DNA methylation in insects

Cytosine DNA methylation has been demonstrated in numerous eukaryotic organisms and has been shown to play an important role in human disease. The function of DNA methylation has been studied extensively in vertebrates, but establishing its primary role has proved difficult and controversial. Analysing methylation in insects has indicated an apparent functional diversity that seems to argue against a strict functional conservation. To investigate this hypothesis, we here assess the data reported in four different insect species in which DNA methylation has been analysed more thoroughly: the fruit fly Drosophila melanogaster, the cabbage moth Mamestra brassicae, the peach-potato aphid Myzus persicae and the mealybug Planococcus citri.     

DNA methylation plays a crucial role during early Nasonia development

Although the role of DNA methylation in insect development is still poorly understood, the number and role of DNA methyltransferases in insects vary strongly between species. DNA methylation appears to be widely present among the social hymenoptera and functional studies in Apis have suggested a crucial role for de novo methylation in a wide variety of developmental processes. The sequencing of three parasitoid Nasonia genomes revealed the presence of three Dnmt1 (Dnmt1a, Dnmt1b and Dnmt1c) genes and one Dnmt2 and Dnmt3 gene, suggesting a role of DNA methylation in Nasonia development. In the present study we show that in Nasonia vitripennis all Dnmt1 messenger RNAs (mRNAs) and Dnmt3 mRNA are maternally provided to the embryo and, of these, Dnmt1a is essential during early embryogenesis. Lowering of maternal Dnmt1a mRNA results in embryonic lethality during the onset of gastrulation. This dependence on maternal Dnmt1a during embryogenesis in an organismal group outside the vertebrates, suggests evolutionary conservation of the function of Dnmt1 during embryogenesis.     

限制性酶切扫描技术测定DNA甲基化组的方法建立

目的优化试验条件，建立用于DNA甲基化组测定的限制性酶切扫描技术（RLGS）。方法选取冰冻胃癌组织及其周围非癌组织各2份，提取基因组大分子DNA（〉50000bp），用甲基化敏感的限制性内切酶Not Ⅰ等对DNA进行多重酶切、同位素”P标记、二维电泳、扫描分析，并且根据已有的位点DNA序列数据库，确定所得扫描图谱上位点所对应的序列信息。结果成功得到RLGS扫描图；有效点平均在1200个左右，标本质量较好的图谱平均可获得有效点1800个左右，与国外实验室的平均水平相当，经过比对可找出放射自显影信号强度减弱或增强的点，结果可重复，并能在Not Ⅰ-EcoR V克隆文库中找到这些点所对应的序列信息。结论成功建立了RLGS技术平台，并能够稳定工作。     

DNA甲基转移酶在急性髓系白血病中作用的研究进展

急性髓系白血病(AML)的病因目前尚未完全阐明,研究发现其与异常的表观遗传学改变密切相关。DNA甲基化是由DNA甲基转移酶(DNMT)介导的表观遗传过程。DNMT由DNMT1、DNMT3A和DNMT3B组成,是高度保守的DNA修饰酶,其在表观遗传调控中发挥重要作用,并与AML的临床特征与治疗效果的关系十分密切。深入了解DNMT在AML中的作用,有助于阐明AML的发生机制,并为其临床治疗提供潜在的靶点。     

Sox30基因在大鼠睾丸发育中的表达与甲基化分析

目的分析Sox30基因在大鼠胚胎发育以及性腺中的表达和甲基化情况。方法取大鼠胚胎发育不同时期组织、出生后至73 d雌雄大鼠性腺和成年不同组织,利用RT-PCR分析该基因的表达情况。利用亚硫酸氢钠处理后测序法(BSP)分析Sox30基因甲基化发生情况。结果 Sox30基因在大鼠胚胎发育10.5 d就开始表达;出生后Sos30主要在睾丸中表达,且表达逐渐增加,而在卵巢、子宫中不表达;成年大鼠的脑、心、肝、肾、脾、胰、肺、垂体、肠、肌肉、海马等组织中该基因低表达或者不表达。Sox30表达受甲基化调控,低表达或者不表达组织中该基因明显高甲基化。结论 Sox30基因表达受到其启动子区域甲基化调控,成年大鼠中该基因仅在睾丸中高表达,表明该基因与雄性性别发育以及睾丸的功能维持有关。     

Association of interleukin-6 methylation in leukocyte DNA with serum level and the risk of ischemic heart disease

Background Interleukin-6 (IL-6), a multifunctional cytokine, plays an important role in the development of ischemic heart disease (IHD), and DNA hypomethylation of 2 CpGs, located downstream in the proximity of the IL-6 gene promoter, has been associated with risk factor for IHD. This study was to examine the association of blood leukocyte DNA methylation of the 2 CpGs in IL-6 with the risk of IHD and the serum IL-6 level. Methods IL-6 methylation levels of 582 cases and 673 controls were measured using the bisulfite pyrosequencing technology. Serum level of IL-6 was measured using enzyme-linked immunosorbent assay. Results The IL-6 methylation was significantly lower in IHD cases than in the controls, irrespective of CpG site. After multivariate adjustment, lower (< median) average IL-6 methylation was associated with an increased risk of IHD (OR 1.57, 95% CI 1.22–2.02, p?β?=??1.02?pg/mL per increase in IL-6 methylation, p?=?0.002) among IHD cases. This significant relationship was not observed among controls. Conclusions DNA hypomethylation of IL-6 gene measured in blood leukocytes was associated with increased risk of IHD. IL-6 demethylation may upregulate its expression, whereby exerting its risk effect on the development of IHD.     

Alternative migratory locust phenotypes are associated with differences in the expression of genes encoding the methylation machinery

Despite the importance of locust density‐dependent polyphenism as a model system for understanding phenotypic plasticity, there is still much to be learnt about its underlying molecular control. Here we describe the first investigation into the expression of genes encoding the DNA methylation machinery in the migratory locust (Locusta migratoria). We show that the alternative solitarious and gregarious phenotypic states induced by different locust rearing densities are associated with significant differences in the expression of the target genes DNA methyltransferase 1, DNA methyltransferase 2 and methyl‐CpG‐binding domain protein 2/3. This variation was most pronounced in the embryos of solitarious vs. gregarious mothers. We mapped the embryonic methylation profiles of several intragenic regions and a Long Interspersed Nuclear Element (LINE), each of which is known to be differentially expressed between alternative locust phenotypes or has been directly implicated in phase change. LmI and three genes, adenyl cyclase‐associated binding protein 2, choline kinase alpha‐like and henna, were methylated. Our results set the stage for future studies investigating the specific role of DNA methylation in the maternal transfer of migratory locust phase polyphenism.     

白血病细胞WT1基因启动子区DNA甲基化研究

目的：研究白血病细胞WT1启动子区DNA甲基化与其表达的关系。方法：采用RT-PCR技术、硫化PCR结合限制性内切酶技术检测白血病细胞系及正常人外周血单个核细胞WT1基因的表达及其启动子区DNA甲基化水平。结果：正常人外周血单个核细胞及U937细胞不表达WT1基因，而HL-60、K562和KG1细胞高表达WT1基因，HL-60细胞WT1基因无甲基化。结论：WT1基因启动子区DNA甲基化不能抑制其表达，尚存在其他调节WT1基因表达的因素。     

胃癌细胞系中甲基化与叶酸干预对多种基因的调控研究

目的：胃癌的发生从分子发病水平上说是一个涉及多种癌基因、抑癌基因、凋亡相关基因等的异常和积累的多步骤的过程。DNA甲基化在基因表达中起调控作用已成为共识[1]。各种肿瘤的发生过程中有癌基因的低甲基化和抑癌基因的高甲基化，因为甲基化程度与基因表达呈负相关，所以引起抑癌基因的表达减弱或缺失及癌基因的表达增强。5-氮脱氧胞苷（5-aza-2’-deoxycytidine, 5-aza-dC）通过抑制甲基化酶使基因去甲基化，通过5-aza-dC干预能够观察甲基化对基因表达的影响。胃癌发生中也有抑癌基因的高甲基化及癌基因的低甲基化现象，但是迄今为止未见同时研究同一细胞系的多种基因的甲基化对基因表达和细胞周期的影响。本研究对此作了有益的探讨，以期为以后的胃肠道肿瘤的治疗提供分子水平的依据。本文主要探讨不同分化的胃癌细胞系在不同浓度的5-氮脱氧胞苷（5-aza-2’-deoxycytidine, 5-aza-dC）和甲基化食物供体叶酸的化学干预下癌基因、抑癌基因和与凋亡有关的基因与甲基化调控的关系。方法： 培养高分化、中分化和未分化的胃癌细胞MKN－45、MKN－28、HGC－27，分别以不同浓度的5-aza-dC干预细胞，以同一浓度的叶酸干预细胞，部分细胞48小时后再以不同浓度的5-aza-dC干预。提取细胞的RNA，用RT-PCR的方法检测p16INK4A、p21WAF1、p73、c-myc、c-Ha-ras等多种基因的表达情况。结果：在不同分化的三种胃癌细胞中基因表达受甲基化调控的程度也不同。抑癌基因中p16INK4A在干预前MKN－45和HGC－27细胞表达，而在用5-aza-dC干预后表达增强，且不同的胃癌细胞株表达增强与5-aza-dC干预的时间与浓度不同有关，p21WAF1、p73没有明显变化；癌基因中的c-myc、c-Ha-ras变化不明显；所有胃癌细胞系中叶酸干预及叶酸联合5-aza-dC干预前后所以基因的mRNA表达没有明显变化。结论：不同分化人胃癌细胞中，甲基化修饰对癌基因、抑癌基因、与凋亡有关的基因的调控差异明显。其中的机制研究是否与个体差异有关。但是在肿瘤细胞中的叶酸干预后对甲基化的调控不敏感，可能是因为肿瘤细胞对叶酸的甲基化供体已经不起作用，与癌前病变补充叶酸不同。     

氨甲蝶呤耐药细胞内整体DNA甲基化水平检测方法的建立及其应用

目的 建立一种简便快速检测细胞内整体DNA甲基化水平的方法,以用于临床甲基化诊断.方法 采用高效毛细管电泳技术,以pH 9.6的48 mmol/L碳酸氢钠溶液[含60 mmoL/L十二烷基硫酸钠(SDS)]为分离缓冲液,检测波长为256 nm,在20 kV电压下,0.7 psi压力进样时间5 s,对2'-脱氧胞苷(dC)、5-甲基-2'-脱氧胞苷(mdC)、2'-脱氧腺苷(dA)、2'-脱氧胸苷(dT)、2'-脱氧鸟苷(dG)5种物质进行分离.在此基础上检测氨甲蝶呤(MTX)诱导的肺癌A549耐药细胞株内整体DNA甲基化水平.结果 通过不断优化分离缓冲液中SDS浓度(40、60、80 mmol/L)、pH值(9.4、9.6、9.8)、分离电压(15、17、19、20、22 kV)、进样时间(5、10、15、20、30 s)和毛细管温度(15、20、25、30℃),建立高效毛细管电泳检测细胞内整体DNA甲基化水平的方法,可在10 min内实现dC、mdC、dA、dT、dG的完全分离,其日内变异系数(CV)<0.2%,日间CV<2%,最低检出限为2μmoL/L.检测肺癌A549亲本细胞甲基化水平为(4.80±0.52)%;而不同浓度(15、30、40 μmol/L)MTX诱导耐药A549细胞株的甲基化水平分别为(4.20±0.44)%、(3.70±0.36)%、(3.10±0.35)%.结论 建立高效毛细管电泳检测整体DNA甲基化水平的方法具有高效、快速、简单、灵敏的特点;MTX耐药细胞株内整体DNA甲基化水平随着耐药浓度的增加明显降低.     

超高效液相色谱-荧光法检测精液中DNA甲基化水平

目的建立超高效液相色谱-荧光法(UPLC-FLD)定量分析精液标本中DNA甲基化水平。方法精液样本DNA提取、水解后,以100 mmol/L 2-溴苯乙酮为衍生试剂,在0.51 mol/L乙酸乙腈溶液中80℃加热60 min,生成脱氧胞嘧啶核苷(d C)和5-甲基脱氧胞嘧啶核苷(5md C)的荧光衍生物(λex=306 nm,λem=378 nm)。取1μL上清液进样,经Agilent C18色谱柱(2.1×100 mm、1.8μm)分离[流动相为28%甲醇+10%乙腈+62%水溶液(含0.1%甲酸),柱温40℃,流速0.3 m L/min]及统计学分析,并用临床样本进行验证。结果在上述最佳衍生反应与色谱分离条件下,7 min即可将精液DNA中的d C与5md C完全分离,无需消除RNA干扰;本法中5md C的检测限为2.5 pg/μL,线性范围为0.025~0.75 ng/μL,r0.99,日间、日内变异系数(CV)5.6%,衍生化合物室温条件放置30 h内比较稳定。临床精液样本结果表明,少弱畸精子症患者的DNA甲基化水平低于健康男性。结论建立的UPLC-FLD法可快速、简便、准确、稳定地检测基因组DNA甲基化水平。     

结直肠癌Septin9基因甲基化检测质控品制备及其应用

目的制备可模拟临床标本的血浆Septin9基因甲基化(mSEPT9)质控品并用于室间质量评价。方法选择Septin9区域已发生/未发生甲基化的HeLa/Jurkat细胞进行培养,收集并抽提细胞基因组DNA,经mSEPT9试剂盒检测,依据检测Ct值用阴性血浆稀释分装成含有不同浓度的质控品,对质控品均匀性和稳定性进行评价后,将5个样品随机编号后发送至参评实验室,分析评价回报结果。结果抽提得到的细胞基因组DNA纯度和浓度均可满足使用需求,且可用作mSEPT9检测的质控品。均匀性和稳定性均符合中国合格评定国家认可委员会对能力验证样品要求。部分参评实验室出现假阳性和假阴性情况,仅有约55.6%实验室的检测结果(Ct值)呈良好线性相关。9家参评实验室中,7家实验室(77.8%)成绩优秀,1家实验室(11.1%)成绩合格,1家实验室(11.1%)成绩不合格。结论成功研制血浆mSEPT9检测的质控品并应用于室间质评,有利于评估并提升临床实验室检测能力。     

Association between CpG island DNA methylation in the promoter region of RELN and positive and negative types of schizophrenia

ObjectiveTo explore the association between CpG island methylation in the promoter region of RELN and positive (type I) and negative (type II) types of schizophrenia, and investigate serum interleukin (IL)-1β, IL-6, and myelin basic protein (MBP) in schizophrenia.MethodsLevels of CpG island methylation in the promoter region of RELN were detected in peripheral blood of patients with schizophrenia (experimental group) and healthy individuals (control group), and serum IL-1β, IL-6, and MBP were measured.ResultsThe positive rate of CpG island methylation in the promoter region of RELN was higher in the experimental group than in the control group; however, there were no significant differences between type I and II patients. There were differences in Positive and Negative Syndrome Scale (PANSS) scores and serum IL-1β, IL-6, and MBP between type I and II patients. Furthermore, there were positive correlations between serum IL-1β, IL-6, and MBP and PANSS scores (negative symptoms) in type II patients.ConclusionCpG island methylation in the promoter region of RELN was associated with schizophrenia, but not with its clinical type. There may be different pathological mechanisms in type I and II schizophrenia, and type II schizophrenia may be associated with serum IL-1β, IL-6, and MBP.     

Evidence for a functional role of endogenously produced somatomedinlike peptides in the regulation of DNA synthesis in cultured human fibroblasts and porcine smooth muscle cells.

Cultured porcine aortic smooth muscle cells and human fibroblasts produce somatomedinlike peptides and secrete them into the surrounding microenvironment. This production has been linked to their ability to replicate. The objective of this study was to determine if a specific anti-somatomedin-C (Sm-C) monoclonal antibody that binds the somatomedinlike peptides could inhibit replication by porcine aortic smooth muscle cells and human fibroblasts. To determine if the antibody could inhibit the effect of endogenously produced somatomedinlike peptide, increasing concentrations of antibody were co-incubated with platelet-derived growth factor, a known stimulant of somatomedinlike peptide secretion, and Sm-C-deficient platelet-poor plasma. Addition of the antibody reduced fibroblast [3H]thymidine incorporation from 35,100 +/- 500 to 10,600 +/- 700 cpm (P less than 0.001), and in smooth muscle cells from 29,600 +/- 1,800 to 10,800 +/- 1,100 cpm (P less than 0.001). Co-incubation of exogenously added Sm-C (20 ng/ml) with maximally inhibitory dilutions of antibody increased [3H]thymidine incorporation in fibroblasts from 7,800 +/- 1,000 to 18,900 +/- 800 cpm (P less than 0.01), and in smooth muscle cells from 9,800 +/- 1,200 to 17,200 +/- 1,100 cpm (P less than 0.01). Insulin, which can substitute for Sm-C as a mitogen and does not bind to the antibody, stimulated DNA synthesis when co-incubated with the antibody, thereby excluding the possibility of nonspecific cytotoxicity. These results strengthen the hypothesis that the rate of DNA synthesis of these two cell types in vitro is directly linked to their capacity to produce somatomedinlike peptides. They further support the cellular production of somatomedinlike peptides as examples of the autocrine model of growth regulation.     

Downregulation of SNCA Expression by Targeted Editing of DNA Methylation: A Potential Strategy for Precision Therapy in PD

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