Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs.     

QM/MM MD and free energy simulations of G9a-like protein (GLP) and its mutants: understanding the factors that determine the product specificity

Certain lysine residues on histone tails could be methylated by protein lysine methyltransferases (PKMTs) using S-adenosyl-L-methionine (AdoMet) as the methyl donor. Since the methylation states of the target lysines play a fundamental role in the regulation of chromatin structure and gene expression, it is important to study the property of PKMTs that allows a specific number of methyl groups (one, two or three) to be added (termed as product specificity). It has been shown that the product specificity of PKMTs may be controlled in part by the existence of specific residues at the active site. One of the best examples is a Phe/Tyr switch found in many PKMTs. Here quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) and free energy simulations are performed on wild type G9a-like protein (GLP) and its F1209Y and Y1124F mutants for understanding the energetic origin of the product specificity and the reasons for the change of product specificity as a result of single-residue mutations at the Phe/Tyr switch as well as other positions. The free energy barriers of the methyl transfer processes calculated from our simulations are consistent with experimental data, supporting the suggestion that the relative free energy barriers may determine, at least in part, the product specificity of PKMTs. The changes of the free energy barriers as a result of the mutations are also discussed based on the structural information obtained from the simulations. The results suggest that the space and active-site interactions around the ε-amino group of the target lysine available for methyl addition appear to among the key structural factors in controlling the product specificity and activity of PKMTs.     

植物组蛋白赖氨酸甲基化建立过程及其遗传性研究进展

表观遗传学主要包括DNA甲基化、组蛋白修饰和非编码RNA,组蛋白甲基化作为组蛋白修饰中的一种重要修饰,在植物体的发育和环境适应中发挥着重要作用。组蛋白甲基化主要发生在赖氨酸残基上,同时根据不同的赖氨酸位点和每个赖氨酸位点甲基化程度的不同,形成了不同的赖氨酸甲基化修饰。根据对基因的不同功能,通常将组蛋白赖氨酸甲基化修饰分为2大类:(1)能够促进基因表达的,如H3K4me3和H3K36me3;(2)能够抑制基因表达的,如H3K9me2和H3K27me3。不同的组蛋白赖氨酸甲基化去甲基化过程需要相应的阅读(reader)、书写(writer)和擦除(eraser)3种蛋白。同时,组蛋白赖氨酸甲基化的遗传性质目前还不是很清楚。综述了植物中组蛋白赖氨酸甲基化建立与去除过程,以及对组蛋白赖氨酸甲基化可遗传性的探讨。     

Epigenetic regulation: methylation of histone and non-histone proteins

Histone methylation is believed to play important roles in epigenetic memory in various biological processes. However, questions like whether the methylation marks themselves are faithfully transmitted into daughter cells and through what mechanisms are currently under active investigation. Previously, methylation was considered to be irreversible, but the recent discovery of histone lysine demethylases revealed a dynamic nature of histone methylation regulation on four of the main sites of methylation on histone H3 and H4 tails (H3K4, H3K9, H3K27 and H3K36). Even so, it is still unclear whether demethylases specific for the remaining two sites, H3K79 and H4K20, exist. Furthermore, besides histone proteins, the lysine methylation and demethylation also occur on non-histone proteins, which are probably subjected to similar regulation as histones. This review discusses recent progresses in protein lysine methylation regulation focusing on the above topics, while referring readers to a number of recent reviews for the biochemistry and biology of these enzymes     

Epigenetic gene regulation by plant Jumonji group of histone demethylase

Histone methylation plays an important role in epigenetic regulation of gene expression. Reversible methylation/demethylation of several histone lysine residues is mediated by distinct histone methyltransferases and histone demethylases. Jumonji proteins have been characterized to be involved in histone demethylation. Plant Jumonji homologues are found to have important functions in epigenetic processes, gene expression and plant development and to play an essential role in interplay between histone modifications and DNA methylation. This article is part of a Special Issue entitled: Epigenetic Control of cellular and developmental processes in plants.     

TET家族DNA羟化酶与5-hmC在肿瘤中的作用机制的研究进展

DNA甲基化失调引起基因表达异常是表观遗传学的一个显著特点。目前已知,由DNA甲基转移酶（DNA methyltransferases,DMNTs）催化DNA甲基化,其酶基因突变或表达异常引起DNA甲基化水平的改变。近期研究发现了一种DNA去甲基化酶--TET（Ten-Eleventranslocation）家族DNA羟化酶,能通过多种途径催化5-甲基胞嘧啶（5．methylcytosine,5-mC）去甲基化,从而调控DNA基化的平衡。5-羟甲基胞嘧啶（5-hydroxymethylcytosine,5-hmC）作为DNA去甲基化多重步骤中重要的中间产物,其水平在肿瘤的发生和发展时期发生显著变化。该文从TET家族蛋白展开,介绍TET蛋白的结构、功能及作用机制以及多种人类肿瘤中丁E丁家族基因与5-hmC水平的相关性及其对肿瘤发生发展、诊断预后等临床意义的研究进展。     

Oligomerization and Auto-methylation of the Human Lysine Methyltransferase SETD6

Signaling via lysine methylation by protein lysine methyltransferases (PKMTs), has been linked to diverse biological and disease processes. The mono-methyltransferase SETD6 (SET-domain-containing protein 6) is a member of the PKMT family and was previously shown to regulate essential cellular processes such as the NF-κB, WNT and the oxidative stress pathways. However, on the biochemical level, little is known about the enzymatic mode of action of SETD6. Here we provide evidence that SETD6 forms high-molecular-weight structures. Specifically, we demonstrate that SETD6 monomeric, dimeric and trimeric forms are stabilized by the methyl donor, S-adenosyl-l-methionine. We then show that SETD6 has auto-methylation activity at K39 and K179, which serves as the major auto-methylation sites with a moderate auto-methylation activity toward K372. A point mutation at K179 but not at K39 and K372, located at the SET domain of SETD6, impaired SETD6 ability to form a trimer, strongly implying a link between the auto-methylation and the oligomerization state. Finally, by radioactive in vitro methylation experiments and biochemical kinetics analysis, we show that the auto-methylation at K39 and K179 increases the catalytic rate of SETD6. Collectively, our data support a model by which SETD6 auto-methylation and self-interaction positively regulate its enzymatic activity in vitro and may suggest that other PKMTs are regulated in the same manner.     

Structural Basis of a Histone H3 Lysine 4 Demethylase Required for Stem Elongation in Rice

Histone lysine methylation is an important epigenetic modification in regulating chromatin structure and gene expression. Histone H3 lysine 4 methylation (H3K4me), which can be in a mono-, di-, or trimethylated state, has been shown to play an important role in gene expression involved in plant developmental control and stress adaptation. However, the resetting mechanism of this epigenetic modification is not yet fully understood. In this work, we identified a JmjC domain-containing protein, JMJ703, as a histone lysine demethylase that specifically reverses all three forms of H3K4me in rice. Loss-of-function mutation of the gene affected stem elongation and plant growth, which may be related to increased expression of cytokinin oxidase genes in the mutant. Analysis of crystal structure of the catalytic core domain (c-JMJ703) of the protein revealed a general structural similarity with mammalian and yeast JMJD2 proteins that are H3K9 and H3K36 demethylases. However, several specific features were observed in the structure of c-JMJ703. Key residues that interact with cofactors Fe(II) and N-oxalylglycine and the methylated H3K4 substrate peptide were identified and were shown to be essential for the demethylase activity in vivo. Several key residues are specifically conserved in known H3K4 demethylases, suggesting that they may be involved in the specificity for H3K4 demethylation.     

组蛋白去甲基化酶与肿瘤发生

组蛋白甲基化修饰是一个可逆的动态的调节过程。甲基化和/或去甲基化状态与表观遗传、转录调控和维持基因组完整性等密切相关。组蛋白甲基化状态异常会直接或间接影响各种生理和病理过程。已知组蛋白去甲基化酶包括赖氨酸特异性去甲基化酶（LSD）家族和含JmjC结构域的JMJD家族。研究发现,两者与肿瘤的发生均有着密切的关系。本文总结了组蛋白去甲基化酶在组蛋白甲基化修饰及肿瘤研究方面的最新进展,为组蛋白修饰的功能及肿瘤诊断、治疗、预后监测等研究提供新思路。在胃癌、乳腺癌、结肠癌等常见肿瘤中,组蛋白去甲基化酶可改变组蛋白的甲基化水平或者直接作用于癌基因,也可调节microRNA或转录因子等,促进或抑制肿瘤的发生发展与影响肿瘤的预后。     

Mammalian DNA demethylation

DNA cytosine methylation is a reversible epigenetic mark regulating gene expression. Aberrant methylation profiles are concomitant with developmental defects and cancer. Numerous studies in the past decade have identified enzymes and pathways responsible for active DNA demethylation both on a genome-wide as well as gene-specific scale. Recent findings have strengthened the idea that 5-methylcytosine oxidation catalyzed by members of the ten-eleven translocation (Tet1–3) oxygenases in conjunction with replication-coupled dilution of the conversion products causes the majority of genome-wide erasure of methylation marks during early development. In contrast, short and long patch DNA excision repair seems to be implicated mainly in gene-specific demethylation. Growth arrest and DNA damage-inducible protein 45 a (Gadd45a) regulates gene-specific demethylation within regulatory sequences of limited lengths raising the question of how such site specificity is achieved. A new study identified the protein inhibitor of growth 1 (Ing1) as a reader of the active chromatin mark histone H3 lysine 4 trimethylation (H3K4me3). Ing1 binds and directs Gadd45a to target sites, thus linking the histone code with DNA demethylation.     

PGC7/Stella protects against DNA demethylation in early embryogenesis

DNA methylation is an important means of epigenetic gene regulation and must be carefully controlled as a prerequisite for normal early embryogenesis. Although global demethylation occurs soon after fertilization, it is not evenly distributed throughout the genome. Genomic imprinting and epigenetic asymmetry between parental genomes, that is, delayed demethylation of the maternal genome after fertilization, are clear examples of the functional importance of DNA methylation. Here, we show that PGC7/Stella, a maternal factor essential for early development, protects the DNA methylation state of several imprinted loci and epigenetic asymmetry. After determining that PGC7/Stella binds to Ran binding protein 5 (RanBP5; a nuclear transport shuttle protein), mutant versions of the two proteins were used to examine exactly when and where PGC7/Stella functions within the cell. It is likely that PGC7/Stella protects the maternal genome from demethylation only after localizing to the nucleus, where it maintains the methylation of several imprinted genes. These results demonstrate that PGC7/Stella is indispensable for the maintenance of methylation involved in epigenetic reprogramming after fertilization.     

Structural and Functional Coordination of DNA and Histone Methylation

One of the most fundamental questions in the control of gene expression in mammals is how epigenetic methylation patterns of DNA and histones are established, erased, and recognized. This central process in controlling gene expression includes coordinated covalent modifications of DNA and its associated histones. This article focuses on structural aspects of enzymatic activities of histone (arginine and lysine) methylation and demethylation and functional links between the methylation status of the DNA and histones. An interconnected network of methyltransferases, demethylases, and accessory proteins is responsible for changing or maintaining the modification status of specific regions of chromatin.     

Nutrient-dependent methylation of a membrane-associated protein of Escherichia coli.

Starvation of a mid-log-phase culture of Escherichia coli B/r for nitrogen, phosphate, or carbon resulted in methylation of a membrane-associated protein of about 43,000 daltons (P-43) in the presence of chloramphenicol and [methyl-3H]methionine. The in vivo methylation reaction occurred with a doubling time of 2 to 5 min and was followed by a slower demethylation process. Addition of the missing nutrient to a starving culture immediately prevented further methylation of P-43. P-43 methylation is not related to the methylated chemotaxis proteins because P-43 is methylated in response to a different spectrum of nutrients and because P-43 is methylated on lysine residues. The characteristics of P-43 are similar to those of a methylated protein previously described in Bacillus subtilis and B. licheniformis (R. W. Bernlohr, A. L. Saha, C. C. Young, B. R. Toth, and K. J. Golden, J. Bacteriol. 170:4113-4118, 1988; K. J. Golden and R. W. Bernlohr, Mol. Gen. Genet. 220:1-7, 1989) and are consistent with the proposal that methylation of this protein functions in nutrient sensing.     

Large-scale global identification of protein lysine methylation in vivo

Lysine methylation mediated by methyltransferase enzymes is present on multiple proteins throughout the cell; however, methods to uncover and characterize global protein lysine methylation patterns do not readily exist. Here we developed pan-specific methyl lysine antibodies that we utilized in immunoprecipitation experiments coupled with mass spectrometry to yield one of the first large-scale surveys of protein lysine methylation in vivo. In total, 552 different lysine methylation sites were determined, making this one of the most comprehensive global studies published to date. The large majority of these sites have not been yet reported. These sites showed significantly enriched sequence motifs and resided in proteins that are involved in diverse biological processes, particularly in chromatin organization. Our data provide a comprehensive view of lysine methylation in human cells and a powerful resource to facilitate investigations into the function of lysine methylation on non-histone proteins.     

DNA羟甲基化修饰在消化系统肿瘤中的研究进展

DNA羟甲基化修饰是基因组表观遗传学的重要调控方式,指5-甲基胞嘧啶(5-m C)在TET蛋白家族的催化作用下氧化生成5-羟甲基胞嘧啶(5-hm C),完成DNA胞嘧啶的去甲基化过程。基因组甲基化异常导致了多种肿瘤的发生,羟甲基化修饰作为去甲基化的一种,同样与肿瘤发生密不可分。在消化系统肿瘤发生发展过程中存在5-hm C含量的变化,其原因可能与TET蛋白家族、IDH突变等密切相关,提示DNA羟甲基化修饰参与了消化系统肿瘤的发生发展过程。本文围绕DNA羟甲基化修饰与消化系统肿瘤之间的关系进行综述,旨在为消化系统肿瘤羟甲基化修饰研究提供新方向。