Remodeling of nucleosome-dimer particles with yIsw2 promotes their association with ALL-1 SET domain in vitro

Functioning of histone lysine methyltransferases (HKMTs) involves interactions of their catalytic domain "SET" with the N-termini of histone H3. However, these interactions are restricted in canonical nucleosomes due to the limited accessibility of H3 termini. Here we investigated whether nucleosome remodeling with the yeast Isw2 affects nucleosome affinity to the SET domain of ALL-1 HKMT. Reconstitution of mononucleosomes by salt dilutions also produces some nucleosome-dimer particles (self-associated mononucleosomes, described by: Tatchell and van Holde (1977) Biochemistry, 16, 5295-5303). The GST-tagged SET-domain polypeptide of ALL-1 was assayed for binding to assembled mononucleosomes and nucleosome-dimer particles, either intact or remodeled with purified yeast Isw2. Remodeling of mononucleosomes does not noticeably affect their affinity to SET domain; however, yIsw2 remodeling of nucleosome-dimer particles facilitated their association with GST-SET polypeptide. Therefore, it is conceivable that nucleosome interactions in trans could be implicated in the maintenance of chromatin methylation patterns in vivo.     

Computational characterization of substrate and product specificities,and functionality of S‐adenosylmethionine binding pocket in histone lysine methyltransferases from Arabidopsis,rice and maize

Histone lysine methylation by histone lysine methyltransferases (HKMTs) has been implicated in regulation of gene expression. While significant progress has been made to understand the roles and mechanisms of animal HKMT functions, only a few plant HKMTs are functionally characterized. To unravel histone substrate specificity, degree of methylation and catalytic activity, we analyzed Arabidopsis Trithorax‐like protein (ATX), Su (var)3‐9 h omologs protein (SUVH), Su(var)3‐9 related protein (SUVR), ATXR5, ATXR6, and E(Z) HKMTs of Arabidopsis, maize and rice through sequence and structure comparison. We show that ATXs may exhibit methyltransferase specificity toward histone 3 lysine 4 (H3K4) and might catalyse the trimethylation. Our analyses also indicate that most SUVH proteins of Arabidopsis may bind histone H3 lysine 9 (H3K9). We also predict that SUVH7, SUVH8, SUVR1, SUVR3, ZmSET20 and ZmSET22 catalyse monomethylation or dimethylation of H3K9. Except for SDG728, which may trimethylate H3K9, all SUVH paralogs in rice may catalyse monomethylation or dimethylation. ZmSET11, ZmSET31, SDG713, SDG715, and SDG726 proteins are predicted to be catalytically inactive because of an incomplete S‐adenosylmethionine (SAM) binding pocket and a post‐SET domain. E(Z) homologs can trimethylate H3K27 substrate, which is similar to the Enhancer of Zeste homolog 2 of humans. Our comparative sequence analyses reveal that ATXR5 and ATXR6 lack motifs/domains required for protein‐protein interaction and polycomb repressive complex 2 complex formation. We propose that subtle variations of key residues at substrate or SAM binding pocket, around the catalytic pocket, or presence of pre‐SET and post‐SET domains in HKMTs of the aforementioned plant species lead to variations in class‐specific HKMT functions and further determine their substrate specificity, the degree of methylation and catalytic activity.     

Histone lysine methyltransferases and demethylases in Plasmodium falciparum

Dynamic histone lysine methylation, regulated by methyltransferases and demethylases, plays fundamental roles in chromatin structure and gene expression in a wide range of eukaryotic organisms. A large number of SET-domain-containing proteins make up the histone lysine methyltransferase (HKMT) family, which catalyses the methylation of different lysine residues with relatively high substrate specificities. Another large family of Jumonji C (JmjC)-domain-containing histone lysine demethylases (JHDMs) reverses histone lysine methylation with both lysine site and methyl-state specificities. Through bioinformatic analysis, at least nine SET-domain-containing genes were found in the malaria parasite Plasmodium falciparum and its sibling species. Phylogenetic analysis separated these putative HKMTs into five subfamilies with different putative substrate specificities. Consistent with the phylogenetic subdivision, methyl marks were found on K4, K9 and K36 of histone H3 and K20 of histone H4 by site-specific methyl-lysine antibodies. In addition, most SET-domain genes and histone methyl-lysine marks displayed dynamic changes during the parasite asexual erythrocytic cycle, suggesting that they constitute an important epigenetic mechanism of gene regulation in malaria parasites. Furthermore, the malaria parasite and other apicomplexan genomes also encode JmjC-domain-containing proteins that may serve as histone lysine demethylases. Whereas prokaryotic expression of putative active domains of four P. falciparum SET proteins did not yield detectable HKMT activity towards recombinant P. falciparum histones, two protein domains expressed in vitro in a eukaryotic system showed HKMT activities towards H3 and H4, respectively. With the discovery of these Plasmodium SET- and JmjC-domain genes in the malaria parasite genomes, future efforts will be directed towards elucidation of their substrate specificities and functions in various cellular processes of the parasites.     

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.     

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.     

Structural and functional profiling of the human histone methyltransferase SMYD3

The SET and MYND Domain (SMYD) proteins comprise a unique family of multi-domain SET histone methyltransferases that are implicated in human cancer progression. Here we report an analysis of the crystal structure of the full length human SMYD3 in a complex with an analog of the S-adenosyl methionine (SAM) methyl donor cofactor. The structure revealed an overall compact architecture in which the "split-SET" domain adopts a canonical SET domain fold and closely assembles with a Zn-binding MYND domain and a C-terminal superhelical 9 α-helical bundle similar to that observed for the mouse SMYD1 structure. Together, these structurally interlocked domains impose a highly confined binding pocket for histone substrates, suggesting a regulated mechanism for its enzymatic activity. Our mutational and biochemical analyses confirm regulatory roles of the unique structural elements both inside and outside the core SET domain and establish a previously undetected preference for trimethylation of H4K20.     

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.     

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.     

Saturation transfer difference measurements with SU(VAR)3-9 and S-adenosyl-L-methionine

Saturation transfer difference NMR measurements were performed to investigate the interaction of S-adenosyl-l-methionine (AdoMet) with SU(VAR)3-9 from Drosophila melanogaster. SU(VAR)3-9 has a SET domain and plays an important role in methylation of lysine-9 of histone H3 which results in gene silencing. We determined the binding epitope of AdoMet and compared it with a crystal structure of another SET protein.     

敲除里氏木霉hkmt基因可增强纤维素酶的酶活性和表达水平

表观遗传是不涉及DNA序列变化的可遗传变化,包括DNA甲基化、组蛋白修饰和miRNA调控等。在组蛋白甲基化修饰中,主要是组蛋白赖氨酸甲基转移酶（histone lysine methyltransferase,HKMT）参与调控。有文献报道,HKMT蛋白的催化核心为SET结构域,它具有促进或抑制基因表达的作用。在里氏木霉(Trichoderma reesei)中,HKMT对纤维素酶基因的表达调控的机制尚不明确。本文阐述了以里氏木霉为研究对象,利用Split-Maker技术构建了组蛋白赖氨酸甲基转移酶基因敲除表达盒,并转化了里氏木霉T. reesei QM9414。经PCR及Southern印迹验证正确后,显微镜观察到T.reesei Δhkmt菌株菌丝较长,分支较多。检测到突变体菌株连续7d滤纸酶活（filter paper enzyme activity,AFP）和羧甲基纤维素钠酶活 (carboxymethyl cellulose sodium enzyme activity,CMCA)。结果分别比野生型菌株高出5.00 IU·mL^-1、15.00 IU·mL^-1。利用RT-qPCR检测到突变菌株纤维素酶及其相关基因cbh1、egl1和xyr1的表达分别高出野生型4.51、3.87和2.51倍。通过对野生型菌株和突变菌株形态特征、纤维素酶酶活性、纤维素酶相关基因表达量的探索,为进一步研究里氏木霉表观遗传调控对纤维素酶表达的影响提供了新思路和实验资料。     

敲除里氏木霉hkmt基因可增强纤维素酶的酶活性和表达水平

表观遗传是不涉及DNA序列变化的可遗传变化,包括DNA甲基化、组蛋白修饰和miRNA调控等。在组蛋白甲基化修饰中,主要是组蛋白赖氨酸甲基转移酶（histone lysine methyltransferase,HKMT）参与调控。有文献报道,HKMT蛋白的催化核心为SET结构域,它具有促进或抑制基因表达的作用。在里氏木霉(Trichoderma reesei)中,HKMT对纤维素酶基因的表达调控的机制尚不明确。本文阐述了以里氏木霉为研究对象,利用Split-Maker技术构建了组蛋白赖氨酸甲基转移酶基因敲除表达盒,并转化了里氏木霉T. reesei QM9414。经PCR及Southern印迹验证正确后,显微镜观察到T.reesei Δhkmt菌株菌丝较长,分支较多。检测到突变体菌株连续7d滤纸酶活（filter paper enzyme activity,AFP）和羧甲基纤维素钠酶活 (carboxymethyl cellulose sodium enzyme activity,CMCA)。结果分别比野生型菌株高出5.00 IU·mL^-1、15.00 IU·mL^-1。利用RT-qPCR检测到突变菌株纤维素酶及其相关基因cbh1、egl1和xyr1的表达分别高出野生型4.51、3.87和2.51倍。通过对野生型菌株和突变菌株形态特征、纤维素酶酶活性、纤维素酶相关基因表达量的探索,为进一步研究里氏木霉表观遗传调控对纤维素酶表达的影响提供了新思路和实验资料。     

Structure of the SET domain histone lysine methyltransferase Clr4

Methylation of histone H3 lysine 9 is an important component of the 'histone code' for heterochromatic gene silencing. The SET domain-containing Clr4 protein, a close relative of Su(var)3-9 proteins in higher eukaryotes, specifically methylates lysine 9 of histone H3 and is essential for silencing in Schizosaccharomyces pombe. Here we report the 2.3 A resolution crystal structure of the catalytic domain of Clr4. The structure reveals an overall fold rich in beta-strands, a potential active site consisting of a SAM-binding pocket, and a connected groove that could accommodate the binding of the N-terminal tail of histone H3. The pre-SET motif contains a triangular zinc cluster coordinated by nine cysteines distant from the active site, whereas the post-SET region is largely flexible but proximal to the active site. The structure provides insights into the architecture of SET domain histone methyltransferases and establishes a paradigm for further characterization of the Clr4 family of epigenetic regulators.