Activity and crystal structure of human thymine DNA glycosylase mutant N140A with 5-carboxylcytosine DNA at low pH

The mammalian thymine DNA glycosylase (TDG) excises 5-carboxylcytosine (5caC) when paired with a guanine in a CpG sequence, in addition to mismatched bases. Here we present a complex structure of the human TDG catalytic mutant, asparagine 140 to alanine (N140A), with a 28-base pair DNA containing a G:5caC pair at pH 4.6. TDG interacts with the carboxylate moiety of target nucleotide 5caC using the side chain of asparagine 230 (N230), instead of asparagine 157 (N157) as previously reported. Mutation of either N157 or N230 residues to aspartate has minimal effect on G:5caC activity while significantly reducing activity on G:U substrate. Combination of both the asparagine-to-aspartate mutations (N157D/N230D) resulted in complete loss of activity on G:5caC while retaining measurable activity on G:U, implying that 5caC can adopt alternative conformations (either N157-interacting or N230-interacting) in the TDG active site to interact with either of the two asparagine side chain for 5caC excision.     

逆境胁迫下植物DNA甲基化变异的研究进展

胁迫是指生物体持续地暴露在环境的刺激下,并且植物有能力建立保护和适应的机制.逆境胁迫抑制生物体的生长、发育和繁殖,它通常决定了物种的分布,更重要的是它对特定的种群提供了一种选择性进化动力.植物可以通过忍受、抗性和避免或最终逃避这三种不同的策略来应对胁迫.DNA甲基化作为一种表观遗传现象,是指在甲基化酶的作用下,不涉及基因的DNA序列改变,而使基因功能发生变化,以对外界的环境刺激作出应答反应.这种变化常常可以传递给后代,并形成表观遗传记忆,这对培育植物抗性新品种提供了可能.综述了植物响应逆境胁迫中的DNA甲基化修饰的研究进展,旨在深入了解DNA甲基化变化对植物抗逆性的影响.     

线粒体DNA与DNA甲基化

线粒体是除细胞核之外唯一携带遗传物质的细胞器,其线粒体DNA（mitochondrial DNA,mtDNA）控制着线粒体一些最基本的性质,对细胞功能有着重要影响.DNA甲基化是调节基因表达的重要方式之一.研究表明mtDNA存在CpG位点的低甲基化,并且mtDNA基因的表达受核DNA（nuclear DNA,nDNA）及线粒体自身DNA甲基化的调控,mtDNA和nDNA协同作用参与机体代谢调节和疾病发生发展过程.就近年来mtDNA与DNA甲基化的关系作一综述.     

DNA甲基化及其对植物发育的调控

DNA甲基化属于一种表观遗传修饰,主要发生在CpG双核苷酸序列中的胞嘧啶上,是在DNA甲基转移酶催化下,以S-腺苷甲硫氨酸为甲基供体,将甲基转移到胞嘧啶上,生成5-甲基胞嘧啶的一种反应。DNA甲基化在植物生长过程中具有极其重要的作用。综述了植物DNA甲基化的特征、调控机制,及其对植物基因表达影响的研究进展。     

The exploration of N6-deoxyadenosine methylation in mammalian genomes

N6-methyladenine (N6-mA,m6dA,or 6mA),a prevalent DNA modification in prokaryotes,has recently been identified in higher eukaryotes,including mammals.Although 6mA has been well-studied in prokaryotes,the function and regulatory mechanism of 6mA in eukary-otes are still poorly understood.Recent studies indicate that 6mA can serve as an epigenetic mark and play critical roles in various biological processes,from transposable-element suppression to environmental stress response.Here,we review the significant advances in methodology for 6mA detection and major progress in understanding the regulation and function of this non-canonical DNA methylation in eukaryotes,predominantly mammals.     

A Dam methylation mutant of Klebsiella pneumoniae is partially attenuated

In Klebsiella pneumoniae, a chromosomal insertion mutation was constructed in the dam gene, which encodes DNA adenine methylase (Dam), resulting in a mutant unable to methylate specific nucleotides. In some bacteria, the Dam methylase has been shown to play an important role in virulence gene regulation as well as in methyl-directed mismatch repair and the regulation of replication initiation. Disruption of the normal Dam function by either eliminating or greatly increasing expression in several organisms has been shown to cause attenuation of virulence in murine models of infection. In K. pneumoniae, a mutation-eliminating Dam function is shown here to result in only partial attenuation following intranasal and intraperitoneal infection of Balb/C mice.     

DNA甲基化与基因表达调控研究进展

表观遗传修饰是指不改变DNA序列的、可遗传的对碱基和组蛋白的化学修饰,主要包括DNA甲基化、组蛋白修饰、染色质重塑以及非编码RNA等.表观遗传修饰是更高层次的基因表达调控手段.DNA甲基化是一种重要的表观遗传修饰,参与基因表达调控、基因印记、转座子沉默、X染色体失活以及癌症发生等重要生物学过程.近年来随着研究方法和技术的进步,全基因组DNA甲基化的研究广泛兴起,多个物种全基因组甲基化图谱被破译,全局水平对DNA甲基化的研究不仅利于在宏观层面上了解DNA甲基化的特性与规律,同时也为深入分析DNA甲基化的生物学功能与调控奠定了基础.结合最新研究进展综述DNA甲基化在基因组中的分布模式、规律以及和基因转录的关系等.     

Characterization of NLRP12 during the In Vivo Host Immune Response to Klebsiella pneumoniae and Mycobacterium tuberculosis

The majority of nucleotide binding domain leucine rich repeats-containing (NLR) family members has yet to be functionally characterized. Of the described NLRs, most are considered to be proinflammatory and facilitate IL-1β production. However, a newly defined sub-group of NLRs that function as negative regulators of inflammation have been identified based on their abilities to attenuate NF-κB signaling. NLRP12 (Monarch-1) is a prototypical member of this sub-group that negatively regulates both canonical and noncanonical NF-κB signaling in biochemical assays and in colitis and colon cancer models. The role of NLRP12 in infectious diseases has not been extensively studied. Here, we characterized the innate immune response of Nlrp12^−/− mice following airway exposure to LPS, Klebsiella pneumoniae and Mycobacterium tuberculosis. In response to E. coli LPS, Nlrp12^−/− mice showed a slight decrease in IL-1β and increase in IL-6 production, but these levels were not statistically significant. During K. pneumoniae infection, we observed subtle differences in cytokine levels and significantly reduced numbers of monocytes and lymphocytes in Nlrp12^−/− mice. However, the physiological relevance of these findings is unclear as no overt differences in the development of lung disease were observed in the Nlrp12^−/− mice. Likewise, Nlrp12^−/− mice demonstrated pathologies similar to those observed in the wild type mice following M. tuberculosis infection. Together, these data suggest that NLRP12 does not significantly contribute to the in vivo host innate immune response to LPS stimulation, Klebsiella pneumonia infection or Mycobacterium tuberculosis.     

Structural biology-based insights into combinatorial readout and crosstalk among epigenetic marks

Epigenetic mechanisms control gene regulation by writing, reading and erasing specific epigenetic marks. Within the context of multi-disciplinary approaches applied to investigate epigenetic regulation in diverse systems, structural biology techniques have provided insights at the molecular level of key interactions between upstream regulators and downstream effectors. The early structural efforts focused on studies at the single domain-single mark level have been rapidly extended to research at the multiple domain–multiple mark level, thereby providing additional insights into connections within the complicated epigenetic regulatory network. This review focuses on recent results from structural studies on combinatorial readout and crosstalk among epigenetic marks. It starts with an overview of multiple readout of histone marks associated with both single and dual histone tails, as well as the potential crosstalk between them. Next, this review further expands on the simultaneous readout by epigenetic modules of histone and DNA marks, thereby establishing connections between histone lysine methylation and DNA methylation at the nucleosomal level. Finally, the review discusses the role of pre-existing epigenetic marks in directing the writing/erasing of certain epigenetic marks. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function.     

DNA甲基化与植物抗逆性研究进展

DNA甲基化是真核细胞基因组重要修饰方式之一.DNA甲基化通过与转录因子相互作用或通过改变染色质结构来影响基因的表达,从表观遗传水平对生物遗传信息进行调节,在生长发育过程中起着重要的作用,而且植物DNA甲基化还参与了环境胁迫下的基因表达调控过程.本文对植物DNA甲基化的产生机制、功能,以及DNA甲基化在植物应对逆境胁迫中的作用进行综述,以更好地理解植物DNA甲基化及其对环境胁迫的响应,为植物抗逆性研究及作物遗传改良提供理论参照.     

Characterization of the genes encoding urease activity of Klebsiella pneumoniae

Abstract The genes encoding urease activity of Klebsiella pneumoniae were cloned and expressed in Escherichia coli . Transformants containing recombinant plasmids were selected by the antibiotic resistance phenotype and the production of urease in a Urease-test agar. Deletion derivatives of the parental recombinant plasmid were construced, and the relative position of six genes, necessary for urease production, was determined. Using a colorimetric assay it was demonstrated that some of the transformants exhibited ureolytic activity up to six-times greater than that of the original K pneumoniae isolate. Dot-blot DNA hybridization analysis revealed that the urease gene cluster of K. pneumoniae possesses no significant homology with those of Proteus species and Morganella morganii .     

DNA甲基化在植物研究中的应用现状与前景

DNA甲基化是主要发生在CpG双核苷酸序列中的胞嘧啶上的一种表面遗传修饰。它以S-腺苷甲硫氨酸为甲基供体,在DNA甲基酶的催化下,将甲基转移到胞嘧啶上,生成5-甲基胞嘧啶。DNA甲基化在植物的很多生命过程中具有重要的作用。本文就其作用机制、主要研究应用以及未来的前景进行简单阐述,从而为DNA甲基化在植物遗传学研究中的研究提供理论参考。     

Updating In Vivo and In Vitro Phosphorylation and Methylation Sites of Voltage‐Gated Kv7.2 Potassium Channels

Voltage‐gated Kv7.2 potassium channels regulate neuronal excitability. The gating of these channels is tightly controlled by various mediators and neurotransmitters acting via G protein‐coupled receptors; the underlying signaling cascades involve phosphatidylinositol‐4,5‐bisphosphate (PIP₂), Ca²⁺/calmodulin, and phosphorylation. Recent studies found that the PIP₂ sensitivity of Kv7.2 channels is affected by two posttranslational modifications, phosphorylation and methylation, harboured within putative PIP₂‐binding domains. In this study, we updated phosphorylation and methylation sites in Kv7.2 either heterologously expressed in mammalian cells or as GST‐fusion proteins exposed to recombinant protein kinases by using LC–MS/MS. In vitro kinase assays revealed that CDK5, protein kinase C (PKC) alpha, PKA, p38 MAPK, CamKIIα, and GSK3β could mediate phosphorylation. Taken together, we provided a comprehensive map of phosphorylation and methylation in Kv7.2 within protein–protein and protein–lipid interaction domains. This may help to interpret the functional roles of individual PTM sites in Kv7.2 channels. All MS data are available via ProteomeXchange with the identifier PXD005567.     

Epigenetic regulation in the carcinogenesis of cholangiocarcinoma

Cholangiocarcinoma (CCA) is a malignancy arising from the epithelial cells lining the biliary tract. Despite the existence of variation in incidence and etiology worldwide, its incidence is increasing globally in the past few decades. Surgery is the only curative treatment option for a minority of patients presented with early disease; while moderate effective chemotherapy remains the standard care for patients with locally advanced or metastatic diseases. In this article, we briefly review the molecular alterations that have been described in CCAs focusing on the role of epigenetic modification, including promoter methylation inactivation, histone modification and microRNA, in the carcinogenesis and progression of CCAs. This article is part of a Directed Issue entitled: Epigenetics dynamics in development and disease.     

加勒比松种源苗期DNA甲基化多样性分析

为了解加勒比松(Pinus caribaea)种源的遗传多样性,利用甲基化敏感扩增多态性技术对加勒比松3个变种17个种源的DNA甲基化多样性进行了研究。结果表明,56对引物组合共扩增出425条谱带,其中多态性谱带422条,多态性百分率为99.25%。加勒比松种源幼苗半甲基化比率比全甲基化比率稍高,洪都拉斯加勒比松、古巴加勒比松和巴哈马加勒比松的DNA甲基化率分别为22.39%、22.29%和22.35%,差异不显著。加勒比松的DNA序列遗传多样性(H=0.4376)高于DNA甲基化多样性(H=0.3274),Mantel检验表明,基因组遗传变异与表观遗传变异不存在相关性(r=-0.171,P=0.16)。表观聚类与遗传聚类间存在较大差异,两种聚类分析结果均未将3个加勒比松变种分开。这表明加勒比松变种间的表观遗传变异极为丰富,能为加勒比松遗传改良提供优良种质资源。     

Dependency of the Cancer-Specific Transcriptional Regulation Circuitry on the Promoter DNA Methylome

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