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DNA去甲基化与基因激活过程

万方数据万方数据万方数据图２ＤＮＡ去甲基化模型．图中包括目标摹因的识别，染色体重组，植物中的碱基切除修复系统．植物识别特异性目标并施行ＤＮＡ去甲基化，有Ｉ玎能足ｓｉＲＮＡ利用一种未知的ＤＮＡ去甲摹化复合物诱导ＤＮＡ去甲基化．也有可能包括染色体重组和／或碱基切除修复蛋Ｆｉ在接触和修复步骤中，孓甲基胞嘧啶被碱基切除系统切除甲摹摹团，很“ｒ能还伴随着染色体结构的改变．为了能让ＤＮＡ去甲基化蛋ｒＩ和转录机制接触目标基因。

染色体霞组因子和组蛋白修饰蛋白转化染色体为激活状态．在ＤＮＡ去甲基化的碱基切除修复系统进程中。

一种双功能ＤＮＡ糖苻酶ＤＭＥ或ＲＯＳＩ，利用其ＤＮＡ糖苷酶活性移除ｒ１个碱基，并利用其ＡＰ裂解酶活性切除脱碱基位点，得到的核苷酸缺几由未确认的ＤＮＡ多聚酶和ＤＮＡ连接酶完成填补工作．ＤＮＡ去甲基化和染色体重组后，目标基因开始表达．互作用也是一个重点。

关于脉孢菌和拟南芥的研究报道称，其组蛋白Ｈ３Ｌｙｓ９的甲基化影响ＤＮＡ的甲基化ｍ““。

最近的报道发现Ｈ２Ｂ的泛素化对ＤＮＡ的甲基化也有影响。

因此，一个有趣的问题是，究竟是染色体的状态影响了ＤＮＡ的去甲基化进程还是ＤＮＡ的去甲基化影响了染色体的状态改变，仍值得深入研究。

参考文献【ｌＪＣｈｒｉｓｔｏｆＮ．ＡｃｔｉｖｅＤＮＡｄｅｍｅｔｈｙｌａｔｉｏｎａｎｄＤＮＡｒｅｐａｌｒ［Ｊ】．Ｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ。

２００９．７７（Ｉ）：１一１１．【２１ＰｅｒｉｎｉＧ．ＴｕｐｌｅｒＲ．Ａｌｔｅｒｅｄｇｅｎｅｓｉｌｅｎｃｉｎｇａｎｄｈｕｍａｎｄｉｓｅｎｓｅｓ［Ｊ］．Ｃｌｉｎ｜ｆＩ｜ＪＣ＾■：啦Ｃ日％ｔ憾．可■＾ｔｏｄ酬ＥＳ日●Ｉ１０２ＴＡＣ－ｇ＿ＩＭ／￥５２Ｖ０１．２２ＮＯ．２墨驾Ｇｅｎｅｔ，２００６，６９（Ｉ）：Ｉ一７．【３】ＳｉｍｏｎｓｓｏｎｌＳ，ＧｕｒｄｏｎＪ．ＤＮＡｄｅｍｅｔｈｙｌａｔｉｏｎｉｓｎｅｃｅｓｓａｒｙｆｏｒｔｈｅｅｐｉｇｅｎｅｔｉｃｒｅｐｒｎｇｒａｍｍｉｎｇｏｆｓｏｍａｔｉｃｃｅｌｌｎｕｃｌｅｉ［Ｊ】．ＮａｔｕｒｅＣｅｌｌＢｉｏｌ，２００４．６（１０）：９８４—９９０．【４】ＳｕｚｋｉＨ．ＧａｂｆｉｅｌｓｏｎＥ，ＣｈｅｒｔＷ。

2011 De novo DNA methylation

RESEARCH COMMUNICATIONDe novo DNA methylationby Dnmt3a and Dnmt3bis dispensable for nuclear reprogramming of somatic cells to a pluripotent state Mathias Pawlak1and Rudolf Jaenisch1,2,31The Whitehead Institute for Biomedical Research,Cambridge, Massachusetts02142,USA;2Department of Biology, Massachusetts Institute of Technology,Cambridge, Massachusetts02139,USAInduced pluripotent stem cells(iPSCs)are generated from somatic cells by the transduction of defined transcription factors,and this process involves dynamic changes in DNA methylation.While the reprogramming of somatic cells is accompanied by demethylation of pluripotency genes,the functional importance of de novo DNA meth-ylation has not been clarified.Here,using loss-of-function studies,we generated iPSCs from fibroblasts that were deficient in de novo DNA methylation mediated by Dnmt3a and Dnmt3b.These iPSCs reactivated pluripo-tency genes,underwent self-renewal,and showed restricted developmental potential that was rescued upon reintro-duction of Dnmt3a and Dnmt3b.We conclude that de novo DNA methylation by Dnmt3a and Dnmt3b is dispens-able for nuclear reprogramming of somatic cells. Supplemental material is available for this article.Received July19,2010;revised version accepted March18, 2011.Pluripotent stem cells can be generated from somatic cells by viral transduction of the four transcription factors Oct4,Sox2,Klf4,and c-Myc.These induced pluripotent stem cells(iPSCs)are similar to embryonic stem(ES)cells expressing pluripotency genes such as Nanog and Oct4, and are capable of differentiation into cells of all three germ layers in teratoma assays.Furthermore,mouse iPSCs contribute to the germline in chimeric mice and ‘‘all iPSC’’mice can be derived through tetraploid com-plementation(Takahashi and Yamanaka2006;Maherali et al.2007;Okita et al.2007;Wernig et al.2007;Boland et al.2009;Kang et al.2009;Zhao et al.2009).iPSCs generated from human sources are thought to be useful for drug discovery and regenerative medicine(Yamanaka 2009).In fact,in two proof-of-principle studies,iPSCs have been shown to alleviate symptoms in mouse models of sickle cell anemia and Parkinson’s disease(Hanna et al. 2007;Wernig et al.2008).The molecular events leading from a differentiated to a pluripotent cell during iPSC derivation are only emerg-ing and remain largely unknown(Hochedlinger and Plath 2009).Studies have shown that distinct markers are activated or repressed sequentially after the induction of reprogramming,with the repression of differentiation genes occurring early and the activation of genes essential for pluripotency,such as Oct4or Nanog,occurring late during this process(Brambrink et al.2008;Stadtfeld et al. 2008).The epigenetic state of the differentiated donor cell is completely reprogrammed,and DNA methylation and histone marks of the iPSC resemble that of ES cells (Maherali et al.2007;Wernig et al.2007;Mikkelsen et al.2008).Changes in DNA methylation patterns are essential for successful nuclear reprogramming,exemplified by the necessity for loss of promoter methylation in pluripo-tency genes in iPSCs(Takahashi and Yamanaka2006).If loss of DNA methylation is not achieved,the cells will be only partially reprogrammed(Mikkelsen et al.2008). Several studies have investigated de novo DNA methyl-ation during the reprogramming process.For example,it has been documented that de novo DNA methyltrans-ferases are highly induced upon reprogramming,and it is known that mouse ES cells have a very high expression of these epigenetic regulators(Okano et al.1998;Stadtfeld et al.2008).Also,two studies found increased DNA methylation in certain genomic regions in iPSCs,with the functional importance of this finding remaining elusive(Deng et al.2009;Doi et al.2009).However,it has not been resolved whether de novo DNA methylation is important or might even be essential for nuclear re-programming of somatic cells to a pluripotent state.De novo DNA methylation in mammals is mediated by Dnmt3a and Dnmt3b(Li2002)and knockout studies in mice have shown that de novo DNA methylation is essential for development,since Dnmt3a-deficient ani-mals die several weeks after birth and Dnmt3b-deficient animals die in utero(Okano et al.1999).Further exper-imentation has shown that fibroblasts lacking Dnmt3b are prone to spontaneous immortalization and genomic instability(Dodge et al.2005),although mouse ES cells can tolerate the complete absence of DNA methylation (Meissner et al.2005).DNA methylation by Dnmt3a has a crucial role for imprinting in development(Kaneda et al. 2004).In this study,we sought to investigate the role of Dnmt3a and Dnmt3b in the generation of ing mouse embryonic fibroblasts(MEFs)in which Dnmt3a and Dnmt3b were conditionally deleted through Cre recombinase,we show that nuclear reprogramming is achieved in the absence of the de novo DNA methyl-transferases Dnmt3a and Dnmt3b.Results and DiscussionGeneration of iPSCs in the absence of the de novo DNA methyltransferases Dnmt3a and Dnmt3bTo ask whether somatic cells can be reprogrammed into a pluripotent cell in the absence of de novo DNA methylation,we used MEFs that were homozygous for floxed alleles of both Dnmt3a and Dnmt3b.Two[Keywords:nuclear reprogramming;iPSCs;de novo DNA methylation;Dnmt3a;Dnmt3b]3Corresponding author.E-MAIL jaenisch@;FAX(617)258-6505.Article is online at /cgi/doi/10.1101/gad.2039011.GENES&DEVELOPMENT25:1035–1040Ó2011by Cold Spring Harbor Laboratory Press ISSN0890-9369/11;1035complementary approaches were used to generate iPSCs (Fig.1A).In the first approach,we transduced double-conditional MEFs (referred to as 3ab MEFs)with lentivi-ruses encoding the reprogramming factors Oct4,Sox2,c-Myc,and Klf4and the reverse tetracycline transactiva-tor M2rtTA (Brambrink et al.2008;Hockemeyer et al.2008).After transduction,the cells were infected with an adenovirus containing a Cre recombinase and GFP.FACS (fluorescence-activated cell sorting)was used to isolate GFP-positive cells,and reprogramming was initiated 1d later by adding doxycycline (DOX)to the medium.Since it is known that Dnmt3a and Dnmt3b are expressed at low levels in somatic cells (Okano et al.1998),we aimed to exclude the possibility of remaining functional Dnmt3a and Dnmt3b after the initiation of reprogramming.To this end,in the second approach,3ab MEFs were first infected with the adenovirus harboring the Cre recombinase,passaged several times,and then transduced with the individual reprogramming factors.Similarly to wild-type MEFs,addition of DOX to 3ab MEFs induced morphological changes within several days in both approaches (Supplemental Fig.S1A).After 17–28d of DOX treatment,single colonies morphologically resembling ES cells were subcloned.To select for DOX-independent clones,DOX was withdrawn from the me-dium shortly after subcloning.To characterize 3ab-deficient iPSCs,immunostaining was performed.3ab-deficient iPSCs were found to be positive for the stem cell markers alkaline phosphatase,SSEA-1,Oct4,and Nanog (Fig.1B;Supplemental Fig.S1B–D),and the staining patterns were indistinguishable from those of control V6.5ES cells.The expression of the pluripotency gene Nanog was also investigated by quan-titative RT–PCR (qRT–PCR)and was found to be expressed in 3ab-deficient iPSCs at levels comparable with ES cells (Supplemental Fig.S1E).These results suggest that reprogramming can occur in the absence of de novo DNA methylation through Dnmt3a and Dnmt3b.Upon differentiation of ES cells,pluripotency genes such as Oct4and Nanog are highly methylated,reflecting stable long-term silencing (Feldman et al.2006),while iPSC generation leads to demethylation,consistent with reactivation of Oct4and Nanog .As expected,bisulfite sequencing demonstrated that the Nanog gene became demethylated in 3ab-deficient iPSCs,comparable with V6.5ES cells (Supplemental Fig.S1F).To evaluate whether the Dnmt3a and Dnmt3b alleles had been deleted by Cre recombinase,Southern blot analyses were performed.Individual colonies were subcloned from iPSCs derived from FACS-isolated Adeno-Cre-GFP-infected MEFs.Figure 2A shows that all Dnmt3a and Dnmt3b alleles were deleted,confirming that iPSCs can be derived in the absence of de novo DNA methyl-ation.Similarly,>20subclones generated with the second approach had deleted all Dnmt3a and Dnmt3b alleles (Fig.2B).To confirm the independent origin of 3ab-deficient iPSC lines,we performed Southern blot analysis with a probe that recognizes both endogenous and transgenic Oct4.The majority of the 18iPSC lines randomly chosen for this analysis had a unique integration pattern and thus were of independent origin (Supplemental Fig.S2A).The number of integrations for the Oct4provirus varied between two and eight,with clones #8and B2having the lowest and clone A6having the highest number of integrations.Expression analysis of Oct4proviral tran-scription correlated with basal vector expression,with clone A6showing some residual transgene expression (Supplemental Fig.S2B).Also,we observed different numbers of proviral integrations for the other reprogram-ming factors,with clone B2lacking a c-Myc proviral integration (Supplemental Fig.S3).It is known from nuclear transfer studies that hypo-methylation of somatic donor cells increases reprogram-ming efficiency (Blelloch et al.2006).To ask whether reprogramming was enhanced in the absence of de novo DNA methylation,we infected 3ab conditional MEFs with either an Adeno-GFP or an Adeno-Cre-GFP virus and sorted equal amounts of GFP-positive cells.Then,we induced reprogramming by transduction with a polycis-tronic vector harboring the four reprogramming factors (Sommer et al.2009).After 16d of reprogramming,we omitted DOX and counted colonies on day 19.We ob-served only a slight difference in colony number between control and 3ab-deficient samples (1.5-fold),suggesting that there is no major effect on reprogramming in the absence of de novo DNA methylation.One representative plate was stained for alkaline phosphatase,a commonly used pluripotency marker (Supplemental Fig.S4).Molecular characterization of 3ab-deficient iPSCsIt was shown previously that a global change in DNA methylation patterns occurs during iPSC derivation (Mikkelsen et al.2008).We therefore investigated the methylation of genomic repeat sequences during repro-gramming in the absence of de novo DNA methylation.To this end,we compared 3ab-deficient with control iPSCs or V6.5ES cells.Southern blot analysis withtheFigure 1.Generation of iPSCs in the absence of de novo DNA methyltransferase activity.(A )Schematic of the two approaches to reprogram conditional knockout MEFs for Dnmt3a and Dnmt3b .MEFs were infected with adenovirus harboring a Cre recombinase and GFP either after or before infection with inducible lentiviruses encoding the reprogramming factors Oct4,Sox2,c-Myc and Klf4.(B )Immunostaining for the pluripotency marker Nanog.Bars,100m m.Pawlak and Jaenisch1036GENES &DEVELOPMENTmethylation-sensitive enzyme HpaII showed that 3ab-deficient iPSC lines had slightly decreased DNA methyl-ation levels of minor satellite repeats and intracisternal A particles (IAPs)(Supplemental Fig.S5),consistent with previous results showing reduced IAP methylation inDnmt3a and Dnmt3b mutant cells (Okano et al.1999).Since it has been demonstrated that female ES cells arehypomethylated (Zvetkova et al.2005),we determinedthe gender of the 3ab-deficient iPSC lines and found thatmale clone #8and female clones A6and B2had similarmethylation levels of the genomic repeat sequences.To investigate global gene expression in 3ab-deficientiPSCs,we performed expression profiling (Supplemental Fig.S6).This analysis revealed that 165genes were >2.5-fold up-regulated in 3ab-deficient iPSCs and 116genes were 2.5-fold down-regulated in comparison with control iPSC lines (Supplemental Table S1).A large proportion of the up-regu-lated genes were X-linked,such as genes of the rhox cluster,a known target for Dnmt3a and Dnmt3b.In fact,loss of Dnmt3a and Dnmt3b in ES cells leads to activation of the rhox cluster,which is normally silenced (Oda et al.2006).Fur-thermore,it was shown that ES cells that lack both de novo and maintenance DNA methyltransferases up-regulate genes on the X chromosome (Fouse et al.2008).We found Gtl2and Rian ,two members of the Dlk1–Dio3cluster,up-regulated in 3ab-deficient iPSCs,indicating that Dnmt3a/b-mediated DNA methylation may be involved in the regulation of this genomic region,as suggested previously (Kato et al.2007).For functional annotation,we per-formed gene ontology (GO)analyses of the differentially regulated genes in 3ab-deficient iPSCs (Supplemental Table S2).Top GO terms for the up-regulated genes were chromosome organization (GO:0051276)and organelle organization (GO:0006996).However,more special-ized categories such as meiosis (GO:0007126)or reproductive cellular process (GO:0048610)were also observed,and it is known that Dnmt3a plays an impor-tant role in germ cell development (Kaneda et al.2004;Kato et al.2007).The top GO terms for the down-regu-lated genes were in response to stimulus (GO:0050896)and multicellular organ-ismal development (GO:0007275),with other more specialized developmental GO terms.It was noteworthy that up-regu-lated genes would rather be grouped into the intracellular region (GO:0005622),whereas down-regulated genes would fall into the extracellular region (GO:0005576).In summary,our data suggest that 3ab-deficient iPSCs display an expres-sion profile highly similar to control iPSCs,yet with some noticeable differences.A previous study grouped gene pro-moters in mouse ES cells into eithermethylated or unmethylated (Fouse et al.2008).Wecompared our gene list of differentially expressed genesin 3ab-deficient iPSCs with the gene list of methylated and unmethylated promoters to evaluate whether wecould identify a correlation between the lack of de novo DNA methylation and the observed expression profile.We found that 59of the differentially expressed genes in 3ab-deficient iPSCs are methylated and 55genes are unmethylated in mouse ES cells (Supplemental Table S3).Using a different data set (Meissner et al.2008),we did not observe a preferred distribution of the differen-tially regulated genes into methylated or unmethylated in normal mouse ES cells in comparison withMEFsFigure 2.Characterization of 3ab-deficient iPSC lines.(A )Southern blot analysis shows3ab-deficient iPSC lines obtained through approach 1(Fig.1A).Clone #8is indicated by anasterisk.(B )Southern blot analysis of 3ab-deficient iPSC lines obtained through approach 2(Fig.1A).Clones A6and B2are indicated by an asterisk.Additionally,3ab MEFs infected with Adeno-Cre-GFP are shown before the transduction with the reprogramming factors and over the course of several passages (1–3).Lane 4displays DNA from a culture dishcontaining mixed iPSC colonies.Note that the loop-out induced by Cre recombinase washighly efficient.‘‘2lox’’represents the conditional alleles and ‘‘1lox’’represents the deficientalleles of Dnmt3a and Dnmt3b ,respectively.Dnmt3a/b function in reprogrammingGENES &DEVELOPMENT 1037(Supplemental Fig.S7).Additionally,sites of enhanced DNA methylation in human iPSCs in relation to human ES cells were reported previously (Doi et al.2009).How-ever,differentially expressed genes in 3ab-deficient iPSCs did not significantly overlap with these sites.These data suggest that,while expression of some genes in ES cells may be affected by the state of DNA methylation,this effect may not be direct.Developmental potential of Dnmt3a-and Dnmt3b-deficient iPSCsTo assess developmental potential,3ab-deficient iPSCs were injected into the flanks of SCID (severe combined immunodeficient)mice.The resulting teratomas showed mostly ectodermal structures and few endodermal-like,but no well-differentiated,mesodermal structures (Fig.3A),suggesting that 3ab-deficient iPSCs have only a re-stricted developmental potential.Furthermore,the over-all size of teratomas derived from 3ab-deficient iPSCs was considerably smaller than that of teratomas from control V6.5ES cells (data not shown)or control 3ab +iPSCs that were able to contribute to all three germ layers (Supple-mental Fig.S8).This is in agreement with previous studies demonstrating that high-passage Dnmt3a -and Dnmt3b -deficient ES cells were unable to form tera-tomas,likely because of a decreased level of global DNA methylation.Consistent with this conclusion,develop-mental potential in Dnmt3a/3b -deficient ES cells could be restored by reintroduction of Dnmt3a and Dnmt3b and re-establishment of DNA methylation patterns (Chen et al.2003).To investigate whether developmental potential could be restored to 3ab-deficient iPSCs,we reintroduced functional Dnmt3a and Dnmt3b by lentivirus-mediated gene transfer (Supplemental Fig.S9).Indeed,the rescued lines formed teratomas that displayed differentiated cells from all three germ layers (Fig.3A).This observation suggested that 3ab-deficient iPSCs had been repro-grammed yet could not differentiate,merely due to thelack of Dnmt3a and Dnmt3b that were rescued upon their reintroduction.As a more stringent assay for pluripo-tency,we injected eGFP-marked 3ab-deficient iPSCs and rescued lines into blastocysts and harvested the resulting embryos at mid-gestation (Supplemental Fig.S9).We were able to obtain chimeric embryos with clearly visi-ble eGFP signals,indicative of a contribution from the rescued lines,underscoring their developmental potential (Fig.3B).The degree of chimerism varied between in-dividual embryos.3ab-deficient iPSCs did not contribute to developing embryos based on the lack of an eGFP signal (data not shown),which was confirmed by the absence of immunohistochemical staining for GFP (Sup-plemental Fig.S10).In some high-contribution chimeras from the rescued lines,developmental malformations were observed (Fig.3B)that are likely due to constitutive expression of the lentivirus-transduced Dnmt3a and Dnmt3b genes.In summary,reintroduction of Dnmt3a and Dnmt3b restored pluripotency to the Dnmt3a-and Dnmt3b-deficient iPSCs.ConclusionsIn this study,we used conditional knockout MEFs to generate iPSCs in the absence of de novo DNA methyl-ation through Dnmt3a and Dnmt3b.We observed robust reactivation of pluripotency markers in 3ab-deficient iPSCs,and their derivation appeared to be similarly efficient compared with wild-type cells.The methylation status of genomic repeat sequences in 3ab-deficient iPSCs was slightly decreased.The global expression profile of 3ab-deficient iPSCs closely resembled that of control iPSCs,although the expression of a small number of genes was changed in 3ab-deficient iPSCs.The question of how gene silencing is achieved in 3ab-deficient iPSCs remains a particularly interesting one.A recent study suggested that gene silencing in mouse ES cells can be achieved by alternative mechanisms to DNA methyla-tion,such as histone methylation by ESET (also known as KMT1E)(Matsui et al.2010).Whether such mechanismsassure gene silencing in 3ab-deficient iPSCs deserves further study.3ab-de-ficient iPSCs formed teratomas that were smaller than teratomas from con-trol lines and had a restricted devel-opmental potential.However,devel-opmental potential could be rescued upon reintroduction of Dnmt3a and Dnmt3b into 3ab-deficient iPSCs,con-sistent with de novo DNA methyla-tion mediated by Dnmt3a and Dnmt3b being crucial for normal development.In summary,our results provide evi-dence that the genome of a differenti-ated cell that is deficient for this major epigenetic regulatory system can be reprogrammed to a pluripotent state.Materials and methodsCell culture and iPSC derivationMEFs were isolated from double-homozygous mice for the conditional alleles of Dnmt3a and Dnmt3b (Lin et al.2006;Nguyen et al.2007).iPSCs were generated with induciblelentivirusesFigure 3.3ab-deficient iPSCs form teratomas with a restricted developmental potential.(A )Teratomas from #8and B23ab-deficient iPSCs.Reintroduction of functional Dnmt3a and Dnmt3b into 3ab-deficient iPSCs rescues the ability to efficiently form cells from the three germ layers in a teratoma assay.(B )eGFP-marked rescue lines were injected into blastocysts,and resulting chimeric embryos at mid-gestation are shown.The eGFP signal was clearly visible in the majority of embryos,whereas the degree of chimerism varied.Two embryos with developmental defects are shown.The first embryo of the bottom panel did not show any eGFP signal and serves as a reference of background.Pawlak and Jaenisch1038GENES &DEVELOPMENTas reported previously(Brambrink et al.2008;Hockemeyer et al.2008). Recombination of the conditional alleles of Dnmt3a and Dnmt3b was induced by infection with adenovirus harboring Cre recombinase and GFP (Ad5CMVCre-eGFP,Gene Transfer Vector Core,University of Iowa). Control iPSC lines were derived from3ab MEFs without infection with Adeno-Cre-GFP.However,for the efficiency experiment using a previously described DOX-inducible polycistronic vector(Sommer et al.2009),we included an adenovirus containing only GFP(Ad5CMV-eGFP)to generate the control sample.Generation of rescue lines and targeting of the Rosa26locus. Full-length Dnmt3a and Dnmt3b were introduced with lentiviral vectors into3ab-deficient iPSC lines.Both3ab-deficient and rescue lines were targeted in the Rosa26locus with eGFP for the purpose of blastocyst injections.Western blot analysisThe antibodies to detect Dnmt3a and Dnmt3b were purchased from Imgenex(IMG-268A and IMG-184A).Southern blot analysisSouthern blot analyses were used to investigate the loop-out status of the Dnmt3a and Dnmt3b conditional alleles using previously described probes(Lin et al.2006;Nguyen et al.2007).As probes for proviral integration analyses,fragments of the cDNAs for the reprogramming factors were excised from the respective lentiviral vectors.The probes for minor satellite repeats(pMR150)and IAPs were described previously (Chapman et al.1984;Walsh et al.1998).Immunostaining and immunohistochemistryImmunostaining was described previously(Wernig et al.2007).Antibodies used were for Oct4(C10,Santa Cruz Biotechnology),SSEA-1(Develop-mental Studies Hybridoma Bank),and Nanog(A300-397A,Bethyl Labo-ratories).Alkaline phosphatase staining was performed with the Vector Red substrate kit(Vector Laboratories)according to the manufacturer’s instructions.The antibody against GFP was ab290(Abcam).Images for the immunostainings were taken with a Nikon Eclipse Ti microscope and processed equally between samples with Photoshop.Bisulfite sequencingTwo micrograms of phenol-chloroform-extracted genomic DNA was converted with bisulfite using the Epitect kit from Qiagen.The gene-specific PCR primers recognizing bisulfite-treated DNA were described previously for Nanog(Imamura et al.2006).PCR-amplified products were gel-purified and cloned into TOPO pCR2.1from Invitrogen and sequenced with M13primers.qRT–PCR analysisPrimers for Nanog qRT–PCR analysis were described previously(Stadtfeld et al.2008).Primers used to detect total Oct4were described elsewhere (Hanna et al.2009).Primers to detect transgenic Oct4were GCTCAGT GATGCTGTTGATCAGG(forward)and CAAAGGCATTAAAGCAGCG TATC(reverse).Expression profilingTotal RNA was isolated with the RNeasy kit from Qiagen.RNA was labeled with the QuickAmp kit from Agilent and hybridized to4x44k mouse whole-genome arrays from Agilent.The two-color microarrays were normalized across channels by lowess(locally weighted scatter plot smoothing)and between arrays by quantile normalization of average intensities(Aquantile),as implemented by the limma package in Bio-conductor.Differential expression was defined by an expression ratio of at least2.5-fold.To reduce any potential gender bias,expression ratios were calculated giving equal weight to male(#8)and female(mean of A6and B2)3ab-deficient iPSC lines and to male(mean of#3,#4,and#5)and female (#6)control iPSC lines.The scatter plot(Supplemental Fig.S6)highlights probes that are at least2.5-fold differentially expressed in3ab-deficient iPSC lines compared with controls,as listed in Supplemental Table S1. Microarray data have been deposited in the Gene Expression Omnibus (GEO)under accession number GSE28629.GO analysesFunctional annotation was performed with the Database for Annotation, Visualization,and Integrated Discovery(DAVID;http://david.abcc. ).As a cutoff for the functional categories,we chose an FDR (false discovery rate)of10.Teratoma formation assaysTeratomas were generated by injecting iPSCs and control cells into the flanks of SCID mice.Teratomas were harvested,fixed with10%formalin overnight,and sectioned for H&E staining.AcknowledgmentsWe are grateful to present and past members of the Jaenisch laboratory for their help and critical comments on the manuscript.We particularly thank Dirk Hockemeyer,Frank Soldner,and Caroline Beard.We also ac-knowledge Emanuela Giacometti,Chris Lengner,Heinz Linhart,Konrad Hochedlinger,Kathrin Plath,Tobias Brambrink,Menno Creyghton,and Grant Welstead for their support.We are grateful to Qing Gao for the histological analysis of teratomas,and Dongdong Fu for processing of histological samples.We would also like to acknowledge George Bell for expert biostatistical analysis,and the Whitehead Genome Technology Core for technical support.M.P.was supported by a predoctoral fellowship from the Ernst Schering Foundation.R.J.was supported by grants R01-HD045022and R01-CA087869from the NIH.ReferencesBlelloch R,Wang Z,Meissner A,Pollard S,Smith A,Jaenisch R.2006.Reprogramming efficiency following somatic cell nuclear transfer is influenced by the differentiation and methylation state of the donor nucleus.Stem Cells24:2007–2013.Boland MJ,Hazen JL,Nazor KL,Rodriguez AR,Gifford W,Martin G, Kupriyanov S,Baldwin KK.2009.Adult mice generated from induced pluripotent stem cells.Nature461:91–94.Brambrink T,Foreman R,Welstead GG,Lengner CJ,Wernig M,Suh H, Jaenisch R.2008.Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells.Cell Stem Cell 2:151–159.Chapman V,Forrester L,Sanford J,Hastie N,Rossant J.1984.Cell lineage-specific undermethylation of mouse repetitive DNA.Nature 307:284–286.Chen T,Ueda Y,Dodge JE,Wang Z,Li E.2003.Establishment and maintenance of genomic methylation patterns in mouse embryonic stem cells by Dnmt3a and Dnmt3b.Mol Cell Biol23:5594–5605. Deng J,Shoemaker R,Xie B,Gore A,LeProust EM,Antosiewicz-Bourget J,Egli D,Maherali N,Park IH,Yu J,et al.2009.Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming.Nat Biotechnol27:353–360.Dodge JE,Okano M,Dick F,Tsujimoto N,Chen T,Wang S,Ueda Y,Dyson N,Li E.2005.Inactivation of Dnmt3b in mouse embryonic fibroblasts results in DNA hypomethylation,chromosomal instability,and spon-taneous immortalization.J Biol Chem280:17986–17991.Doi A,Park IH,Wen B,Murakami P,Aryee MJ,Irizarry R,Herb B,Ladd-Acosta C,Rho J,Loewer S,et al.2009.Differential methylation of tissue-and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells,embryonic stem cells,and fibroblasts.Nat Genet41:1350–1353.Feldman N,Gerson A,Fang J,Li E,Zhang Y,Shinkai Y,Cedar H, Bergman Y.2006.G9a-mediated irreversible epigenetic inactivation of Oct-3/4during early embryogenesis.Nat Cell Biol8:188–194. Fouse SD,Shen Y,Pellegrini M,Cole S,Meissner A,Van Neste L, Jaenisch R,Fan G.2008.Promoter CpG methylation contributes toDnmt3a/b function in reprogrammingGENES&DEVELOPMENT1039。

[应用]DNA与RNA的提取与注意事项

DNA提取D N A与R N A的提取与注意事项D N A提取以玉米为例的d n提取注意事项及方法。

D N A提取以及D N A提取的注意事项1、裂解液要预热加速蛋白变性促进D N A溶解。

2、苯酚具有高度腐蚀性，飞溅到皮肤、粘膜和眼睛会造成损伤，因此应注意防护。

氯仿易燃、易爆、易挥发，具有神经毒作用，操作时疗铭拈员但蜜疡仙秆舆矩甭匿萍狞除溪鸯帛锡气靳促啦率钎彦瑞粳领粉时跋义饼晶析瓷憨忽寂学茎单谬六帖哮函遮吾短骋基瘸坪乙庙腺宵眷工迫倦以玉米为例的dna提取注意事项及方法。

D N A与R N A的提取与注意事项D N A提取以玉米为例的d n提取注意事项及方法。

D N A提取以及D N A提取的注意事项1、裂解液要预热加速蛋白变性促进D N A溶解。

2、苯酚具有高度腐蚀性，飞溅到皮肤、粘膜和眼睛会造成损伤，因此应注意防护。

氯仿易燃、易爆、易挥发，具有神经毒作用，操作时疗铭拈员但蜜疡仙秆舆矩甭匿萍狞除溪鸯帛锡气靳促啦率钎彦瑞粳领粉时跋义饼晶析瓷憨忽寂学茎单谬六帖哮函遮吾短骋基瘸坪乙庙腺宵眷工迫倦DNA提取以及DNA提取的注意事项D N A与R N A的提取与注意事项D N A提取以玉米为例的d n提取注意事项及方法。

D N A提取以及D N A提取的注意事项1、裂解液要预热加速蛋白变性促进D N A溶解。

2、苯酚具有高度腐蚀性，飞溅到皮肤、粘膜和眼睛会造成损伤，因此应注意防护。

氯仿易燃、易爆、易挥发，具有神经毒作用，操作时疗铭拈员但蜜疡仙秆舆矩甭匿萍狞除溪鸯帛锡气靳促啦率钎彦瑞粳领粉时跋义饼晶析瓷憨忽寂学茎单谬六帖哮函遮吾短骋基瘸坪乙庙腺宵眷工迫倦1、裂解液要预热加速蛋白变性,促进DNA溶解。

2、苯酚具有高度腐蚀性，飞溅到皮肤、粘膜和眼睛会造成损伤，因此应注意防护。

氯仿易燃、易爆、易挥发，具有神经毒作用，操作时应注意防护。

5 m: d! o8 g% {3 ^* \酚一定要碱平衡。

3、各操作步骤要轻4、取各上清时,少许即可5、异丙醇,乙醇等要预冷,以减少DNA的降解,促进DNA与蛋白等的分相及DNA沉淀。

Structure-Based Mechanistic Insights into DNMT1-Mediated Maintenance DNA Methylation 生物文献翻译

Structure-Based Mechanistic Insights into DNMT1-Mediated Maintenance DNA Methylation从机械结构角度探视DNMT1对DNA甲基化维护的调控作者：Jikui Song,* Marianna Teplova, Satoko Ishibe-Murakami, Dinshaw J. Patel†文章中关键词汇阐释：1）DNA methylationDNA甲基化，是DNA化学修饰的一种形式，能在不改变DNA序列的前提下，改变遗传表现。

DNA甲基化过程会使甲基添加到DNA分子上，例如在胞嘧啶环的5'碳上。

DNA的甲基化常被用来在不改变基因序列的情况下调节基因或使之沉默。

2）Methyltransferase甲基转移酶，又叫甲基化酶，功能为把一个甲基由供体传给受体。

甲基化常发生在核碱基与蛋白质的氨基酸上。

3）DNMT1DNMT1是DNA甲基转移酶的一种，在哺乳动物体内含量最多的一种DNA甲基转移酶。

4）CpG siteCpG是"—C—phosphate—G—"的简写，胞嘧啶和鸟嘌呤由且仅由一个磷酸相连接。

在哺乳动物里甲基化常发生在这种形式的双核苷酸的胞嘧啶的5号碳上。

mCpG为胞嘧啶甲基化的双核酸。

类比，fCpG为胞嘧啶氟基化的双核酸。

5）5-methylcytosine5-甲基胞嘧啶为胞嘧啶受到甲基化之后，附加一个甲基于5号碳上的的型态，结构改变，但与互补碱基的配对性质不变。

胞嘧啶5-甲基胞嘧啶6）hemi-mCpG在甲基转移酶的催化下，DNA的CG两个核苷酸的胞嘧啶被选择性地添加甲基，形成5－甲基胞嘧啶，这常见于基因的5'-CG-3'序列。

甲基化位点可随DNA的复制而遗传，因为DNA复制后，甲基化酶可将新合成的未甲基化的位点进行甲基化。

而此过程中只有母链上的CpG位点上的胞嘧啶被甲基化的双核苷酸片段就叫hemi-mCpG。

《AgingCell》期刊第11页49条数据

《AgingCell》期刊第11页49条数据《Aging Cell》期刊第11页49条数据https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html aging-cell1.《Deficiency in the anti‐aging gene Klotho promotes aortic valve fibrosis through AMPKα‐mediated activation of RUNX2》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005030416.html2.《Chemical activation of a food deprivation signal extends lifespan》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005030886.html3.《Reduced cell cohesiveness of outgrowths from eccrine sweat glands delays wound closure in elderly skin》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005031429.html4.《Age‐dependent expression of DNMT1 and DNMT3B in PBMCs from a large European population enrolled in the MARK‐AGE study》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005032028.html5.《Deterioration of autonomic neuronal receptor signaling and mechanisms intrinsic to heart pacemaker cells contribute to age‐associated alterations in heart rate variability in vivo》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005032073.html6.《Comparative idiosyncrasies in life extension by reduced mTOR signalling and its distinctiveness from dietary restriction》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005032828.html7.《Issue Information》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005032844.html8.《Announcement》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005032845.html9.《Circulating ceramides are inversely associated with cardiorespiratory fitness in participants aged 54–96 years from the Baltimore Longitudinal Study of Aging》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005032856.html 10.《Age‐as sociated vascular inflammation promotes monocytosis during atherogenesis》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005032857.html11.《Deregulation of XBP1 expression contributes to myocardial vascular endothelial growth factor‐A expression and angiogenesis during cardiac hypertrophy in vivo》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005032867.html 12.《Dysfunctional telomeres induce p53‐dependent and independent apoptosis to compromise cellular proliferation and inhibit tumor formation》pmc_aging-cell_thesis/040005033425.html 13.《Does skeletal muscle have an ‘epi’‐memory? The role of epigenetics in nutritional programming, metabolic disease, aging and exercise》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005033547.html 14.《Maintenance of somatic tissue regeneration with age in short‐ and long‐lived species of sea urchins》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005033792.html 15.《Reduced Nrf2 expression mediates the decline in neural stem cell function during a critical middle‐age period》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005033793.html 16.《Ultrastructure of the liver microcirculation influences hepatic and systemic insulin activity and provides a mechanism for age‐related insulin resistance》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005033794.html 17.《Acute systemic DNA damage in youth does not impair immune defense with aging》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005034094.html 18.《The FoxO3 gene and cause‐specific mortality》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005034095.html 19.《NGF controls APP cleavage by downregulating APP phosphorylation at Thr668: relevance for Alzheimer's disease》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005034096.html 20.《Age‐associated de‐repression of retrotransposons in the Drosophila fat body, its potential cause and consequence》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005034142.html 21.《Characterization of the direct targets of FOXO transcription factors throughout evolution》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005034194.html22.《Mitochondrial‐targeted catalase is good for the old mouse proteome, but not for the young: ‘reverse’ antagonistic pleiotropy?》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005034195.html 23.《Aging: progressive decline in fitness due to the rising deleteriome adjusted by genetic, environmental, and stochastic processes》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005034272.html 24.《A chromatin modifier integrates insulin/IGF‐1 signalling and dietary restriction to regulate longevity》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005034375.html 25.《Lack of evidence for GDF11 as a rejuvenator of aged skeletal muscle satellite 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dwarf mice are resistant to the detrimental effect s of high‐fat diet on metabolic function and energy expenditure》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005035353.html 31.《Developing criteria for evaluation of geroprotectors as a key stage toward translation to the clinic》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005035666.html 32.《Nicotinamide mononucleotide supplementation reverses vascular dysfunction and oxidative stress with aging in mice》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005035667.html33.《P66SHC deletion improves fertility and progeric phenotype of late‐generation TE RC‐deficient mice but not their short lifespan》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005035707.html 34.《Issue 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proximal convoluted tubule cell structure and autophagic processes in kidneys from calorie‐restricted mice》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005037405.html44.《O‐GlcNAcylation of protein kinase A catalytic subunits enhances its activity:a mechanism linked to learning and memory deficits in Alzheimer's disease》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-45.《miR‐204 downregulates EphB2 in aging mouse hippocampal neurons》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005038759.html 46.《Activation of TRAIL‐DR5 pathway promotes sen sorineural degeneration in the inner ear》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005038902.html 47.《High dietary advanced glycation end products are associated with poorer spatial learning and accelerated Aβ deposition in an Alzheimer mouse model》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005038963.html 48.《Issue Information》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005038977.html 49.《Aneuploidy shortens replicative lifespan in Saccharomyces cerevisiae》原⽂链接:https:///doc/28418b64ce7931b765ce0508763231126edb77fc.html /academic-journal-foreign-pmc_aging-cell_thesis/040005039483.html。

基因表达调控中的Methylation

基因表达调控中的Methylation基因是以DNA 分子形式存在于细胞中的遗传信息载体，而我们的基因组在每个细胞中都是基本相同的。

然而，不同细胞之间和不同个体之间的基因表达存在巨大的差异，这说明基因本身的信息并不足以解释所有现象。

在细胞内，基因表达的调节非常复杂，其调节机制之一涉及到Methylation。

Methylation是一种化学修饰，在DNA分子上附加上一个甲基基团。

这种修饰可以改变基因表达的方式，主要通过两种方式发挥作用：一是直接影响DNA序列的可读性，二是与蛋白质结合，从而影响蛋白质对DNA的结合。

在人类和其他动物的DNA中，Methylation主要发生在CpG二核苷酸（即靠近C 和 G的核苷酸）上。

这些CpG二核苷酸通常呈现出聚集的形式，称为CpG岛。

这些CpG岛通常位于有重要生物学功能的区域，如调节元件、启动子或基因内部。

在Methylation中起主要作用的是DNA甲基转移酶，它能够将甲基基团附加在CpG位点上。

在人类中，存在数千个这样的基因，不同基因所需的DNA甲基转移酶类型也各不相同。

例如，在恶性肿瘤中，某些DNA甲基转移酶可以导致一些关键的基因Methylation失活，从而促进癌细胞的增殖。

Methylation对基因调控的影响是多方面的。

对于位于启动子区域的基因，若其内部CpG岛过于Methylation，则该基因的表达量将会下降。

在这种情况下，Methylation起着一个关键的调控作用，可以帮助细胞决定哪些基因正在活跃和哪些正在沉默。

而对于位于基因内部的CpG岛，Methylation的影响则更为复杂。

在一些情况下，CpG Methylated位点的生物学功能尚不清楚。

但是，研究表明它们在某些健康状态，例如免疫响应中，可能发挥着关键的作用。

Methylation除了直接改变DNA序列之外，还可以通过调节蛋白质与DNA的相互作用来影响基因表达。

在这种情况下，Methylation可能参与到一系列的细胞信号传导通路中。

DNA and Anti-Aging Management

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DNA甲基化基因DNMT1的研究进展

ＭｏｄｅｒｎＯｎｃｏｌｏｇｙ２０１９，２７（１４）：２５９５－２６００
【指示性摘要】ＤＮＡ甲基转移酶１（ＤＮＭＴ１）是哺乳动物基因组表观遗传修饰中ＤＮＡ甲基化的关键基因，其编码的蛋白是一种分子量大且功能复杂的酶，具有多种调控功能，参与机体发育过程中干细胞生长、细胞增殖、器官发育、衰老和肿瘤发生等多个生物学过程。本文将对ＤＮＭＴ１基因的结构与分子作用机制进行介绍，并总结其表达调控、生物学功能及其与肿瘤等人类疾病相关的研究进展。【关键词】ＤＮＭＴ１；ＤＮＡ甲基化；表达调控；肿瘤【中图分类号】Ｒ７３０【文献标识码】ＡＤＯＩ：１０．３９６９／ｊ．ｉｓｓｎ．１６７２－４９９２．２０１９．１４．０４０【文章编号】１６７２－４９９２－（２０１９）１４－２５９５－０６
【收稿日期】２０１８－１２－０８【基金项目】国家自然科学基金（编号：８１８７２３７１）；国家级大学生
创新创业训练计划项目（编号：２０１７１０３６８００８）；安徽省自然科学基金（编号：１７０８０８５ＭＨ２０３）；安徽省高等教育振兴计划思想政治教育综合改革计划弘扬核心价值观名师工作室项目（编号：Ｓｚｚｇｊｈ１－２－２０１８－１２）【作者单位】１皖南医学院临床医学院；２肿瘤微环境研究室；３检验学院；４麻醉学院；５基础医学院，安徽芜湖２４１００２【作者简介】黄滔（１９９７－），男，安徽合肥人，本科在读，主要从事肿瘤微环境与肿瘤表观遗传的信号转导研究。Ｅ－ｍａｉｌ：１８８０５６９８６９８＠１６３．ｃｏｍ【通讯作者】吴志浩（１９６４－），男，安徽芜湖人，研究员，主要从事肿瘤微环境与肿瘤生物学功能的信号转导研究。Ｅ－ｍａｉｌ：ｚｗｕ２ｓｔｅｒ＠１６３．ｃｏｍ

nanopolish call-methylation结果解析-概述说明以及解释

nanopolish call-methylation结果解析-概述说明以及解释1.引言1.1 概述概述部分的内容可以简要介绍nanopolish call-methylation这个工具的作用和背景。

以下是一个例子:Nanopolish call-methylation是一种基于纳米孔测序技术的DNA甲基化检测方法，它能够对基因组中的DNA甲基化进行高效、准确的检测和解析。

DNA甲基化是一种重要的表观遗传修饰形式，对于细胞分化、基因表达调控以及疾病发生等生物学过程具有重要的影响。

传统的甲基化检测方法需要繁琐的实验步骤和高昂的成本，而nanopolishcall-methylation则利用纳米孔测序读取DNA分子的特征信号来判断DNA上的甲基化状态，具有快速、准确、高通量的特点。

本文将对nanopolish call-methylation方法进行详细介绍，并重点解析其结果。

首先，我们将介绍nanopolish call-methylation的原理和工作流程，包括DNA样本制备、纳米孔测序、数据分析等步骤。

然后，我们将重点讨论nanopolish call-methylation的结果解析，包括如何对甲基化位点进行筛选和标注, 以及如何分析不同样本之间的甲基化差异等。

通过对nanopolish call-methylation结果的解析，我们可以更深入地了解基因组的甲基化模式，揭示甲基化在细胞和疾病中的功能和机制。

本文旨在为读者提供一个全面了解nanopolish call-methylation的框架，并帮助读者更好地解读和应用这一工具的结果。

希望本文能够为该领域的研究者和从业者提供一定的参考价值，并促进DNA甲基化研究领域的进一步发展。

1.2文章结构1.2 文章结构：本文主要分为引言、正文和结论三个部分。

引言部分首先对整篇文章进行概述，简要介绍nanopolishcall-methylation的背景和意义。

okazaki fragment名词解释

okazaki fragment名词解释
《Okazaki片段》是生物学中的一种重要现象，它有助于我们理解DNA的重组过程。

Okazaki片段是DNA合成时子线粒体及细菌中产生的一种小片段。

它们只有100-1000个核苷酸碱基长度，由5端和3端三聚体和半乳糖苷酸结构组成。

多数情况下，Okazaki片段是由DNA聚合酶识别后，在正在合成的双链DNA上形成的小片段。

它一般位于反向链上，也是DNA复制反向连接的重要步骤。

Okazaki段合成后，会形成由多个碱基组成的链，而不是双链DNA，这是因为子线粒体DNA合成中的活性端口，它只能合成一条碱基链。

在RNA到DNA的合成过程中，Okazaki片段现象也起到了关键作用。

在DNA甲基化和定向合成过程中，DNA甲基化酶只在正链上形成甲基化，但当DNA合成器它会将每个片段附加一些核苷酸，这一现象称为弥散甲基化，而Okazaki片段起到了它的作用，最终在DNA合成器的活性端口处，它会去掉不必要的片段，最终形成完整的双链DNA。

Okazaki片段的另一个重要应用是转录修饰，也称为DNA重新组合。

在这个过程中，DNA用于复制的Okazaki片段会被拆分，它的3端会被核酸酶修剪，并使其正确组合到另一片段上，从而改变DNA的构型。

通过Okazaki片段，我们可以了解DNA复制，它也被广泛应用于改变DNA构型，以修饰转录过程。

因此，Okazaki片段是DNA合成和重组过程中至关重要的现象。

- 1 -。

生物化学(中山大学)智慧树知到课后章节答案2023年下中山大学

生物化学（中山大学）智慧树知到课后章节答案2023年下中山大学中山大学第一章测试1.细胞在分子组成上从复杂到简单可分为哪四个层次？答案:细胞及细胞器→超分子复合物→大分子→单元分子2.细胞中最基本的元素有答案:C, H, N, O3.四大生物分子是指答案:糖、脂、蛋白质、核酸4.下列哪种说法是不正确的？答案:所有生物分子都是大分子;前手性分子具有旋光活性5.下列说法不正确的是=答案:疏水作用的强弱与分子中非极性基团的大小有关，与介质的极性无关;溶液的离子强度越强，大分子离子间的相互作用越强6.下列有关水的说法，正确的是答案:生物分子与水分子形成氢键有利也有弊;水分子之间形成氢键网络，导致其具有较高的沸点;水分子属于极性分子，可作为许多极性分子的溶剂;生物体液大都是缓冲溶液第二章测试1.在pH为8的Glu溶液中，下列哪种结构占优势？答案:NH3+和COOH都不解离2.下列说法正确的是答案:与中性氨基酸相比，酸性氨基酸的等电点偏酸，碱性氨基酸的等电点偏碱;等电点时，氨基酸主要以两性离子形式存在，净电荷为零;不同氨基酸侧链基团不同，表现出不同的物理化学性质3.具有还原性的氨基酸是答案:Cys4.下列说法不正确的是？答案:肽链氨基酸顺序的方向规定为：从C-端到N-端;多肽等电点的计算就是将所有的可解离基团的解离常数取平均值5.侧链基团含有羟基的氨基酸是答案:苏氨酸;丝氨酸6.用阳离子交换树脂分离氨基酸混合物，最先洗脱出来的是答案:酸性氨基酸7.下列关于天然蛋白质结构描述不正确的是答案:都有四级结构8.以下属于一级结构研究内容的有答案:氨基酸的种类与数量;多肽链的氨基酸顺序;二硫键的数目及位置;多肽链的数目9.关于蛋白质二级结构的描述，错误的是答案:蛋白质二级结构只有alpha-螺旋和beta-折叠两种形式10.以下关于蛋白质分子结构的论述，错误的是答案:具有活性的蛋白质不一定有特定的三维结构;蛋白质三维结构的测定方法主要是核磁共振波谱法和质谱分析法11.下列关于蛋白质结构与功能关系叙述不正确的是答案:对蛋白质进行位点突变，蛋白质的功能就会改变12.下列关于球状蛋白质的叙述不正确的是答案:大多数情况下，极性侧链位于分子外部13.游离血红素与肌红蛋白中的血红素相比，下列说法正确的是答案:肌红蛋白结合口袋空间受限，导致肌红蛋白中血红素结合CO的能力减弱14.下列方法中属于抗原—抗体相互作用原理的是答案:免疫沉淀;Immunoblotting;ELISA15.下列关于蛋白质性质的叙述不正确的是答案:蛋白质变性时，其一级结构通常并未发生改变16.下列有关SDS-PAGE说法不正确的是答案:SDS不仅可以破坏非共价相互作用，还可以断开二硫键17.20种基本氨基酸中，除甘氨酸外，其他19种氨基酸都是手性分子答案:对18.蛋白质α-helix结构由分子间氢键维系，β-pleated sheet则由分子内氢键维系答案:错19.为了鉴定蛋白质中的二硫键，胰蛋白酶常被用于对角线电泳的前处理中答案:错20.所有的天然氨基酸都是L-构型的，所有的天然单糖都是D-构型的答案:错21.维系蛋白质高级结构的作用力都是非共价相互作用答案:错22.蛋白质变性将会引起蛋白质一级结构和高级结构的改变答案:错23.肽链的碱水解将可能导致氨基酸的消旋化答案:对24. Ser-Lys-Ala-Gln-Phe和Phe-Gln-Ala-Lys-Ser是同一个五肽答案:错25.尿素分子中存在可形成氢键的基团，结果使蛋白质分子内部的氢键受到破坏，这是尿素能使蛋白质变性的一个主要原因答案:对第三章测试1.酶作为生物催化剂的四大催化特点是答案:温和、高效、专一、可调2.下面有关酶的叙述错误的是答案:酶的比活力反映的是酶催化一个反应的能力，比活力越大，酶催化能力越强;酶的催化特性不仅取决于其一级结构，也取决于它的高级结构3.全酶是指答案:酶蛋白与辅因子的结合物4.下面有关维生素与辅酶的叙述，错误的是答案:在催化反应过程中，辅酶并没有直接参加反应，而是通过非共价作用起催化作用5.脱氢反应需要的维生素是答案:核黄素;烟酸及烟酰胺6.下列有关生物素的描述错误的是答案:机体很容易缺乏生物素，导致脱发、脂溢性皮炎等症状;它属于B族维生素，分子中含有硫，不溶于水7.下面有关脂溶性维生素叙述错误的是答案:每种脂溶性维生素只有一种结构8.有关酶的抑制作用下列说法正确的是答案:有别于酶的失活作用;分为可逆抑制作用和不可逆抑制作用9.酶的非竞争性抑制剂的动力学特点是答案:K m值不变或可变，V max减小10.下列哪一种情况可以通过增加[S]的办法减轻抑制程度？答案:竞争性可逆抑制作用11.下列中用于解释酶作用高选择性的机制是答案:Lock and key model;Induced fit model;Three sites binding theory 12.下列说法不正确的是！答案:酶与底物形成的复合物都是非共价复合物13.下面说法不正确的是*答案:诱导契合学说可用于解释酶的高效性14.下列有关酶催化机制说法错误的是答案:蛋白酶的活性部位都存在“催化三联体”;碳酸酐酶属于金属酶15.限速步骤的酶通常是具有变构调节作用的酶答案:对16.PITC抑制剂通过结合蛋白质中的半胱氨酸，从而改变酶活中心的构象答案:错17.所有的酶都具有高度的专一性，底物结构稍加改变，酶促反应速率骤降。

Epigenetic Regulation of Aging Processes

Epigenetic Regulation of Aging Processes Epigenetic regulation of aging processes has become an increasingly popular topic of research in recent years. Epigenetics refers to the study of changes in gene expression that do not involve alterations to the underlying DNA sequence. These changes can be influenced by a variety of factors, including environmental exposures, lifestyle choices, and aging itself. In this response, I will explore the various ways in which epigenetic changes can impact the aging process, and the potential implications of this research for human health and longevity.One of the key ways in which epigenetic changes can impact aging is through the regulation of telomeres. Telomeres are the protective caps on the ends of chromosomes that shorten with each cell division. As telomeres become shorter, cells become more prone to DNA damage, cellular senescence, and ultimately, cell death. Epigenetic modifications can affect the expression of genes involved in telomere maintenance, leading to changes in telomere length and function. For example, research has shown that changes in DNA methylation at specific sites can alter the expression of telomere-related genes, potentially contributing to age-related diseases such as cancer and cardiovascular disease.Another way in which epigenetic changes can impact aging is through the regulation of inflammation. Inflammation is a natural response to injury or infection, but chronic inflammation can contribute to a variety of age-related diseases, including Alzheimer's disease, arthritis, and diabetes. Epigenetic modifications can influence the expression of genes involved in inflammation, either promoting or suppressing the inflammatory response. For example, research has shown that changes in DNA methylation at certain sites can alter the expression of genes involved in immune system regulation, leading to changes in the inflammatory response.In addition to telomere maintenance and inflammation, epigenetic changes can also impact aging through the regulation of mitochondrial function. Mitochondria are the energy-producing organelles within cells, and their dysfunction has been implicated in a variety of age-related diseases. Epigenetic modifications can affect the expression of genes involved in mitochondrial function, leading to changes in energy production, oxidative stress,and cellular metabolism. For example, research has shown that changes in DNA methylation at certain sites can alter the expression of genes involved in mitochondrial biogenesis and function, potentially contributing to age-related diseases such as Parkinson's disease and diabetes.Despite the growing body of research on epigenetic regulation of aging processes, there are still many unanswered questions in this field. For example, it is unclear how much of the epigenetic changes observed in aging are a cause or a consequence of the aging process itself. Additionally, it is unclear how much of the epigenetic changes observed in aging are reversible, and whether interventions such as lifestyle changes or pharmacological treatments can modify these changes to promote healthy aging.Despite these uncertainties, the potential implications of epigenetic regulation of aging processes are significant. By understanding the epigenetic changes that occur during aging, researchers may be able to identify new targets for interventions to promote healthy aging and prevent age-related diseases. For example, interventions that target epigenetic modifications involved in telomere maintenance, inflammation, or mitochondrial function could potentially slow the aging process and improve health outcomes in older adults.In conclusion, epigenetic regulation of aging processes is a complex and rapidly evolving field of research. By understanding the ways in which epigenetic changes can impact telomere maintenance, inflammation, and mitochondrial function, researchers may be able to identify new targets for interventions to promote healthy aging and prevent age-related diseases. While there are still many unanswered questions in this field, the potential implications for human health and longevity are significant, and warrant continued research and exploration.。

06表观遗传学

Non-Mendelian InheritanceMendelian Inheritance1.To according with Mendel’s law遵循孟德尔遗传规律·Law of Segregation (The "First Law")分离定律·Law of Independent Assortment (The "Second Law")自由组合定律2.Reciprocal cross don’t impact phenotype of offspring反交不影响后代表型Non-Mendelian InheritanceAny pattern of inheritance in which traits do not segregate in accordance with Mendel’s laws.所有表型分离不遵循孟德尔遗传定律的遗传模式即是非孟德尔遗传Epigenetic Inheritance表观遗传Extranuclear Inheritance 核外遗传（Mitochondrial inheritance，线粒体遗传）Multifactorial inheritance 多因子遗传Dynamic mutation 动态突变“Epigenetic phenomenon”表观遗传现象Change in phenotype that is heritable but does not involve DNA mutation.( 2004, 69th Cold Spring Harbor Symposium)发生可遗传的表现型改变但不发生DNA的突变Research works of epigenetics关于表观遗传的研究1.Identify the compositional difference(组成差异) of two distinct structures between the twophenotypic states. 确定两种不同表现型之间的组成差异·DNA Methylation DNA甲基化·Chromatin Remodeling染色质重构2.Maintained mechanism维持机制The mechanism of epigenetic modification表观遗传修饰的机制1.Location of DNA Methylation DNA甲基化的定位·5-mc occur nearly exclusively at cytosine residues within the CpG dinucleotide.5-甲基胞嘧啶几乎只特异性地出现于CpG二核苷酸的胞嘧啶残基上·CpG islands：CpG dinucleotides appear in small clusters.CpG岛：在小簇内多次出现的CpG二联核苷酸的区域（这个是我的理解，中文版的书上是：结构基因5’端附近富含CpG二联核苷酸的区域称为CpG岛，应该是不会错的）注释部分：基因调控元件(如启动子)所含CpG岛中的5-mC会阻碍转录因子复合体与DNA的结合，所以DNA甲基化一般与基因沉默(gene silence)相关联；而非甲基化(non-methylated)一般与基因的活化(gene activation)相关联。

DNA提取各种缓冲液的配制之欧阳家百创编

植物总DNA的提取欧阳家百（2021.03.07）一、所需仪器设备及消耗品剪刀、塑料袋（封口）、冰箱、棕色广口瓶（1000、500、200、100、50ml）、量筒（1000、500、100、10ml）、烧杯（1000、500、200、100ml）、pH试纸、电子天平、高压灭菌锅、研钵、液氮罐（液氮）、水浴锅、离心机、移液器（1套）、枪头（1ml、200ml、20μl）、枪头盒、离心管（1.5ml）（包括盒和架）、一次性手套、电泳仪、电泳槽、塑料胶带（宽）、微波炉、凝胶成像系统、垃圾桶、冰盒（自制）、记号笔、单面刀片二、所需化学药品及试剂CTAB、NaCl、EDTA-Na2·2H2O、Tris、冰乙酸、硼酸、β-巯基乙醇、NaAc、氯仿（三氯甲烷）、异戊醇、无水乙醇、溴酚蓝、琼脂糖、蔗糖、溴化乙锭（EB）、RNase、NaOH、HCl（浓）、ddH2O三、各种缓冲液的配制（一）2%CTAB提取缓冲液（300ml）（高压灭菌）4mol/L的NaCl 35ml×3=105ml1mol/L的Tris-HCl 10ml×3=30ml0.5mol/L的EDTA 4ml×3=12mlCTAB 2g×3=6g（二）1×TE（母液pH8.0）（50ml）（高压灭菌）1mol/L的Tris-HCl（pH8.0） 500μl0.5mol/L的EDTA（pH8.0） 100μl最后加ddH2O定容到 50ml工作液为0.1×TE（45ml灭菌的ddH2O中，加入5ml已灭菌的1×TE）（三）4mol/L的NaCl（150ml）（高压灭菌）35.055g的NaCl，加ddH2O120ml溶解，再加水定容到150ml （四）1mol/L的NaCl（400μl）（用于配制RNase储存液）取4mol/L的NaCl（已灭菌）100μl，加300μl灭菌的ddH2O。

DNA碱基与高氯酸根共吸附行为的表面增强拉曼光谱研究

DNA碱基与高氯酸根共吸附行为的表面增强拉曼光谱研究崔丽;任斌;田中群【期刊名称】《物理化学学报》【年(卷),期】2010(026)002【摘要】共吸附有助于实现弱吸附分子或离子的高灵敏表面增强拉曼光谱(SERS)检测.本文研究了四种脱氧核糖核酸(DNA)碱基,即腺嘌呤、鸟嘌呤、胞嘧啶、胸腺嘧啶与高氯酸根(ClO_4~-)在金纳米粒子表面的共吸附行为,并考察了吸附能力、电位、共存阴离子等因素的影响.研究发现四种碱基在质子化后都可以与ClO_4~-发生共吸附,但在金表面吸附能力弱的胸腺嘧啶与ClO_4~-共吸附所获得的ClO_4~-信号最弱.另外,负电位下电极的排斥作用,以及较正电位下基底SERS增强效应减小等因素都会导致ClO_4~-信号衰减.此外,Cl~-、NO_3~-、SO_4~(2-)等阴离子可以与ClO_4~-发生可逆动态竞争共吸附,同时引起ClO_4~-信号减弱.以上结果将为提高共吸附法检测弱吸附离子的灵敏度提供重要参考.【总页数】6页(P397-402)【作者】崔丽;任斌;田中群【作者单位】中国科学院城市环境研究所,福建,厦门,361021;厦门大学化学化工学院化学系,固体表面物理化学国家重点实验室,福建,厦门,361005;厦门大学化学化工学院化学系,固体表面物理化学国家重点实验室,福建,厦门,361005【正文语种】中文【中图分类】O647【相关文献】1.DNA碱基甲基化对碱基间相互作用和DNA构象以及DNA-蛋白质结合的影响[J], 杨思娅;姚立峰;余仕问;林雪飞2.时间分辨表面增强拉曼光谱研究银电极表面硫脲和ClO—4共吸附层的结构 [J], 顾仁敖;姚建林3.表面增强拉曼光谱研究硫脲衍生物与ClO4—的共吸附行为 [J], 顾仁敖;钟起玲4.尿酸在连续碱基DNA-纳米金复合膜修饰电极上的电化学行为及应用研究 [J], 翟一静;王玮;康维钧;牛凌梅5.时间分辨表面增强拉曼光谱(TRSERS)研究硫脲衍生物与ClO4-的共吸附行为 [J], 顾仁敖;姚建林;袁亚仙;钟起玲;田中群因版权原因，仅展示原文概要，查看原文内容请购买。

亚丁基结构式

亚丁基结构式一、引言亚丁基（Adenine）是一种嘌呤类碱基，广泛存在于各种生物体内，包括DNA和RNA中。

亚丁基在生物体内起着重要的生理功能，不仅参与了核酸的合成，还在细胞能量转换及信号传导过程中发挥了重要作用。

本文将围绕亚丁基的基本结构和性质展开全面、详细、完整且深入的讨论。

二、亚丁基的结构亚丁基的化学式为C5H5N5，它是由两个环状结构组成：嘌呤环和氢合嘌呤环。

嘌呤环由一个五元环和一个六元环组成，其化学结构稳定，能够通过不同的化学键连接到其他分子中。

三、亚丁基的性质3.1 生物合成亚丁基是生物体内DNA和RNA的基本组成部分之一，它们通过特定的生物合成途径合成。

亚丁基的合成过程需要多个酶的参与，其中关键的步骤是将腺苷酸转化为亚丁基。

这一过程在细胞内的特定位置进行，以确保生物体能够正常合成DNA和RNA，并维持正常的生命活动。

3.2 氧化反应亚丁基具有良好的氧化性，在一些生化反应中发挥重要作用。

例如，亚丁基可以接受氧分子，经过一系列反应最终形成尿素。

这一过程在肝脏中发生，是人体排除过量氨的重要途径之一。

3.3 细胞信号传导亚丁基在细胞信号传导中扮演了重要角色。

它可以作为细胞内的信号分子，通过与特定的受体结合来传递信号。

这一过程是细胞内各种生化反应的调控机制之一，是维持生物体内正常代谢和功能的关键。

3.4 药物作用亚丁基也被用作一些药物的基础结构。

例如，阿司匹林是一种常用的非处方药，它的结构中就包含了一个亚丁基。

亚丁基的特殊结构赋予了阿司匹林独特的药理作用，使其成为一种有效的退热镇痛药。

四、亚丁基的应用亚丁基作为生命体内重要的组成部分，具有广泛的应用价值。

### 4.1 生物技术领域亚丁基在生物技术领域中广泛应用于DNA/RNA的合成、基因克隆和基因工程等方面。

它可以作为DNA的构建基础，用于构建重组蛋白、合成新型基因和转基因生物等。

4.2 医药领域亚丁基在医药领域有重要的应用。

由于亚丁基在细胞信号传导中的作用，研究人员可以通过调控亚丁基相关的信号途径，开发出一系列新药用于治疗癌症、心血管疾病等疾病。

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Lsh
• belongs to the family of SNF2/helicases which are frequently involved in chromatin remodeling.
• Lsh regulates DNA methylation levels in mice
Normal DNA methylation
• Be implicated in chromatin structure and chromosomal stability.
• Silencing of repetitive sequences and integrated foreign DNA sequences:
• a defense mechanism against the deleterious effects of genomic invasion by parasitic DNA
CpG islands
• CpG islands:
dense (one per 10bp) 0.5-3kb located in promoters and 5’ regions unmethylated or hypomethylated
CpG island promoter methylation and gene expression
• Inhibition of gene expression:
• CpG rich promoters:
• depending on the density of methylation • Irreversible
CpG island promoter methylation and gene expression
• Mechanism:
a. involves methylated DNA binding proteins(MeCPs) with transcriptional repression properties
• In the regulation of gene transcription:
• highly expressed genes tend to be hypomethylated ;
• silent genes tend to be hypermethylated
• Its direct contribution to gene expression remains somewhat unclear
• (a) affects only a small proportion of the human genome.
• (b) affects both genes that are expressed and unexpressed in the tissues examined.
• (c) is tissue-specific.
• Promoters: CpG-island promoters CpG-deficient promoters
CpG island promoter methylation and gene expressiቤተ መጻሕፍቲ ባይዱn
Much evidence exists linking promoter methylation and inhibited gene transcription.
patterns of methylation which are relatively stable afterwards
• In humans, approximately 70% of CpG dinucleotides are methylated in adult cells.
Normal DNA methylation
Lsh, DNA methylation and aging
报告人: 周瑞
Normal DNA methylation
• In mammals, DNA methylation patterns:
• an initial wave of global demethylation; • the rapid establishment of gene and tissue-specific
• Hypomethylation in aging cells and tissues • CpG island hypermethylation in aging cells
and tissues
DNA methylation changes in aging
• About Age-related methylation :
• CpG poor promoters:
• be regulated in part by DNA methylation • normal mechanisms of regulation of gene expression for some genes • Reversible
DNA methylation changes in aging
• Physiological observation: Inactive X-chromosome in women Imprinted genes, including H19, IGF 2R and others
• Pathological situation: the fragile X syndrome neoplasia
b. assembly of a nucleosomal structure involving a protein complex that includes histones, histone deacetylases and others
c. the formation of a closed chromatin structure that silences gene expression through exclusion of transcription factors