Epigenetic regulation of planarian stem cells by the SET1/MLL family of histone methyltransferases

Chromatin regulation is a fundamental mechanism underlying stem cell pluripotency, differentiation, and the establishment of cell type-specific gene expression profiles. To examine the role of chromatin regulation in stem cells in vivo, we study regeneration in the freshwater planarian Schmidtea mediterranea. These animals possess a high concentration of pluripotent stem cells, which are capable of restoring any damaged or lost tissues after injury or amputation. Here, we identify the S. mediterranea homologs of the SET1/MLL family of histone methyltransferases and COMPASS and COMPASS-like complex proteins and investigate their role in stem cell function during regeneration. We identified six S. mediterranea homologs of the SET1/MLL family (set1, mll1/2, trr-1, trr-2, mll5–1 and mll5–2), characterized their patterns of expression in the animal, and examined their function by RNAi. All members of this family are expressed in the stem cell population and differentiated tissues. We show that set1, mll1/2, trr-1, and mll5–2 are required for regeneration and that set1, trr-1 and mll5–2 play roles in the regulation of mitosis. Most notably, knockdown of the planarian set1 homolog leads to stem cell depletion. A subset of planarian homologs of COMPASS and COMPASS-like complex proteins are also expressed in stem cells and implicated in regeneration, but the knockdown phenotypes suggest that some complex members also function in other aspects of planarian biology. This work characterizes the function of the SET1/MLL family in the context of planarian regeneration and provides insight into the role of these enzymes in adult stem cell regulation in vivo.     

Cancers and the NSD family of histone lysine methyltransferases

Both genetic and epigenetic alterations are responsible for the stepwise initiation and progression of cancers. Only epigenetic aberrations can be reversible, allowing the malignant cell population to revert to a more benign phenotype. The epigenetic therapy of cancers is emerging as an effective and valuable approach to both the chemotherapy and the chemoprevention of cancer. The utilization of epigenetic targets that include histone methyltransferase (HMTase), Histone deacetylatase, and DNA methyltransferase, are emerging as key therapeutic targets. The nuclear receptor binding SET domain (NSD) protein is a family of three HMTases, NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1, and plays a critical part in chromatin integrity as evidenced by a growing number of conditions linked to the alterations and/or amplification of NSD1, NSD2, and/or NSD3. NSD1, NSD2 and NSD3 are associated with multiple cancers. The amplification of either NSD1 or NSD2 triggers the cellular transformation and thus is key in the early carcinogenesis events. In most cases, reducing the levels of NSD proteins would suppress cancer growth. NSD1 and NSD2 were isolated as genes linked to developmental diseases, such as Sotos syndrome and Wolf-Hirschhorn syndrome, respectively, implying versatile aspects of the NSD proteins. The NSD pathways, however, are not well understood. It is noteworthy that the NSD family is phylogenetically distinct compared to other known lysine-HMTases, Here, we review the current knowledge on NSD1/NSD2/NSD3 in tumorigenesis and prospect their special value for developing novel anticancer drugs.     

Product specificity and mechanism of protein lysine methyltransferases: insights from the histone lysine methyltransferase SET8

Molecular dynamics simulations employing a molecular mechanics (MM) force field and hybrid quantum mechanics (QM) and MM (QM/MM) have been carried out to investigate the product specificity and mechanism of the histone H4 lysine 20 (H4-K20) methylation by human histone lysine methyltransferase SET8. At neutral pH, the target lysine is available to only the enzyme in the protonated state. The first step in the methylation reaction must be deprotonation of the lysine target which is followed by the (+)AdoMet methylation of the neutral lysine [Enz.Lys-CH(2)-NH(3)(+).(+)AdoMet --> H(+) + Enz.Lys-CH(2)-NH(2).(+)AdoMet -->--> Enz.Lys-CH(2)-N(Me)H(2)(+).AdoHcy]. The electrostatic interactions between two positive charges on (+)AdoMet and Lys20-NH(3)(+) decrease the pK(a) of Lys20-NH(3)(+). Upon formation of Enz.Lys-NH(3)(+).(+)AdoMet, a water channel by which the proton escapes to the outer solvent phase is formed. The formation of a water channel for the escape of a proton from Lys20-N(Me)H(2)(+) in Enz.Lys20-N(Me)H(2)(+).(+)AdoMet is not formed because the methyl substituent blocks the starting of the water channel. Thus, a second methylation does not take place. The dependence of the occurrence of methyl transfer on the formation of a water channel in SET8 is in accord with our previous reports on product specificity by histone lysine monomethyltransferase SET7/9, large subunit lysine dimethyltransferase (LSMT), and viral histone lysine trimethyltransferase (vSET). The average value of the experimental DeltaG(E)() for the six lysine methyl transfer reactions catalyzed by vSET, LSMT, and SET7/9 with p53 as a substrate is 22.1 +/- 1.0 kcal/mol, and the computed average (DeltaG(C)()) is 22.2 +/- 0.8 kcal/mol. In this study, the computed free energy barrier of the methyl transfer reaction [Lys20-NH(2) + (+)AdoMet --> Lys20-N(Me)H(2)(+) + AdoHcy] catalyzed by SET8 is 20.8 kcal/mol. This is in agreement with the value of 20.6 kcal/mol calculated from the experimental rate constant (0.43 +/- 0.02 min(-1)). Our bond-order computations establish that the H4-K20 monomethylation in SET8 is a concerted linear S(N)2 displacement reaction.     

Molecular recognition of histone H3 by the WD40 protein WDR5

The WD40-repeat protein WDR5 is a conserved subunit of Trithorax (TRX) histone methyltransferase complexes. WDR5 has been reported to selectively bind dimethylated Lys4 (K4me2) in histone H3 to promote K4 trimethylation by TRX. To elucidate the basis of this binding specificity, we have determined the crystal structure of WDR5 bound to a histone H3 peptide bearing K4me2. The structure reveals that the N terminus of histone H3 binds as a 3(10)-helix in the central depression formed by the WD40 repeats. R2 in histone H3 is bound in the acidic channel in the protein's core, whereas K4me2 is solvent exposed and does not engage in direct interactions with WDR5. Functional studies confirm that WDR5 recognizes A1, R2 and T3 in histone H3 but has virtually identical affinities for the unmodified and mono-, di- and trimethylated forms of K4, demonstrating that it does not discriminate among different degrees of methylation of this residue.     

The DNA methyltransferases associate with HP1 and the SUV39H1 histone methyltransferase

The DNA methyltransferases, Dnmts, are the enzymes responsible for methylating DNA in mammals, which leads to gene silencing. Repression by DNA methylation is mediated partly by recruitment of the methyl-CpG-binding protein MeCP2. Recently, MeCP2 was shown to associate and facilitate histone methylation at Lys9 of H3, which is a key epigenetic modification involved in gene silencing. Here, we show that endogenous Dnmt3a associates primarily with histone H3-K9 methyltransferase activity as well as, to a lesser extent, with H3-K4 enzymatic activity. The association with enzymatic activity is mediated by the conserved PHD-like motif of Dnmt3a. The H3-K9 histone methyltransferase that binds Dnmt3a is likely the H3-K9 specific SUV39H1 enzyme since we find that it interacts both in vitro and in vivo with Dnmt3a, using its PHD-like motif. We find that SUV39H1 also binds to Dnmt1 and, consistent with these interactions, SUV39H1 can purify DNA methyltransferase activity from nuclear extracts. In addition, we show that HP1β, a SUV39H1-interacting partner, binds directly to Dnmt1 and Dnmt3a and that native HP1β associates with DNA methyltransferase activity. Our data show a direct connection between the enzymes responsible for DNA methylation and histone methylation. These results further substantiate the notion of a self-reinforcing repressive chromatin state through the interplay between these two global epigenetic modifications.     

E2F1 mediates DNA damage and apoptosis through HCF‐1 and the MLL family of histone methyltransferases

E2F1 is a key positive regulator of human cell proliferation and its activity is altered in essentially all human cancers. Deregulation of E2F1 leads to oncogenic DNA damage and anti‐oncogenic apoptosis. The molecular mechanisms by which E2F1 mediates these two processes are poorly understood but are important for understanding cancer progression. During the G1‐to‐S phase transition, E2F1 associates through a short DHQY sequence with the cell‐cycle regulator HCF‐1 together with the mixed‐lineage leukaemia (MLL) family of histone H3 lysine 4 (H3K4) methyltransferases. We show here that the DHQY HCF‐1‐binding sequence permits E2F1 to stimulate both DNA damage and apoptosis, and that HCF‐1 and the MLL family of H3K4 methyltransferases have important functions in these processes. Thus, HCF‐1 has a broader role in E2F1 function than appreciated earlier. Indeed, sequence changes in the E2F1 HCF‐1‐binding site can modulate both up and down the ability of E2F1 to induce apoptosis indicating that HCF‐1 association with E2F1 is a regulator of E2F1‐induced apoptosis.     

组蛋白甲基转移酶的研究进展

组蛋白的甲基化修饰主要是由一类含有SET结构域的蛋白来执行的, 组蛋白甲基化修饰参与异染色质形成、基因印记、X染色体失活和转录调控等多种主要生理功能, 组蛋白的修饰作用是表观遗传学研究的一个重要领域。组蛋白甲基化的异常与肿瘤发生等多种人类疾病相关, 可以特异性地激活或者抑制基因的转录活性。研究发现, 组蛋白甲基转移酶的作用对象不仅仅限于组蛋白, 某些非组蛋白也可以被组蛋白甲基转移酶甲基化, 这将为探明细胞内部基因转录、信号转导、甚至个体的发育和分化机制提供更广阔的空间。     

WRAD: enabler of the SET1-family of H3K4 methyltransferases

Methylation of histone H3 at lysine 4 (H3K4) is a conserved feature of active chromatin catalyzed by methyltransferases of the SET1-family (SET1A, SET1B, MLL1, MLL2, MLL3 and MLL4 in humans). These enzymes participate in diverse gene regulatory networks with a multitude of known biological functions, including direct involvement in several human disease states. Unlike most lysine methyltransferases, SET1-family enzymes are only fully active in the context of a multi-subunit complex, which includes a protein module comprised of WDR5, RbBP5, ASH2L and DPY-30 (WRAD). These proteins bind in close proximity to the catalytic SET domain of SET1-family enzymes and stimulate H3K4 methyltransferase activity. The mechanism by which WRAD promotes catalysis involves elements of allosteric control and possibly the utilization of a second H3K4 methyltransferase active site present within WRAD itself. WRAD components also engage in physical interactions that recruit SET1-family proteins to target sites on chromatin. Here, the known molecular mechanisms through which WRAD enables the function of SET1-related enzymes will be reviewed.     

Structural basis for molecular recognition and presentation of histone H3 by WDR5

Histone methylation at specific lysine residues brings about various downstream events that are mediated by different effector proteins. The WD40 domain of WDR5 represents a new class of histone methyl-lysine recognition domains that is important for recruiting H3K4 methyltransferases to K4-dimethylated histone H3 tail as well as for global and gene-specific K4 trimethylation. Here we report the crystal structures of full-length WDR5, WDR5Delta23 and its complexes with unmodified, mono-, di- and trimethylated histone H3K4 peptides. The structures reveal that WDR5 is able to bind all of these histone H3 peptides, but only H3K4me2 peptide forms extra interactions with WDR5 by use of both water-mediated hydrogen bonding and the altered hydrophilicity of the modified lysine 4. We propose a mechanism for the involvement of WDR5 in binding and presenting histone H3K4 for further methylation as a component of MLL complexes.     

The trxG family histone methyltransferase SET DOMAIN GROUP 26 promotes flowering via a distinctive genetic pathway

Histone methylation is a major component in numerous processes such as determination of flowering time, which is fine‐tuned by multiple genetic pathways that integrate both endogenous and environmental signals. Previous studies identified SET DOMAIN GROUP 26 (SDG26) as a histone methyltransferase involved in the activation of flowering, as loss of function of SDG26 caused a late‐flowering phenotype in Arabidopsis thaliana. However, the SDG26 function and the underlying molecular mechanism remain largely unknown. In this study, we undertook a genetic analysis by combining the sdg26 mutant with mutants of other histone methylation enzymes, including the methyltransferase mutants Arabidopsis trithorax1 (atx1), sdg25 and curly leaf (clf), as well as the demethylase double mutant lsd1‐like1 lsd1‐like2 (ldl1 ldl2). We found that the early‐flowering mutants sdg25, atx1 and clf interact antagonistically with the late‐flowering mutant sdg26, whereas the late‐flowering mutant ldl1 ldl2 interacts synergistically with sdg26. Based on microarray analysis, we observed weak overlaps in the genes that were differentially expressed between sdg26 and the other mutants. Our analyses of the chromatin of flowering genes revealed that the SDG26 protein binds at the key flowering integrator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1/AGAMOUS‐LIKE 20 (SOC1/AGL20), and is required for histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 36 trimethylation (H3K36me3) at this locus. Together, our results indicate that SDG26 promotes flowering time through a distinctive genetic pathway, and that loss of function of SDG26 causes a decrease in H3K4me3 and H3K36me3 at its target gene SOC1, leading to repression of this gene and the late‐flowering phenotype.     

Dynamic behavior of the post‐SET loop region of NSD1: Implications for histone binding and drug development

NSD1 is a SET‐domain histone methyltransferase that methylates lysine 36 of histone 3. In the crystal structure of NSD1, the post‐SET loop is in an autoinhibitory position that blocks binding of the histone peptide as well as the entrance to the lysine‐binding channel. The conformational dynamics preceding histone binding and the mechanism by which the post‐SET loop moves to accommodate the target lysine is currently unknown, although potential models have been proposed. Using molecular dynamics simulations, we have identified potential conformations of the post‐SET loop differing from those of previous studies, as well as proposed a model of peptide‐bound NSD1. Our simulations illustrate the dynamic behavior of the post‐SET loop and the presence of a few distinct conformations. In every case, the post‐SET loop remains in an autoinhibitory position blocking the peptide‐binding cleft, suggesting that another interaction is required to optimally position NSD1 in an active conformation. This finding provides initial evidence for a mechanism by which NSD1 preferentially binds nucleosomal substrates.