消退训练对条件恐惧记忆模型大鼠海马DNA甲基化蛋白及新生神经元的影响

目的探讨不同消退训练方式对成年大鼠恐惧记忆、DNA甲基化蛋白及海马新生神经元的影响。方法雄性SD大鼠随机分成4组:正常对照组、消退对照组、传统消退组和提取消退组。大鼠给予"声音-电击"刺激建立条件性恐惧记忆模型,再进行传统消退或提取消退训练,在第1天、第4天和第7天检测不僵直时间百分比(即消退保持成绩),利用免疫印迹、免疫组织化学检测海马DNA甲基化蛋白表达和新生神经元情况。结果消退训练后第4天,不僵直时间百分比传统消退组[(28.06±11.33)%]和提取消退组[(30.28±11.48)%]高于消退对照组[(21.35±9.45)%],但低于正常对照组[(75.65±8.69)%](t=2.204,2.517,7.955,7.023,P<0.05)。消退训练后第7天,不僵直时间百分比提取消退组[(69.72±13.62)%]高于消退对照组[(24.27±11.67)%,t=7.052,P<0.01]和传统消退组[(50.64±12.51)%,t=2.451,P<0.05],但与正常对照组[(72.03±9.36)%]差异无统计学意义(t=0.251,P>0.05)。消退训练后第4天,传统消退组和提取消退组大鼠海马Dnmt-1、MBD-2表达低于消退对照组,但高于正常对照组(P<0.05)。消退训练后第7天,提取消退组的海马Dnmt-1、MBD-2表达明显低于消退对照组和传统消退组(P<0.05),但与正常对照组差异无统计学意义。消退训练后第4天,传统消退组和提取消退组大鼠海马Brdu阳性细胞表达高于消退对照组,但低于正常对照组(P<0.05)。消退训练后第7天,提取消退组的海马Brdu阳性细胞表达明显高于消退对照组和传统消退组(P<0.05),但与正常对照组差异无统计学意义。结论消退训练可以降低大鼠的条件性恐惧记忆,并且提取消退范式优于传统消退范式,该作用可能是通过降低海马DNA甲基化和增加新生神经元来实现。

DNA甲基化结合蛋白

DNA甲基化是哺乳动物细胞中最重要的表观遗传学修饰之一,大约70%—80%的CpG发生这种甲基化修饰.异常的甲基化在许多癌症中频发,启动子CpG岛的高甲基化作为普遍的失活机制介导抑癌基因沉默.甲基化信号由甲基化结合蛋白来转译,它们能够特异性识别并结合至甲基化位点通过募集辅阻遏复合物例如组蛋白去乙酰化酶(Histone Deacetylase,HDAC)等建立沉默的染色质,从而在DNA甲基化和基因沉默中起桥梁作用.目前,哺乳动物中已鉴定出的甲基化结合蛋白有三类,分别是:MBD(Methyl-CpG-Binding Domain)、Kaiso以及SRA(Set and Ring finger-associated)家族.本文就这三大家族(以MBD为主)各自的结构、功能、结合甲基化DNA的特性以及它们在某些疾病发生中的作用做一综述.

冠心病患者MMP-9基因DNA去甲基化及其异常表达的研究

目的冠心病患者中金属基质蛋白酶-9异常表达和冠心病发表机制和病情进展密切相关,但是其基因水平的调节机制还不清楚。本研究为明确表观遗传学调节机制是否参与冠心病患者中MMP-9的异常表达。方法纳入明确诊断稳定型心绞痛32例、急性心肌梗死患者20例以及冠脉造影正常对照39例,从外周血单个核细胞提取基因组DNA,亚硫酸氢钠处理DNA,采用甲基化敏感的PCR,通过设计特异性的针对甲基化和非甲基化的MMP-9启动子序列的引物,进行PCR扩增后检测PCR产物的浓度并结合基因测序以测定其甲基化程度,分组进行比较和统计学分析,同时ELISA测定血浆中MMP-9的浓度。结果和对照组相比,稳定型心绞痛患者和急性心肌梗死患者MMP-9基因启动子区域甲基化密度明显降低(6.1±2.5%vs 42.3±5.2%,P<0.05;25.6±4.7%vs42.3±5.2%,P<0.05),且在急性心肌梗死中去甲基化集中在部分位点;SCHD组以及AMI组患者和对照组相比,其血浆MMP-9的浓度都显著升高(11.4±1.7vs0.72±0.27,21.6±3.2 vs0.72±0.27,P<0.05)。结论动脉粥样硬化患者MMP-9的基因启动子区域部分CpG位点甲基化水平降低,并和患者血浆MMP-9的浓度升高一致,提示DNA甲基化可能参与动脉粥样硬化中金属基质蛋白酶的表达调节。

Wnt通路中蓬松蛋白DNA甲基化对骨质疏松患者骨髓间充质干细胞成骨分化影响

目的:探讨蓬松蛋白(DVL)DNA甲基化对骨质疏松(OP)患者骨髓间充质干细胞(BMSCs)成骨分化及Wnt通路的影响。方法:分离、培养OP患者的BMSCs,成骨诱导培养BMSCs 0、7、14、21天,观察BMSCs细胞形态变化,检测碱性磷酸酶(ALP)染色及活性,检测茜素红染色及钙结节形成,荧光定量聚合酶链式反应(RT-PCR)及免疫印迹检测远端缺失同源盒5(Dlx5)、核心结合蛋白因子2(Runx2)、成骨细胞特异性转录因子(OSX)、Ⅰ型胶原蛋白(CollaⅠ)表达。检测DVL1、Wnt、糖原合成酶激酶3(GSK3)、β连环蛋白(β-catenin)表达及DVL DNA甲基化水平。在成骨诱导培养基中加入甲基转移酶抑制剂5-Aza,将BMSCs分为对照组(Control组)、甲基转移酶抑制剂组(5-Aza组)、甲基转移酶抑制剂+si-NC组(5-Aza+si-NC组)、甲基转移酶抑制剂+si-Wnt组(5-Aza+si-Wnt组),依次进行ALP活性测定,茜素红染色及钙结节形成测定。RT-PCR检测Dlx5、Runx2、OSX、Colla 1水平,免疫印迹检测Dlx5、Runx2、OSX、CollaⅠ、DVL1、Wnt、GSK3、β-catenin的蛋白表达量,并检测DVL DNA甲基化水平。结果:成骨诱导后BMSCs具有强的ALP活性和矿化结节生成能力,且随着培养时间的增长,BMSCs细胞ALP活性和矿化结节生成能力增强,Dlx5、Runx2、OSX、CollaⅠm RNA水平和Wnt、GSK3、β-catenin、DVL1表达升高,DVL DNA甲基化水平降低(P<0.05)。5-Aza组较Control组ALP染色加深,活性增强,钙结节形成增多(P<0.05),Dlx5、Runx2、OSX、CollaⅠm RNA及蛋白表达、Wnt、GSK3、β-catenin、DVL1表达升高,DVL DNA甲基化水平降低(P<0.05)。5-Aza+si-Wnt组较5-Aza+si-NC组ALP染色变浅,活性降低,钙结节形成减少(P<0.05),Dlx5、Runx2、OSX、CollaⅠm RNA及蛋白表达、Wnt、GSK3、β-catenin、DVL1表达降低,DVL DNA甲基化水平升高(P<0.05)。结论:DVL DNA甲基化可以通过抑制Wnt/β-catenin信号通路抑制OP患者BMSCs成骨分化。

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.

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.

DNA甲基化结合蛋白与心血管疾病

心血管疾病目前不仅是发达国家及发展中国家人口死亡和患病的主要原因,也是世界范围内最突出的健康问题。其病因为高血压、吸烟、血脂异常、糖尿病、肥胖、大气污染等。近年来,DNA甲基化与心血管疾病的研究成为热点,DNA甲基化结合蛋白在DNA甲基化介导的基因沉默中起桥梁作用,DNA甲基化结合蛋白与心血管疾病的关系逐渐揭开面纱。本文就DNA甲基化结合蛋白在心血管疾病中的作用展开讨论。

氢醌致DNA甲基化改变及可能机制

苯是一种广泛使用的工业原料,同时是一种确定的遗传毒性致癌物,主要由其活性代谢产物——氢醌发挥毒性作用。氢醌本身普遍应用于黑白显影剂、橡胶防老剂、稳定剂和抗氧剂。氢醌可引起机体多系统毒性损伤,长期试验表明氢醌还是一种致癌剂。近年来有文献报道氢醌暴露可导致DNA甲基化水平发生改变,本文综述了氢醌可能通过调节DNA甲基化转移酶的活力、氧化应激损伤干扰、DNA损伤修复蛋白调控以及其他表观遗传学修饰的参与等影响DNA甲基化状态,旨在为预防氢醌乃至苯中毒提供参考依据。

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.

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.

Psammaplin A对离体结肠癌SW480细胞增殖、凋亡的影响及机制

目的探讨组蛋白去乙酰化3(HDAC3)抑制剂Psammaplin A对离体结肠癌细胞增殖、凋亡的影响及机制。方法取处于对数生长期结肠癌SW480细胞,随机分为Psammaplin组与对照组,Psammaplin组给予不同浓度(0.5、5、50、500、5 000μg/m L)Psammaplin A干扰48 h,对照组不给予任何干预。采用Western blotting法检测HDAC3及DNMT3a蛋白表达:MTT法检测不同细胞培养时间(24、48、72、96 h)、不同浓度梯度Psammaplin A对SW480细胞增殖的影响。流式细胞术检测Psammaplin A对SW480细胞周期及凋亡的影响。结果 Psammaplin组0.5、5、50、500、5 000μg/m L的Psammaplin A干扰SW480细胞48 h后,HDAC3蛋白相对表达量分别为23.28±8.91、21.72±9.18、18.63±7.26、14.17±5.64、10.58±7.22,对照组HDAC3蛋白相对表达量为24.84±7.65,与对照组比较,Psammaplin组在500及5 000μg/m L时差异有统计学意义(P均<0.05)。Psammaplin组0.5、5、50、500、5 000μg/m L的Psammaplin A干扰SW480细胞48 h后,DNMT3a蛋白相对表达量分别为18.36±8.43、17.51±6.29、16.12±6.54、11.27±5.31、10.54±4.26,对照组为20.15±6.31,与对照组比较,Psammaplin组在500及5 000μg/m L时差异有统计学意义(P均<0.05)。50μg/m L的Psammaplin A干预SW480细胞培养24、48、72及96 h后,Psammaplin组细胞存活率为时间依赖性下降,与对照组比较,在48、72及96 h时差异均有统计学意义(P均<0.05);Psammaplin组0.5、5、50、500、5 000μg/m L的Psammaplin A作用48 h后,与对照组比较,SW480细胞存活率呈剂量依赖性下降,在50、500、5 000μg/m L时差异有统计学意义(P均<0.05)。Psammaplin组与对照组G1期细胞所占比例分别为(69.27±0.93)%、(81.25±0.89)%,G2期细胞所占比例分别为(4.72±1.83)%、(9.62±1.34)%,S期细胞所占比例分别为(27.61±1.65)%、(10.43±0.97)%,两组各期细胞所占比例比较,P均<0.05。Psammaplin组与对照组细胞凋亡率分别为56.98%、31.67%,两组比较,P<0.01。结论 Psammaplin A可通过抑制HDAC3及DNMT3a表达降低结肠癌细胞存活率及增殖活性,诱导凋亡。

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＇.

精子发生的表观遗传学变化及其对后代的影响

目的通常的观点认为,卵细胞因为提供了母方基因组和大部分甚至全部的细胞质而对受精卵的形成起到更加重要的作用,而精子仅仅带来了父方的基因组。近期的研究表明,精子发生过程中的表观遗传学变化也对其后代的胚胎形成及发育有着重要的影响。因此,了解精子发生的表观遗传学变化及其研究的最新动态对于研究精子发生过程本身、精子发生对于子代胚胎的影响以及辅助生殖技术等精细胞临床应用研究都有十分重要的意义。方法本文将从DNA甲基化、鱼精蛋白取代作用及留守组蛋白修饰、小RNAs的调控作用三大方面概括说明哺乳动物精子发生过程中的表观遗传学研究最新进展。结论简要说明精子发生过程中这种非基因组序列的改变对后代胚胎形成及发育造成的影响。

Engineered SNAP-MBD2b proteins for specific recognition of methylated DNA

Methyl-CpG-binding domain （MBD） proteins can specifically recognize and bind methylated CpG sites of DNA, thus repress gene transcription. In this study, we designed and expressed two recombinant proteins, MBD2b and SNAP-MBD2b, in E. coli. An optimized protocol was developed to purify the proteins using Ni-NTA affinity cartridge and cation exchange resin. The engineered proteins purified by this method exhibited more than 93% purity and high binding avidity. We found that both SNAP-MBD2b and MBD2b were prone to aggregate during dialysis. However, this could be prevented by the use of 0.3 mol/L NaCI. The fusion of SNAP-tag with MBD2b significantly enhanced the expression of MBD2b protein in E. coli and reduced the adsorption of MBD2b on solid interfaces involved in protein purification and immobilization. The engineered proteins can be used for the study of interaction with methylated DNA and the assays for DNA methylation.

Epigenetic changes associated with oocyte aging

It is well established that the decline in female reproductive outcomes is related to postovulatory aging of oocytes and advanced maternal age.Poor oocyte quality is correlated with compromised genetic integrity and epigenetic changes during the oocyte aging process.Here,we review the epigenetic alterations,mainly focused on DNA methylation,histone acetylation and methylation associated with postovulatory oocyte aging as well as advanced maternal age.Furthermore,we address the underlying epigenetic mechanisms that contribute to the decline in oocyte quality during oocyte aging.

Epigenetics of human melanoma： promises and challenges

Melanoma is the deadliest form of skin cancer with rising incidence and mortality rates. Although early-stage melanoma is highly curable, advanced-stage melanoma is refractory to treatment. This underscores the importance of prevention and early detection as well as the need to improve treatment and prognostication of human melanoma. Elucidating the underlying mechanisms of the initi- ation and progression of human melanoma can help identify potential targets of intervention for prevention, diagnosis, therapy, and prognosis of this disease. Aberrant DNA methylation and histone modifications are the best-established epigenetic mechanisms of carcinogenesis. The occurrence of epigenetic changes prior to clinical diagnosis of cancer and their reversibility through pharmaco-logic/genetic approaches offer a promising avenue for basic and translational research on human melanoma. Candidate gene（s） or genome-wide aberrant DNA methylation and histone modifications have been observed in human melanoma tumor tissues and cell lines, and correlated to cellular and functional characteristics and/or clinicopathologicai features of this malignancy. The present review summarizes the published researches on aberrant DNA methylation and histone modifications in connection with human melanoma. Representative studies are highlighted to set forth the current state of knowledge, gaps in the knowledgebase, and future directions in these epigenetic fields of research. Examples of epigenetic therapy applied for human melanoma in vitro, and the challenges of its in vivo application for clinical treatment of solid tumors are discussed.

Histone methyltransferase TXRl is required for both H3 and H3.3lysine 27 methylation in the well-known ciliated protist Tetrahymena thermophila

DNA replication elongation is tightly controlled by histone-modifying enzymes.Our previous studies showed that the histone methytransferase TXRl(Tetrahymena Trithorax related protein 1) specifically catalyzes H3K27 monomethylation and affects DNA replication elongation in Tetrahymena thermophila.In this study,we investigated whether TXRl has a substrate preference to the canonical H3 over the replacement variant H3.3.We demonstrated by histone mutagenesis that K27 Q mutation in H3.3further aggravated the replication stress phenotype of K27 Q mutation in canonical H3,supporting H3.3 as a physiologically relevant substrate of TXRl.This result is in apparent contrast to the strong preference for canonical H3 recently reported in Arabidopsis homologues ATXR5 and ATXR6,and further corroborates the role of TXRl in DNA replication.

The influence of mitochondria in epigenetics revealed through naturally occurring fish cybrids

Epigenetic processes are important mechanisms for phenotypic changes that occur in response to the environment. As such, it is expected that the alteration of cytoplasmic composition （the immediate environment of nuclei） results in the modifica- tion of the methylome and the expression of the nuclear genome. Cytoplasmic hybrids （or cybrids） are an ideal model to study the influence of mitochondria on gene expression. In this study, we take advantage of the natural of two biotypes that have a similar nuclear genome type Chrosomus eos, but harbor mitochondria from different species （C. eos in wild type or C. neogaeus in cybrids） to assess the effects of mitochondria on DNA methylation profiles and protein expression of the nuclear ge- nome. Comparison between these biotypes is particularly relevant given their recent divergence and their low level of genetic dif- ferentiation. Variations of DNA methylation assessed on tissues from different embryonic origins revealed the distinct profiles of cybrid and wild type populations. Differences are more pronounced between wild type and cybrids than between populations of a given biotype. The proteome is also more different between biotypes than within a given biotype. These results indicate a strong influence of mitochondria on the nuclear genome, which remains detectable in different genetic and environmental contexts. These changes in the methylome and proteome of cybrids are expected to reflect the adjustments imposed by the coexistence of nuclear and mitochondrial genomes from different species [Current Zoology 58 （1）： 138-145, 2012].