activity and suggests a new regulatory mechanism.