原核DNA甲基化的表观调控作用的研究进展

自从2009年第三代DNA测序技术平台商业化以来,测序、绘制原核DNA甲基化组飞速发展,10余年来已完成甲基化组测序的细菌超过4000种,极大推动了原核表观遗传学的研究.越来越多的研究表明,DNA甲基化修饰不仅仅局限于宿主的防御功能,而且广泛参与各种细胞过程及基因的表达调控,在染色体的复制起始、细胞周期、致病性、抗生素抗性等方面起到了重要的作用.在简要回顾原核DNA甲基转移酶及其甲基化修饰的相关研究的基础上,着重对近期原核甲基化修饰的调控作用的研究进展进行综述,以期推动原核甲基化修饰表观遗传的研究.

三种四膜虫全基因组N^(6)-甲基腺嘌呤比较研究

为了揭示N^(6)-甲基腺嘌呤(6mA)在近缘物种间的分布规律和物种进化过程中的变化,研究在分离2种四膜虫(Tetrahymena malaccensis和Tetrahymena pyriformi)大核的基础上,利用三代测序技术绘制了其全基因组单碱基分辨率6mA图谱,结合公共数据库中已有的模式种Tetrahymena thermophila数据,开展了3种四膜虫比较6mA甲基化组分析,发现:(1)3种四膜虫6mA甲基化位点分布特征类似,包括呈约200 bp周期性分布和具有保守AT基序;(2)6mA主要分布于基因的5′端,在种间直系同源基因上的分布模式具有区域近似性,但在单碱基水平不完全保守;(3)在种内近期复制的并系同源基因中,6mA分布在单碱基水平具有较高的保守性;(4)结合3种四膜虫的分化时间,估算出了四膜虫中6mA动态变化过程,6mA位点建立的速度大约为每Mb每百万年69.9—226个位点。

Characterization of Two Groups of Low_copy and Specific DNA Sequences Isolated from Chromosome 7B of Common Wheat

Recent work revealed that, in the genomes of polyploid wheat, there exists a class of low_copy and chromosome_specific sequences that are labile upon polyploid formation. This class of sequences was proposed to play a critical role in the stabilization and establishment of nascent plant polyploids as new species. To further study this issue, five wheat chromosome 7B_specific sequences, isolated from common wheat (Triticum aestivum L.) by chromosome microdissection, were characterized. The sequences were studied by genomic Southern hybridizations on a collection of polyploid wheats and their diploid progenitors. Four sequences hybridized to all polyploid species, but at the diploid level to only species closely related to the B_genome of polyploid wheat. This indicates that these sequences originated with the divergence of the diploid species, and was then vertically transmitted to polyploids. One sequence hybridized to all species at both the diploid and polyploid levels, suggesting its elimination after the polyploid wheat formation. The hybridization of this sequence to two synthetic polyploid wheats indicated that sequence elimination is a rapid event and probably related to methylation status of the sequence. Based on the above results, we suggest that selective changes of low_copy sequences occur rapidly after polyploid formation, which may contribute to the differentiation of chromosomes in newly formed allopolyploid wheats.

Epigenetic modification regulates both expression of tumor-associated genes and cell cycle progressing in human colon cancer cell lines: Colo-320 and SW1116

The aim of this study is to assess the effects of DNA methylation and historic acetylation, alone or in combination, on the expression of several tumor-associated genes and cell cycle progression in two established human colon cancer cell lines: Colo-320 and SW1116. Treatments with 5-aza-2'-deoxycytidine (5-aza-dC) and trichostatin A, alone or in combination, were applied respectively. The methylation status of the CDKN2A promoter was determined by methyla-tion-specific PCR, and the acetylated status of the histones associated with the p21WAF1 and CDKN2A genes was examined by chromatin immunoprecipitation. The expression of the CDKN2A, p21WAF1, p53, p73, APC, c-myc, c-Ki-ras and survivin genes was detected by real-time RT-PCR and RT-PCR. The cell cycle profile was established by flow cytometry. We found that along with the demethylation of the CDKN2A gene promoter in both cell lines induced by 5-aza-dC alone or in combination with TSA, the expression of both CDKN2A and APC genes increased. The treatment of TSA or sodium butyrate up-regulated the transcription of p21WAF1 significantly by inducing the acetylation of histones H4 and H3, but failed to alter the acetylation level of CDKN2A-associated histones. No changes in transcription of p53, p73, c-myc, c-Ki-ras and survivin genes were observed. In addition, TSA or sodium butyrate was shown to arrest cells at the G1 phase. However, 5-aza-dC was not able to affect the cell cycle progression. In conclusion, regulation by epigenetic modification of the transcription of tumor-associated genes and the cell cycle progression in both human colon cancer cell lines Colo-320 and SW1116 is gene-specific.

DNA methylation in hepatocellular carcinoma

As for many other tumors,development of hepatocellular carcinoma(HCC)must be understood as a multistep process with accumulation of genetic and epigenetic alterations in regulatory genes,leading to activation of oncogenes and inactivation or loss of tumor suppressor genes(TSG).In the last decades,in addition to genetic alterations,epigenetic inactivation of(tumor suppressor) genes by promoter hypermet hylation has been recognized as an important and alternative mechanism in tumorigenesis.In HCC,aberrant methylation of promoter sequences occurs not only in advanced tumors, it has been also observed in premalignant conditions just as chronic viral hepatitis B or C and cirrhotic liver. This review discusses the epigenetic alterations in hepatocellular carcinoma focusing DNA methylation.

Bioinformatics Analysis on Histone H3-lys-4 Methyltransferase MLL3

[Objective] This study aimed to conduct bioinformatics analysis of histone H3-1ys-4 （H3K4） methyltransferase MLL3 in animals, thus exploring its relatively conservative evolution to reveal the role of histon H3K4 trimethyltransferase MLL3 in human cancers. [Method] By using bioinformatics method, gene structure, amino acid sequences, phylogenetic tree, chromosomal localization and synteny of mouse MLL3 were analyzed. [Result] Primary structure of the encoded mouse MLL3 protein con- tained seven zinc finger domains, an HMG-box （High mobility group-box protein）, a FYRN （F/Y-rich N-terminus） domain, a FYRC （F/Yrich C-terminus） domain, a SET domain and a postSET domain. Results of sequence comparison and homology showed that 19 animal species in this study all had these structures basically, which indicated that these structures were relatively conserved in the evolution; specifically, the SET domain was highly conserved and was necessary to maintain the activity of histone methyltransferases. Results of phylogenetic analysis showed that the loca- tions of the 19 animal species in evolutionary tree were consistent with the taxo- nomic status. Results of synteny analysis showed that there were the same gene in the upstream and downstream of the mouse and human MLL3 gene which were located on different chromosomes, indicating that the mouse and human MLL3 gene had collinearity. [Conclusion] This study had revealed the primary structure of MLL3 nucleotide sequence and amino acid sequence, which had not only laid the foundation for the future research of high-level structure and function of MLL3 protein but also provided the basis for the follow-up study of primer design, promoter analysis, gene cloning and regulation patterns of localization and expression of mouse MLL3 gene.

Transcriptional changes in epigenetic modifiers associated with gene silencing in the intestine of the sea cucumber,Apostichopus japonicus(Selenka),during aestivation

The sea cucumber, Apostichopusjaponicus, undergoes aestivation to improve survival during periods of high-temperature. During aestivation, the metabolic rate is depressed to reduce the consumption of reserved energy. We evaluated the role of epigenetic modification on global gene silencing during metabolic rate depression in the sea cucumber. We compared the expression of epigenetic modifiers in active and aestivating sea cucumbers. The expression of three genes involved in DNA methylation and chromatin remodeling （DNA （cytosine-5）-methyltransferase l, Methyl-CpG-binding domain protein 2）, and Chromodomain-helicase-DNA-binding protein 5） was significantly higher during aestivation （Days 20 and 40）. Similarly, we observed an increase in the expression of genes involved in histone acetylation （Histone deacetylase 3） and Histone-binding protein RBBP4） during the early （Days 5 and 10） and late phases （Days 20 and 40） of aestivation. There was no change in the expression of KAT2B, a histone acetyltransferase. However, the expression of histone methylation associated modifiers （Histone-arginine methyltransferase CARMER and Histone-lysine N-methyltransferase MLL5） was significantly higher after 5 d in the aestivating group. The results suggest that the expression of epigenetic modifiers involved in DNA methylation, chromatin remodeling, histone acetylation, and histone methylation is upregulated during aestivation. We hypothesize that these changes regulate global gene silencing during aestivation in A. japonicus.

Folate levels in mucosal tissue but not methylenetetrahydrofolate reductase polymorphisms are associated with gastric carcinogenesis

AIM: To evaluate whether folate levels in mucosal tissue and some common methylenetetrahydrofolate reductase (MTHFR) variants are associated with the risk of gastric cancer through DNA methylation. METHODS: Real-time PCR was used to study the expression of tumor related genes in 76 mucosal tissue samples from 38 patients with gastric cancer. Samples from the gastroscopic biopsy tissues of 34 patients with chronic superficial gastritis (CSG) were used as controls. Folate concentrations in these tissues were detected by the FOL ACS: 180 automated chemiluminescence system. MTHFR polymorphisms were analyzed by PCR-RFLP, and the promoter methylation of tumor-related genes was determined by methylation-specific PCR (MSP). RESULTS: Folate concentrations were significantly higher in CSG than in cancerous tissues. Decreased expression and methylation of c-myc accompanied higher folate concentrations. Promoter hypermethylation and loss of p16INK4A in samples with MTHFR 677CC were more frequent than in samples with the 677TT or 677CT genotype. And the promoter hypermethylation and loss of p21WAF1 in samples with MTHFR 677CT were more frequent than when 677CC or 677TT was present. The 677CT genotype showed a non-significant higher risk for gastric cancer as compared with the 677CC genotype. CONCLUSION: Lower folate levels in gastric mucosal tissue may confer a higher risk of gastric carcinogenesisthrough hypomethylation and overexpression of c-myc.

JMJD3 is a histone H3K27 demethylase

Histone methylation is an important epigenetic phenomenon that participates in a diverse array of cellular processes and has been found to be associated with cancer. Recent identification of several histone demethylases has proved that histone methylation is a reversible process. Through a candidate approach, we have biochemically identified JMJD3 as an H3K27 demethylase. Transfection of JMJD3 into HeLa cells caused a specific reduction oftrimethyl H3K27, but had no effect on di-and monomethyl H3K27, or histone lysine methylations on H3K4 and H3K9. The enzymatic activity requires the JmjC domain and the conserved histidine that has been suggested to be important for a cofactor binding. In vitro biochemical experiments demonstrated that JMJD3 directly catalyzes the demethylation. In addition, we found that JMJD3 is upregulated in prostate cancer, and its expression is higher in metastatic prostate cancer. Thus, we identified JMJD3 as a demethylase capable of removing the trimethyl group from histone H3 lysine 27 and upregulated in prostate cancer.

Genomic Imprinting—The Story of the Other Half and the Conflicts of Silencing

G-enomic imprinting is an epigenetic mechanism that produces functional differences between the paternal and mammal genomes and plays an essential role in mammalian development and growth. There are a number of genes in our genomes that are subject to genomic imprinting where one parent＇s copy of the gene is expressed while the other is silent. Silencing of one allele predetermines that any function ascribed to that gene are now dependant on the single active copy. Possession of only a single active allele can lead to deleterious health consequences in humans. If imprinted genes are crucial in mammalian development, one would also expect mutations in these genes to cause diseases. Since imprinting is an epigenetic mechanism, mistakes in maintaining epigenetic mark also cause imprinting disorders. Here we in this review focus on the current understanding of this unique genetic mechanism more than two decades after the first description of the imprinting phenomenon was given by McGrath and Solter. Although the possible molecular mechanisms by which imprinting is imposed and maintained are being identified, we have a long way to go in understanding the molecular mechanisms that regulate the expression of these oddly behaving genes, the function of imprinting and the evolution. Post genomic technologies might ultimately lead to a better understanding of the ＇imprinting effects＇.

Characterization of MyDNMT1 and Changes of Global DNA Methylation during the Embryonic Development in Mizuhopecten yessoensis

DNA methylation is a critical epigenetic mechanism that influences gene transcription, genomic stability, X-chromosome inactivation and other factors, and appropriate DNA methylation is crucial in development. DNA methyltransferase 1 （DNMT1） plays an important role in maintaining the established methylation pattern during DNA replication. Although the effect of DNA methylation on embryonic development has been well known in vertebrates, little research has been carried out in invertebrates, especially in marine bivalves. In this study, the DNMT1 gene （MyDNMT1） was firstly identified from Mizuhopecten yessoensis. The full-length cDNA of MyDNMT1 was 5 039 bp, consisted of a 5＇ untranslated region （5＇-UTR） of 79 bp, a 3＇ untranslated region （3＇-UTR） of 199 bp, and a 4 761 bp open reading frame （ORF） encoding a peptide of 1 586 amino acids without a putative signal peptide. The relative mRNA expression level of MyDNMT1 was measured during the embryonic development of M. ydssoensis using real-time PCR, which revealed that the level at stage zygote and trochophore were significantly higher than that at other stages. We further examined the global DNA methylation during development by colorimetric method. The results showed that the methylation level was increased and reached the peak at blastula stage, then dramatically decreased, and fluctuated at early D-shaped larva stage. This study provided greater insight into the DNA methylation of embryonic development, which obtained a better understanding of the relationship between the DNA methylation and the embryonic development in bivalve mollusks.

Methylation of TIMP3 in esophageal squamous cell carcinoma

AIM: To measure the frequency of DNA methylation of the tissue inhibitor of metalloproteinase 3 (TIMP3) promoter and relate this to any change of gene expression in esophageal squamous cell carcinoma in patients from a region of high incidence in China.METHODS: Cancer cell lines were treated with or without the demethylating reagent 5-aza-2’-deoxycytidine. Methylation of the TIMP3 promoter was assessed in three regions by melt curve analysis and its expression was assessed by real-time RT-PCR. Tumors and proximal resection margins were obtained from 64 patients with esophageal squamous cell carcinoma from a region of high incidence in China. Methylation was assessed by melt curve analysis and expression by immunohistochemistry.RESULTS: Methylation in one of the three promoter regions assessed correlated with gene silencing in esophageal cell lines. A degree of methylation of TIMP3 was found in only four esophageal squamous cell carcinomas, and partial loss of TIMP3 protein expression in just one.CONCLUSION: Methylation and loss of expression of TIMP3 occurs infrequently in esophageal squamous cell carcinoma in a region of high incidence in China.

Effects of histone acetylation and DNA methylation on p21^(WAF1)regulation

Cell cycle progression is regulated by interactions between cyclins and cyclin-dependent kinases (CDKs). p21(WAF1) is one of the CIP/KIP family which inhibits CDKs activity. Increased expression of p21(WAF1) may play an important role in the growth arrest induced in transformed cells. Although the stability of the p21( WAF1) mRNA could be altered by different signals, cell differentiation and numerous influencing factors. However, recent studies suggest that two known mechanisms of epigenesis, i.e.gene inactivation by methylation in promoter region and changes to an inactive chromatin by histone deacetylation, seem to be the best candidate mechanisms for inactivation of p21( WAF1). To date, almost no coding region p21(WAF1) mutations have been found in tumor cells, despite extensive screening of hundreds of various tumors. Hypermethylation of the p21(WAF1) promoter region may represent an alternative mechanism by which the p21(WAF1/CIP1) gene can be inactivated. The reduction of cellular DNMT protein levels also induces a corresponding rapid increase in the cell cycle regulator p21(WAF1) protein demonstrating a regulatory link between DNMT and p21(WAF1) which is independent of methylation of DNA. Both histone hyperacetylation and hypoacetylation appear to be important in the carcinoma process, and induction of the p21(WAF1) gene by histone hyperacetylation may be a mechanism by which dietary fiber prevents carcinogenesis. Here, we review the influence of histone acetylation and DNA methylation on p21(WAF1) transcription, and affection of pathways or factors associated such as p 53, E2A, Sp1 as well as several histone deacetylation inhibitors.

P16 gene hypermethylation and hepatocellular carcinoma:A systematic review and meta-analysis

AIM:To quantitatively investigate the effect of p16 hypermethylation on hepatocellular carcinoma (HCC) and hepatocirrhosis using a meta-analysis of available casecontrol studies.METHODS:Previous studies have primarily evaluated the incidence of p16 hypermethylation in HCC and corresponding control groups,and compared the incidence of p16 hypermethylation in tumor tissues,pericancer liver tissues,normal liver tissues and non-tumor liver tissues with that in other diseases.Data regarding publication information,study characteristics,and incidence of p16 hypermethylation in both groups were collected from these studies and summarized.RESULTS:Fifteen studies,including 744 cases of HCC and 645 non-tumor cases,were identified for meta-analysis.Statistically significant odds ratios (ORs) of p16 hypermethylation were obtained from tumor tissues and non-tumorous liver tissues of HCC patients (OR 7.04,95% CI:3.87%-12.78%,P < 0.0001),tumor tissues of HCC patients and healthy liver tissues of patients with other diseases (OR 12.17,95% CI:6.64%-22.31%,P < 0.0001),tumor tissues of HCC patients and liver tissues of patients with non-tumorous liver diseases (OR 6.82,95% CI:4.31%-10.79%,P < 0.0001),and cirrhotic liver tissues and non-cirrhotic liver tissues (OR 4.96,95% CI:1.45%-16.96%,P=0.01).The pooled analysis showed significantly increased ORs of p16 hypermethylation (OR 6.98,95% CI:4.64%-10.49%,P < 0.001) from HCC tissues and cirrhotic tissues.CONCLUSION:P16 hypermethylation induces the inactivation of p16 gene,plays an important role in hepatocarcinogenesis,and is associated with an increased risk of HCC and liver cirrhosis.

Coordinated regulation of active and repressive histone methylations by a dual-specificity histone demethylase ceKDM7A from Caenorhabditis elegans

H3K9me2 and H3K27me2 are important epigenetic marks associated with transcription repression, while H3K4me3 is associated with transcription activation. It has been shown that active and repressive histone methylations distribute in a mutually exclusive manner, but the underlying mechanism was poorly understood. Here we identified ceKDM7A, a PHD （plant homeodomain）- and JmjC domain-containing protein, as a histone demethylase specific for H3K9me2 and H3K27me2. We further demonstrated that the PHD domain of ceKDM7A bound H3K4me3 and H3K4me3 co-localized with ceKDM7A at the genome-wide level. Disruption of the PHD domain binding to H3K4me3 reduced the demethylase activity in vivo, and loss of ceKDM7A reduced the expression of its associated target genes. These results indicate that ceKDM7A is recruited to the promoter to demethylate H3K9me2 and H3K27me2 and activate gene expression through the binding of the PHD domain to H3K4me3. Thus, our study identifies a dual-specificity histone demethylase and provides novel insights into the regulation of histone methylation.

The X-linked mental retardation gene PHF8 is a histone demethylase involved in neuronal differentiation

Recent studies have identified mutations in PHF8, an X-linked gene encoding a JmjC domain-containing protein, as a causal factor for X-linked mental retardation （XLMR） and cleft lip/cleft palate. However, the underlying mechanism is unknown. Here we show that PHF8 is a histone demethylase and coactivator for retinoic acid receptor （RAR）. Although activities for both H3K4me3/2/1 and H3K9me2/1 demethylation were detected in cellularbased assays, reeombinant PHF8 exhibited only H3K9me2/1 demethylase activity in vitro, suggesting that PHF8 is an H3K9me2/1 demethylase whose specificity may be modulated in vivo. Importantly, a mutant PHF8 （phenylalanine at position 279 to serine） identified in the XLMR patients is defective in enzymatie activity, indicating that the loss of histone demethylase activity is causally linked with the onset of disease. In addition, we show that PHF8 binds specifically to H3K4me3/2 peptides via an N-terminal PHD finger domain. Consistent with a role for PHF8 in neuronal differentiation, knockdown of PHF8 in mouse embryonic carcinoma P19 cells impairs RA-induced neuronal differentiation, whereas overexpression of the wild-type but not the F279S mutant PHF8 drives PI9 cells toward neuronal differentiation. Furthermore, we show that PHF8 interacts with RAR~ and functions as a coactivator for RARa. Taken together, our results suggest that histone methylation modulated by PHF8 plays a critical role in neuronal differentiation.

Severity of ulcerative colitis is associated with a polymorphism at diamine oxidase gene but not at histamine N-methyltransferase gene

AIM： To analyse the role of two common polymorphisms in genes coding for histamine metabolising enzymes as it relates to the risk to develop ulcerative colitis （UC） and the clinical course of these patients. METHODS： A cohort of 229 unrelated patients with UC recruited from a single centre and 261 healthy volunteers were analysed for the presence of Thr105Ile and His645Asp amino acid substitutions at histamine N-methyltransferase （HNMT） and diamine oxidase （ABP1） enzymes, respectively, by amplification-restriction procedures. All patients were phenotyped and followed up for at least 2 years （mean time 11 years）. RESULTS： There were no significant differences in the distribution of ABP1 alleles between ulcerative colitis patients and healthy individuals [OR （95% CI） for variant alleles = 1.22 （0.91-1.61）]. However, mutated ABP1 alleles were present with higher frequency among the 58 patients that required immunosuppresive drugs [OR （95 % CI） for carriers of mutated alleles 2.41 （1.21-4.83; P=0.006）], with a significant gene-dose effect （P= 0.0038）. In agreement with the predominant role of ABP1 versus HNMT on local histamine metabolism in human bowel, the frequencies for carriers of HNMT genotypes or mutated alleles were similar among patients,regardless clinical evolution, and control individuals. CONCLUSION： The His645Asp polymorphism of the histamine metabolising enzyme ABP1 is related to severity of ulcerative colitis.

Structural insights into a novel histone demethylase PHF8

Dynamic regulation of histone methylation/demethylation plays an important role during development. Mutations and truncations in human plant homeodomain （PHD） finger protein 8 （PHF8） are associated with X-linked mental retardation and facial anomalies, such as a long face, broad nasal tip, cleft lip/cleft palate and large hands, yet its molecular function and structural basis remain unclear. Here, we report the crystal structures of the catalytic core of PHF8 with or without α-ketoglutarate （α-KG） at high resolution. Biochemical and structural studies reveal that PHF8 is a novel histone demethylase specific for di- and mono-methylated histone H3 lysine 9 （H3K9me2/1）, but not for H3K9me3. Our analyses also reveal how human PHF8 discriminates between methylation states and achieves sequence specificity for methylated H3K9. The in vitro demethylation assay also showed that the F279S mutant observed in clinical patients possesses no demethylation activity, suggesting that loss of enzymatic activity is crucial for pathogenesis of PHF8 patients. Taken together, these results will shed light on the molecular mechanism underlying PHF8-associated developmental and neurological diseases.

Recent advances in fixation of CO_(2) into organic carbamates through multicomponent reaction strategies

Carbon dioxide(CO_(2)) is the main greenhouse gas and also an ideal C1 feedstock in organic synthesis because it is abundant,nontoxic,nonflammable,and renewable.The synthesis of organic carbamates using CO_(2) as a phosgene alternative has attracted extensive attention because of the importance of carbamates in organic synthesis and in the pharmaceutical and agrochemical industries.In recent decades,many multicomponent reaction strategies have been designed for constructing different types of organic carbamate molecules.Most of these methods rely on the in situ generation of carbamate anions from CO_(2) and amines,followed by reactions with other coupling partners.Synthetic strategies for acyclic carbamates include nucleophile‐electrophile coupling,nucleo‐phile‐nucleophile oxidative coupling,difunctionalization of unsaturated hydrocarbons,and C–H bond functionalization.Strategies for the synthesizing cyclic carbamates include carboxylative cyclization of in situ‐generated unsaturated amines and difunctionalization of unsaturated amines with CO_(2) and other electrophilic reagents.This review summarizes the recent advances in the synthesis of organic carbamates from CO_(2) using different multicomponent reaction strategies.Future perspectives and challenges in the incorporation of CO_(2) into carbamates are also presented.