2010 核酸研究 Chromatin methylation activity of Dnmt3a and
表观遗传学学时详解演示文稿
DNA甲基化，组蛋白修饰等功能 ), 及基因型对表型的决定作用。
第四页，220共002225222222页年年年年。11月11月月8月8日8日8日日
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表观遗传修饰机制
第七页，220共00225222222页年2年年。年11月1月18月月8日日88日日
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特点概括
表观遗传学的特点：
可遗传的，即这类改变通过有丝分裂或减数分裂，能在细 胞或个体世代间遗传；
可逆性的基因表达调节，也有较少的学者描述为基 因活性或功能的改变；
没有DNA序列的改变或不能用DNA序列变化来解释。
和表达受抑的基因相关联。
第二十七页，共52页。
染色质重塑
第二十八页，共52页。
Chromosome
Chromatid
5X
Super Solenoid
40X
Solenoid
6X
Nucleosome
7X
DNA
Total: 7 x 6 x 40 x 5=8400
Model for methylation-dependent gene silencing. The structural element of chromatin is the nucleosomal core, which consists of a 146-bp DNA sequence wrapped around core histones. Acetylation of the histones causes an open chromatin config-uration that is associated with transcriptional activity. Methylated cytosines are recognized by methyl-CpG-binding proteins (MBDs), which in turn recruit histone deacetylases (HDACs) to the site of methylation, convert-ing the chromatin into a closed structure that can no longer be accessed by the 第二tr十a九n页，s共c52r页ip。tional machinery.
DNA甲基化检测 ppt课件
PPT课件
19
(３） Total input DNA样品作为模板，用待测基因特异性引物 进行 qPCR检测得到 Ct值(Input）。ChIP DNA样品作为模板， 用待测基因特异性引物进行 qPCR检测得到 Ct值(ChIP ）。样 品 ＤＮＡ 甲基化相对量（ChIP/Total input）通过以下公式计 算: ChIP/Total input=2^ [Ct(Input)-Ct(ChIP)] 通过结果可见脊髓损伤后,大鼠腓肠肌 eif5a基因 DNA甲基 化水平降低。此结果与我们之前得到的 eif5a在脊髓损伤大鼠 腓肠肌中高表达结果相吻合(图8-3）。
甲基化 DNA免疫沉淀法是一种高效富集甲基化 DNA的方法 ꎮ。在该方法中，可与 5mC特异性结合的抗体加入到变性的 基因组 DNA片段中，从而使甲基化的基因组片段免疫沉淀， 形成富集，通过与已有DNA微芯片技术相结合，从而进行 大规模DNA甲基化分析。 该方法简便、特异性高，适合 DNA甲基化组学的分析。 高密度的甲基化芯片是研究全基因甲基化状态的有力工具。
单链DNA在亚硫酸氢钠存在的条件下,能有效地将未甲 基化的脱氧 胞嘧啶(C)转化为脱氧尿嘧啶(U),随后作PCR 扩 增。此过程进一步将模板上的尿嘧啶(U),进一步转化为脱氧 胸腺嘧啶(T),而甲基化的胞嘧啶(mC)不受亚硫酸氢钠影响保 持不变。使DNA片段甲基化状态的差异转化为碱基序列的 差异，根据这种差异即可以获得目的片段的甲基化信 息.MSP法的原理是用亚硫酸氢盐处理目的 DNA,然后设计针 对甲基化和非甲基化序列的引物并进行PCR 扩增，最后通 过琼脂糖凝胶电泳确定与引物互补的 ＤＮＡ 序列的甲基化 PPT课件 状态。
表观遗传学是一种可遗传的调节基因表达的机制。不同于 经典遗传学，表观遗传学不涉及基因序列的改变，而且表观遗 传修饰是可逆的，在基因调控中起重要作用。 DNA甲基化是表观遗传学主要调控机制之一，在维持正常 细胞功能、遗传印记、胚胎发育、染色质结构改变以及人类疾 病的发生、发展中起着重要作用。 DNA甲基化是指，在DNA甲基转移酶（DNMT)作用下以S腺苷甲硫氨酸（SAM)作为甲基供体，将甲基转移到DNA脱氧胞 嘧啶的第五位C，形成5-甲基胞嘧啶（5-mC）的化学修饰过 程 这一过程通常发生在二核苷酸CpG中的胞嘧啶，是最常见 的哺乳动物基因组DNA后天修饰。 PPT课件
2012 RNA modulation of the human DNA methyltransferase 3A
RNA modulation of the human DNA methyltransferase 3ACeleste Holz-Schietinger 1and Norbert O.Reich 1,2,*1Interdepartmental Program in Biomolecular Science and Engineering and 2Department of Chemistry and Biochemistry,University of California,Santa Barbara,CA 93106-9510,USAReceived April 7,2012;Revised April 30,2012;Accepted May 11,2012ABSTRACTDNA methyltransferase 3A (DNMT3A)is one of two human de novo DNA methyltransferases essen-tial for transcription regulation during cellular devel-opment and differentiation.There is increasing evidence that RNA plays a role in directing DNA methylation to specific genomic locations within mammalian cells.Here,we describe two modes of RNA regulation of DNMT3A in vitro .We show a single-stranded RNA molecule that is antisense to the E-cadherin promoter binds tightly to the cata-lytic domain in a structurally dependent fashion causing potent inhibition of DNMT3A activity.Two other RNA molecules bind DNMT3A at an allo-steric site outside the catalytic domain,causing no change in catalysis.Our observation of the potent and specific in vitro modulation of DNMT3A activity by RNA supports in vivo data that RNA interacts with DNMT3A to regulate transcription.INTRODUCTIONDNA methylation,in conjunction with other epigenetic controllers including histone modifications and non-coding-RNA (ncRNA),is critical to the hierarchical regulation of transcription (1,2).In mammals,de novo 5-methylcytosine (5mC)patterning is established by DNA methyltransferase 3A (DNMT3A)and the closely related DNMT3B (1,3,4)and the patterning is maintained by DNMT1(5).The mechanisms that control genomic site selection by the de novo methyltransferases are not under-stood,but there is increasing evidence that binding partners,both RNA and protein,play a major role in controlling DNA methylation (6–9).Non-coding RNAs play an essential role in transcrip-tional regulation,including the formation of ternary complexes (protein/RNA/DNA)that modulate genomic targeting by directing hybridization and providing scaffolding for histone modifying enzymes and transcrip-tion factors (10–13).In plants,fission yeast and flies,interaction with ncRNA directs the DNMTs to specific targets (14).Similar mechanisms have been proposed for mammalian de novo DNMTs,which are supported by recent data showing piwi RNA-directed DNA methyla-tion in germ cells (15)and a short non-coding RNA is necessary for some DNMT3b-mediated methylation through the formation of a sequence-specific triplex struc-ture (16).Antisense RNA to the EF1a promoter causes transcriptional silencing in cells by inducing DNA methy-lation (17).Additionally,RNA transcripts are essential for genomic imprinting and X-chromosome inactivation,both of which occur via DNA methylation (18).This includes Tsix RNA,which regulates X-inactivation and binds to DNMT3A in vivo but neither DNMT3B nor DNMT1(19).Further evidence is shown by DNMTs binding various single-stranded (ss)forms of RNA and DNA,in some cases at an allosteric site (20–25).However,how RNA modulates the catalytic properties of the de novo DNMTs is not known.We probed the interactions between the human de novo DNMT3A and three RNA transcripts previously shown to either associate with DNMT3A or change promoter-specific methylation.These include a siRNA duplex to the NUP153mRNA (20),a ssRNA antisense to the EF1a promoter (EFS1)(26),and a single-stranded antisense RNA to the E-cadherin (CHD )promoter (27).We find these three RNA molecules bind DNMT3A in two differ-ent fashions.NUP153duplex RNA and ss EF1a RNA bind allosterically outside the catalytic domain,causing no measurable change in DNMT3A activity.The CHD RNA can bind to the catalytic domain and is a potent inhibitor of DNMT3A,and its modulation of DNMT3A activity is dependent on the RNA structure.MATERIALS AND METHODS MaterialsThe DNA used as substrates,poly-dIdC ($1900bp;Sigma-Aldrich)and GCbox2(GGGAATTCAAGGGG CGGGGCAATGTTAGGG)duplex were purchased from Midland Certified Reagent Company.Annealing was con-firmed by 12%polyacrylamide gels.The recognition site*To whom correspondence should be addressed.Tel:+18058938368;Fax:+18058934120;Email:reich@8550–8557Nucleic Acids Research,2012,Vol.40,No.17Published online 22June 2012doi:10.1093/nar/gks537ßThe Author(s)2012.Published by Oxford University Press.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (/licenses/by-nc/3.0),which permits unrestricted non-commercial use,distribution,and reproduction in any medium,provided the original work is properly cited.for DNMT3A is underlined.The human promoter substrates were described previously(28).RNA oligo-nucleotides are listed in Table1.CHD RNA(antisense E-cadherin promoter)was purchased from IDT and HPLC purified.EFS1(antisense EF1a promoter)and NUP153antisense and sense were purchased from Dharmacon,desalted and ethanol precipitated.Con-centrations are given in number of molecules.RNA purity was visualized by12%denaturing(12M Urea) PAGE gel and duplex formation was confirmed by12% native PAGE.RNA preparationRNA was prepared by adding1mM MgCl2,then heating to85 C for5min and slow cooling for5h unless otherwise noted(heating slow cooled).Otherwise the RNA was fast cooled by placed directly on ice after the heating step or unheated RNA solutions were thawed by allowing them to equilibrate to room temperature before being used. In methylation and binding assays,the MgCl2was diluted to50m M.The RNA after preparation was visualized on a12%native PAGE gel in0.5ÂTBE.The gel was run at4 C,for10h,visualized by SYBR Gold (Invitrogen)and imaged on a Storm840phosphorimager. Protein expression and purificationThe plasmids used for E.coli recombinant protein expression include pET28-hDNMT3ACopt,pET28-DNMT3A2Copt and pET28-hDNMT3A_catalytic_ domain.Plasmid construction,expression and purification were previously described(28).Native gel mobility RNA shift assaysFull-length DNMT3A,DNMT3A2and catalytic domain and M.HhaI(concentration as indicated),were incubated at room temperature for15min with100nM RNA. Binding occurred in reaction buffer with10m M S-Adenosyl-L-homocysteine(AdoHcy).Samples were run on native6%polyacryamide gels in0.5ÂTBE pH 7.8at250V for30min,stained with SYBR Gold and data were analyzed using ImageJ(29).K D values where determined from a single exponential using Prism V4.0 (GraphPad).Error bars represent the mean standard deviation of at least two replicates.Methylation assaysMethylation assays were carried out in RNase free reaction buffer with50mM Tris–Cl,1mM EDTA,1mM DTT,0.2mg/ml BSA,20mM NaCl and2m M AdoMet at pH7.8.Reactions were initiated by addition of pre-mixed DNA and RNA,and then quenched after 1h.Details of the assay are the same as those previously described(28).IC50assays used enzyme at30nM,DNA was either poly-dIdC at5m M or human promoters10m M, and RNA concentrations were varied as indicated in the figure legends.The RNAse control used5mg/ml of RNAse A pre-incubated with DNA and RNA for5min at37 C before carrying out the methylation assay. Fraction Inhibition was calculated by1À(product formed with RNA/product formed without RNA).The data werefit to a non-linear regression(one phase decay),where half life indicates IC50using Prism.Error bars represent the mean standard deviation of at least three replicates.IC50values for the RNA on each promoter were determined by measuring the amount product formed at varying RNA concentrations. Mechanism of inhibition studies used full-length DNMT3A and the catalytic domain at30nM;poly-dIdC and CHD RNA were used at concentrations indi-cated in thefigure legends.The data werefit using Sigma Plot6.1enzyme kinetics modular1.0,by modeling a single substrate single inhibitor for competitive,non-com-petitive,uncompetitive and mixed type.Modeling using partial inhibition approaches resulted in the same R2as the full-type inhibition.The models were ranked by R2 value.Error bars represent the mean standard deviation of at least three replicates.Data are graphically repre-sented in Lineweaver–Burk form using Sigma Plot6.1 enzyme kinetics modular1.0.Processivity assaysProcessivity assays were carried out as previously described(28)with DNMT3A at50nM.Reactions were initiated by the addition of substrate DNA,poly-dIdC (5m M).A total of200nM CHD RNA was added after 21min.The control is concurrent addition of RNA and DNA at the start of the reaction.Data werefit to a non-linear regression(one phase decay).Data werefit separately before the experimental reaction had CHD RNA added(time0–21min)and after the CHD RNA was added(21–260min),using Prism.Error bars represent the mean standard deviation of at least three replicates. The number of turnovers is calculated by the amount of production formation(nM)divided by the amount of catalytic active sites(nM)total protein concentration times percent active sites(18%)as previously described(22).Table1.RNA sequences used in this studyRNA name Sequence DescriptionCHD50GGGGUGA CG GGGACAGGCGGGGCUG30Antisense E-cadherin promoter EFS150GAGUUUACCC CGCG CCACCUU30Antisense EF1a promoter Nup153duplex50ACCAAAUAAAACUGGCAAAdTdT30siRNA30dTdTUGGUUUAUUUUGACCGUUU50Nuclear pore complex mRNA Nup153-as50ACCAAAUAAAACUGGCAAAdTdT30Nup153-s50UUUGCCAGUUUUAUUUGGUTdTd30Nucleic Acids Research,2012,Vol.40,No.178551M.HhaI methylation IC 50assays were carried out like DNMT3A assays,but with M.HhaI (30nM),AdoMet (5.5m M)and reactions were run for 20min.M.HhaI ex-pression and purification are described in Matje et al .(30).RESULTSThe three RNA sequences studied in this study were pre-viously shown to directly or indirectly associate with one or more of the DNMT enzymes or have been implicated in the epigenetic regulation of specific genes (Table 1).The antisense but not the sense strand of RNA to the human EF1a promoter (EFS1)binds DNMT3A in cells,whereas DNMT1showed no such binding (26).The EF1a RNA also caused transcriptional silencing in cells by inducing DNA methylation (17).The CHD RNA was implicated in DNA methylation changes localized to the E-cadherin promoter (27);although this study was retracted (31),the mitigating circumstances suggest that the body of work was correct.Indeed,the CHD RNA was successfully used to direct transcriptional gene silencing in subsequent work (32).The siRNA duplex to the NUP153mRNA binds to mouse DNMT3A and DNMT3B but not to DNMT1or DNMT2(20).Thus,two of the RNA mol-ecules tested in this study regulate transcriptional gene silencing in cells but the mechanism for regulation is not known.We tested the binding of these RNA to DNMT3A and test if these RNA molecules can modulate the activity of DNMT3A.The secondary structures of the ssRNAs were chara-cterized by melting curve analysis and native PAGE (Figure 1A).Three RNA preparation protocols were used for all RNA samples and were found to generate identical products for ss NUP153and ss EF1a RNA mol-ecules as seen by native PAGE.In contrast,the method of sample preparation dictates CHD RNA structure and ag-gregation (Figure 1A).Figure 1A shows the differencebetween unheated (lane 3),heated to 85 C then slow cooled (lane 1)or heated to 85 C followed by snap cooling on ice (lane 2).The unheated sample (lane 3)shows several forms:a highly aggregated (a )and a multimeric or secondary form of RNA (b and c ).When the RNA was heated to 85 C followed by fast cooling on ice,the majority of the RNA is in the single-stranded un-structured form (lane 2).Heating the RNA to 85 C followed by slow cooling results in multiple bands (lane 1).Much of the RNA is located higher in the gel than the single-stranded form,with a band indicated as b,and smearing at c and some single-stranded unstructured form.There is also a loss in aggregation at the top of the gel (a ),which occurred in the unheated sample.We found that the forms CHD RNA adopts under different preparation conditions have significant impact on the mechanisms and degree of inhibition.We sought to examine the effect of CHD RNA on DNMT3A methylation activity using filter binding assays to measure changes in the amount of tritiated methyl groups transferred from cofactor to poly-dIdC DNA by the enzyme (28).All three preparation conditions of the CHD RNA caused inhibition on full-length DNMT3A (Figure 1B).Unheated CHD RNA shows the greatest inhibition with an IC 50of 24±4.5nM and 100%inhibition.The slow cooled CHD RNA has an IC 50of 33±6.6nM with 89%inhibition and the snap cooled sample showed the least amount of inhibition with 31%inhibition and an IC 50of 67±31nM (Supplementary Table S1).The maximal inhibition by CHD RNA on DNMT3A activity was observed with the unheated and slow cooled RNA correlating with these samples having mostly secondary bands in the native gel (Figure 1A).All subsequent CHD RNA experiments used slowly cooled RNA (Figure 1A).CHD RNA is a potent inhibitor of DNMT3A.The in-hibition seen by the CHD RNA occurs on the full-length DNMT3A (Figure 2A)with an IC 50of 40±6.6nM.We tested if the N-terminal domains mediate this inhibition,such as the PWWP domain,which was previously shown to bind DNA (22).DNMT3A2is a truncated isoform ex-pressed in early development (33),while the catalytic domain of DNMT3A (cDNMT3A)has been demonstrated to have kinetic properties that are essen-tially identical to the full length (22).The IC 50of CHD RNA on DNMT3A2was found to be 24±6.5nM (Figure 2B)and 12±1.4nM against cDNMT3A (Figure 2C and D and Supplementary Table S2).We sought to determine if the CHD RNA-mediated inhibition of DNMT3A was sequence dependent via com-parison to single-stranded and duplex NUP153RNA and EF1a ssRNA.These RNAs were also prepared by slow cooling but additional experiments were done that showed preparation did not change the results for other RNA sequences tested.EF1a RNA was tested up to 2000nM with no inhibition observed (Figure 2E).The sense,anti-sense and duplex NUP153RNA also showed inhibition (Figure 2F and G).No changes in enzyme activity with EF1a or NUP153(antisense,sense or duplex)RNA were observed with DNMT3A2or the DNMT3A catalytic domain (data notshown).Figure 1.Conformational and functional characterization of the CHD RNA.The CHD RNA takes on secondary structure that controls DNMT3A inhibition.(A )Inhibition curve for the CHD RNA on DNMT3A.Arrow bars represent standard deviation from three reac-tions.Black circle =CHD RNA unheated;grey circle =CHD RNA heated slowly cooled;grey square =CHD RNA heated fast cooled.(B )Native-PAGE gel of CHD RNA after different treatments,same amount of RNA loaded for each ne1:RNA heated slow cooled,2:heated fast cooled and 3:unheated,a points to aggregated RNA still in the well,b and c are higher order structures and d is single-stranded unstructured.8552Nucleic Acids Research,2012,Vol.40,No.17To confirm the inhibition observed with CHD RNA is specific to DNMT3A,this RNA was tested against M.HhaI,a bacterial homolog with high structural conservation in the catalytic domain.CHD RNA does not inhibit M.HhaI tested up to 2000nM,150-fold greater than the DNMT3A IC 50(Figure 2H).To confirm that the CHD RNA causes the potent inhibition of DNMT3A,we treated the RNA and DNA (substrate)with RNase for 5min prior to assaying for methylation activity.This pre-treatment resulted in complete loss of the CHD RNA-mediated inhibition (Figure 2G).We confirmed the duplex NUP153RNA binds full-length DNMT3A,as previously shown by Jeffery and Nakielny (20).The binding affinity of the RNA to DNMT3A was determined to be 520±69nM (Figure 3A).The ss EF1a RNA showed very similar results as the NUP153RNA,binding to the full-length DNMT3A with a K D of 690±89nM (Figure 3B),but not binding to the catalytic domain.Minimal to no RNA binding was observed for the DNMT3A catalytic domain.However,the truncated isoform DNMT3A2showed similar binding as the full-length protein (Figure 3C).We also tested RNA binding to the bacterial homolog M.HhaI up to four times the K D of NUP153and EF1a with minimal to no binding for either RNA (Figure 3C).EF1a and NUP153RNA bind the enzymes but do not cause inhibition like the CHD RNA.To determine if the CHD RNA-mediated inhibition is dependent on the nature of the DNA substrate,we tested the inhibitory effect on methylation of seven human pro-moters (28).RNA inhibition was observed to varying degrees with all human promoters.The extent of inhib-ition ranged from 60%to 91%and the IC 50values ranged from 22±4nM to 68±22nM (Figure 4A–D,Supplementary Table S3and Supplementary Figure S1).We observed that the IC 50values is inversely related to the human promoters K m for DNMT3A (Figure 4D),this suggests that the substrate and RNA are binding to the same form of the enzyme.The 3-fold variation in RNA inhibition potency observed with different DNA sub-strates suggests that at least some of the inhibition involves a complex between the enzyme,DNA and RNA (seebelow).Figure 2.Inhibition by the ss CHD RNA.The modulation of DNMT3A activity was tested with four single-stranded and one double-stranded RNA,and only the CHD RNA altered DNMT3A activity.No inhibition was seen on the bacterial homolog.Inhibition curves for CHD RNA with the (A )full-length DNMT3A,(B )DNMT3A2or (C )catalytic domain.(D )CHD RNA IC 50values for DNMT3A truncations.Inhibition curves for EFS1RNA (E )and duplex NUP153(F )on full-length DNMT3A.(G )The extent of DNMT3A and M.HhaI inhibition with different RNA molecules including RNase treatment of CHD RNA prior to DNMT3A activity assay.RNA was varied from 0nM to 2000nM with largest affect on activity given in the bar graph.Control has no RNA added.RNA and DNA (5m M)were pre-mixed and combined with enzyme (50nM)and AdoMet (2m M)to start the reactions.(H )CHD RNA had no affect on M.HhaI activity.Nucleic Acids Research,2012,Vol.40,No.178553Methylation assays were carried out to probe the mech-anism of inhibition by varying the substrate concentration at multiple RNA concentrations.The results are shown in Figure 5.The inhibition data on the full-length DNMT3A best fit a non-competitive or mixed type model (R 2=0.933;Figure 5and Supplementary Table S4),however,the fit to a competitive model was not that dif-ferent (R 2=0.933).The catalytic domain showed com-petitive and mixed type (R 2=0.951)as the best-fit models (Supplementary Figure 2and Supplementary Table S4).Since mixed type inhibition involves an extra variable,non-competitive model best fits the data for the full-length DNMT3A and competitive for the catalytic domain.The non-competitive model best fits the data for the full-length DNMT3A and a competitive model best fits the catalytic domain data.Uncompetitive inhib-ition was the worst fit for both enzymes.Although the difference in fit between each model was not great enough to conclusively determine the mechanism ofinhibition,that data suggest the RNA interacts with the same form of the enzyme as DNA,in addition to binding the binary form (enzyme–DNA complex).Since conventional modeling of the mechanism was in-conclusive,we used another approach to determine if RNA and DNA (substrate)bind to the same form of the enzyme by testing if the RNA can inhibit the enzyme once bound to DNA.Our previous demonstration that DNMT3A acts processively on substrates with multiple sites of methylation provides the basis for this approach (28).An initial control was carried out by adding the CHD RNA and DNA substrate at the same time,which gave the expected 80%inhibition.When RNA is added 25min into the reaction no inhib-ition was seen for 45min,at which point a 20%decrease in inhibition is observed.A similar level of inhibition is seen when DNA lacking CpG sites is added as the chase (28),suggesting that the CHD RNA does not inhibit the enzyme when bound to DNA (Figure 6).These results are consistent with the RNA-mediated inhibition occurring largely as a result of the enzyme dissociating from the DNA and then binding the RNA.However,consideration of all of the inhibition data suggest that DNMT3A inhib-ition occurs through multiple complexes,as discussed below.DISCUSSIONAlthough compelling evidence supports direct and indirect roles of RNA in the regulation of DNA methylation (15,16),we know very little about how RNA–DNMT interactions alter the function of these enzymes.Of the four ssRNA sequences we studied,only the CHD RNA altered the function of DNMT3A (Figure 3).The potency and extent of inhibition is very dependent on the conform-ation of the RNA (Figure 2),further suggesting that the RNA–DNMT interface is quite specific.The CHD RNA did not inhibit the highly structurally homologous bacter-ial DNMT M.HhaI (34),providing further evidence of a unique RNA–DNMT3A interaction (Figure 3H).Two other RNA molecules did bind DNMT3A,the NUP153duplex and the ss EF1a ,with no measurable catalytic changes to DNMT3A,suggesting there may be several mechanisms of interaction with DNMT3A alone.Cellular data also show multiple types of RNA can direct transcriptional gene silencing through alterations in DNA methylation (16,17,32,35).Our data suggest the events observed in the cells may occur through a direct interaction of RNA to DNMT3A.DNMT3A binds CHD RNA with similar affinity as duplex DNA,whereas small molecule inhibitors show much weaker binding (36).The full-length DNMT3A data best fit a model invoking CHD RNA binding to at least one form of the enzyme already bound to the DNA substrate (e.g.non-competitive or mixed type inhibition).This form of allosteric inhibition is similar to previously characterized inhibition of DNMT1by ssDNA (24).The catalytic domain results are best fit to a competitive inhibition model,which is inconsistent with the full-length data,so we utilized the fact that DNMT3AcatalyzesFigure 3.RNA binds to DNMT3A allosterically.RNA-binding curve for DNMT3A full-length with duplex NUP153(A ).or ss EF1a ,(B )error bars are standard error for at least two independent samples insert is Native PAGE gel showing the loss of a RNA (100nM)band as an indicator of DNMT3A binding (varied from 0.2m M–2m M).(C ).The amount of RNA binding to DNMT3A full-length,truncations and M.HhaI at 1m M,RNA (100nM),native PAGE of the unbound RNA.RNA type as indicated,data from three independent gel shift samples.8554Nucleic Acids Research,2012,Vol.40,No.17multiple turnovers on a polymeric substrate (28)to test if the CHD RNA can inhibit the enzyme once bound to DNA.The lack of immediate inhibition upon addition of the RNA (Figure 6)shows that the dissociation of DNMT3A from its DNA substrate is required for CHD RNA inhibition.Thus,inhibition by CHD RNA during catalysis on poly-dIdC is largely or exclusively mediated by the RNAbinding to the same form of the enzyme,which binds the DNA.The most straightforward interpretation is that this involves the same active site,although this is not formally required.The catalytic domain of DNMT3A forms oligomeric complexes with itself (37),where all monomers are most likely not active.The CHDRNAFigure 4.The potency of RNA inhibition on DNMT3A is dependent on DNA substrate.Inhibition curves on the full-length DNMT3A (50nM)with CHD RNA,with different DNA substrates (10m M),(A )Survinin human promoter,(B )p15human promoter and (C )IC 50values for CHD RNA with different substrates,inhibition curves are in Supplementary Figure S1.(D )Inverse relationship of the substrate K m to the IC 50values from the CHD RNA on DNMT3A,log–log linear regression R 2=0.82.Figure 5.Inhibition Model for CHD RNA on full-length DNMT3A.The difference between models was not substantial,but the data best fit a non-competitive or mixed type model.Data shown fit to a non-competitive model R 2=0.933(mixed type =R 2=0.933,competi-tive R 2=0.928,uncompetitive R 2=0.92).CHD RNA inhibitor 0,10,30,50and 70nM.Inhibition model for CHD RNA on the catalytic domain of DNMT3A is shown in Supplementary FigureS2.Figure 6.The CHD RNA does not inhibit the enzyme when bound to DNA.DNMT3A was previously shown to catalyze multiple turnovers on a polymeric substrate,so RNA was added after DNMT3A bound to the polymeric substrate,to test if the enzyme is inhibited when bound to DNA.RNA-mediated inhibition occurs largely as a result of the enzyme dissociating from the DNA and then binding the RNA.CHD RNA (200nM)was added 25min after the full-length DNMT3A (50nM)was combined with the DNA (poly-dIdC at 5m M).Filled square =DNA,open circle =DNA then RNA added at 25min,filled triangle =DNA and RNA added at the start of reaction.Arrow indicates when CHD RNA was added.Nucleic Acids Research,2012,Vol.40,No.178555may bind to an inactive subunit and or bind where it alters the stability of such a complex.For example,RNA binding may disrupt oligomerization,which may not occur once the enzyme is bound to DNA.The somewhat poorfitting of the inhibition data with the full-length enzyme may be due to the CHD RNA binding to both the catalytic site and an allosteric site where the other RNA molecules are binding.The NUP153and EF1a RNA also bound DNMT3A but no changes in activity were observed.These two RNAs,unlike the CHD,only bound to the full-length and DNMT3A2and not to the catalytic domain alone, indicating allosteric binding outside the catalytic domain. The binding affinity was also weaker than the CHD or DNA by about8-fold,also indicating a different binding site.The PWWP domain of DNMT3A and DNMT3B has previously been shown to bind nucleic acids and this domain is needed for heterochromatin association (22,38,39).Recent studies of several chromatin modifica-tion complexes revealed that thousands of RNAs including antisense ncRNAs bind to chromatin modifying complexes(12,40–43).Histone modifying enzymes bind RNA to associate with chromatin or other binding partners.The RNA acts as a scaffold bridging two inde-pendent complexes together,but no changes in catalysis has been observed(44).The RNA molecules that bind DNMT3A suggest a similar scaffolding mechanism may be occurring.Regulatory implications of RNA–DNMT3A interactions We showed that the RNA-mediated inhibition of DNMT3A is highly sequence-specific,involves the cata-lytic domain,and involves the RNA binding to the same form of DNMT3A which binds the DNA substrate as well as to the DNMT3A:DNA complex.What is the potential relevance of these observations to the regulation of DNMT3A function in vivo?RNA may regulate DNMT3A in vivo but with distinct functional conse-quences as a result of occurring in a more complex context involving other proteins or factors.DNMT3A is well known to interact with numerous partner proteins (6–9),and the impact of RNA on DNMT3A in such settings could alter the function of the RNA.Inhibitory roles for RNA in vivo are uncommon but not without precedent(45).DNMT3A inhibition may serve to limit enzymatic activity at particular loci until the enzyme is either activated or directed to a particular genomic position.RNA-mediated inhibition of DNMT3A is pre-vented once the enzyme initiates methylation(Figure6), which may reconcile the inhibition with localized activity. This situation is supported by our observation that DNMT3A inhibition varies with different DNA sub-strates.Our in vitro observations may provide a mechan-ism for the recent demonstration that knockdown of two antisense ssRNAs derived from CpG islands induce methylation at the corresponding promoters(46). Alternatively,our studies were designed to reveal the functional impact of RNA molecules on DNMT3A, whereas the primary role of the RNA may be as a scaffold for DNMT3A and its partner proteins(16).This model is consistent with some RNA sequences binding DNMT3A without functional impact.RNA scaf-folding is also the mechanism used by chromatin modify-ing complexes and transcription factors to direct site specific binding(47).The extensive transcription of the human genome(48–50)may provide RNAs,which either directly module DNMT3A function,and/or provide a scaffold to localize the enzyme,leading to proximal methylation(16).We suggest that the potent and sequence-specific in vitro modulation of DNMT3A function by RNA supports the in vivo mechanisms in which RNA is postulated to regulate transcription(32).SUPPLEMENTARY DATASupplementary Data are available at NAR Online: Supplementary Tables1–4and Supplementary Figures 1and2.ACKNOWLEDGEMENTSWe thank Matt Purdy for help in the original design of the project and Douglas Matje for M.HhaI and critical reading of the manuscript.FUNDINGFunding for open access charge:California Cancer Coordinating Committee.Conflict of interest statement.None declared.REFERENCES1.Bird,A.(2002)DNA methylation patterns and epigenetic memory.Genes Dev.,16,6–21.2.Reik,W.,Dean,W.and Walter,J.(2001)Epigenetic reprogrammingin mammalian development.Science,293,1089–1093.3.Okano,M.,Bell,D.W.,Haber,D.A.and Li,E.(1999)DNAmethyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development.Cell,99,247–257.4.Chen,T.,Ueda,Y.,Dodge,J.E.,Wang,Z.and Li,E.(2003)Establishment and maintenance of genomic methylation patterns in mouse embryonic stem cells by Dnmt3a and Dnmt3b.Mol.Cell.Biol.,23,5594–5605.5.Pradhan,S.,Bacolla,A.,Wells,R.D.and Roberts,R.J.(1999)Recombinant human DNA(cytosine-5)methyltransferase.I.Expression,purification,and comparison of de novo andmaintenance methylation.J.Biol.Chem.,274,33002–33010.6.Denis,H.,Ndlovu,M.N.and Fuks,F.(2011)Regulation ofmammalian DNA methyltransferases:a route to new mechanisms.EMBO Rep.,12,647–656.7.Hata,K.,Okano,M.,Lei,H.and Li,E.(2002)Dnmt3L cooperateswith the Dnmt3family of de novo DNA methyltransferases toestablish maternal imprints in mice.Development,129,1983–1993.8.Klose,R.J.and Bird,A.P.(2006)Genomic DNA methylation:themark and its mediators.Trends Biochem.Sci.,31,89–97.9.Li,Y.Q.,Zhou,P.Z.,Zheng,X.D.,Walsh,C.P.and Xu,G.L.(2007)Association of Dnmt3a and thymine DNA glycosylase links DNA methylation with base-excision repair.Nucleic Acids Res.,35,390–400.10.Zhao,J.,Sun,B.K.,Erwin,J.A.,Song,J.J.and Lee,J.T.(2008)Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome.Science,322,750–756.11.Wang,X.,Arai,S.,Song,X.,Reichart,D.,Du,K.,Pascual,G.,Tempst,P.,Rosenfeld,M.G.,Glass,C.K.and Kurokawa,R.(2008)8556Nucleic Acids Research,2012,Vol.40,No.17。
P53网络的控制机理与建模
P53网络的控制机理与建模张启新北京理工大学 100081E-mail：zhangqixin@摘 要： P53蛋白是一种重要的肿瘤抑制因子，作为转录因子在转录网络之中发挥核心作用。
在细胞受到刺激时P53被激活进而调控其下游基因转录，通过下游基因的表达来调控其原始的细胞周期检查点、DNA损伤修复、细胞凋亡等生物学功能。
P53肿瘤抑制蛋白在细胞内的活性通过包括其转录目标Mdm2、Cop1和Pirh2的反馈环来调控的。
在把各种不同类型细胞置于离子射线，P53 和 Mdm2 的表达水平发生振荡。
这种振荡可以让细胞修复受损的DNA而不会有P53持续过度活化的风险。
据此，我们可以建立P53作用网络的模型来预测不同的刺激对P53网络的影响。
关键词： P53 网络 建模 反馈环 振荡1979年两个不同实验室在研究人的家族腺瘤样息肉病时发现用SV40病毒转化的细胞中有一种蛋白能与SV40的大T抗原相互作用并形成寡聚复合体[1，2]。
后用免疫共沉淀法测定其分子量为53KD，相应的这种蛋白被命名为P53。
1989年P53 野生型基因的鉴定证实野生型 P53 基因被定为肿瘤抑制基因或抗癌基因。
关于P53蛋白质，国内已经发表过很好的综述文章。
在此仅做简要介绍。
P53基因位于17号染色体的P臂上，长度约20Kb。
它编码的人P53蛋白由393个氨基酸组成，分为N-末端的酸性转录活化区（1-50位氨基酸）、信号区（60-94位氨基酸）、中央核心DNA连接区（100-290位氨基酸）、同源寡聚区（323-355位氨基酸）和C-末端的非专一DNA结合区（370-393）氨基酸。
天然 P53 蛋白在溶液中为四聚体，X- 衍射结晶学研究表明，P53 蛋白三级结构拥有 2 个 α 螺旋、10 个β 折叠和 3 个环，两个P53 蛋白可以利用单体间的反向 β- 折叠和反向 α- 螺旋间的相互作用形成二聚体，两个二聚体借助平行的螺旋 - 螺旋接触面形成四聚体。
2010年博士研究生入学考试生化与分子生物学
2010年博士研究生入学考试生化与分子生物学2010年博士研究生入学考试生化与分子生物学考试大纲第一部分考试说明一、考试性质生化与分子生物学是华中科技大学生命科学与技术学院生化与分子生物学等相关专业博士研究生入学考试的专业基础课。
生化与分子生物学入学考试在考查考生基本知识、基本理论的基础上，注重考查考生的试验设计和综合分析问题的能力。
考生应能准确地掌握生化与分子生物学的理论知识和实验方法；并对近期国内外有关生化与分子生物学杰出成果及前沿知识有所了解。
它的评价标准是高等学校优秀毕业生所能达到的及格或及格以上水平，以保证被录取者有较好的生物学理论实验基础。
考试对象为参加2010年全国博士研究生入学考试的准考考生。
二、评价目标生化与分子生物学入学考试在考查基本知识、基本理论的基础上，注重考查考生的试验设计能力，综合分析问题的能力。
考生应能：1、准确地再现生化与分子生物学方面的基础知识。
2、掌握基本的生化与分子生物学实验方法。
3、运用有关知识，解释和设计实验论证某种生化与分子生物学实验现象。
4、对近期国内外有关生化与分子生物学杰出成果及前沿知识应有所了解。
5、准确、恰当地使用本学科的专业术语，文字通顺，层次清楚，有论有据，合乎逻辑地表达。
三、考试形式与试卷结构1、答卷方式：闭卷，笔试。
2、答题时间：180分钟3、各部分内容的考查比例（满分为100分）基础知识约60%综合能力约40%4、题型比例面角，二级结构的基本类型，超二级结构，常见的纤维蛋白质）3、三级结构和四级结构（球状蛋白质三维结构的特征，亚基缔合和四级结构）（六）蛋白质结构和功能的关系1、肌红蛋白、血红蛋白的结构和功能，血红蛋白分子病的机理2、免疫球蛋白，免疫系统的识别，免疫球蛋白的结构和类别（七）蛋白质的分离、纯化和表征1、如何确定蛋白质分子的大小和形状2、蛋白质的胶体性质和沉淀性质3、蛋白质分离纯化的原则和方法以及含量和纯度的测定（八）酶通论1、酶是生物催化剂，酶作为生物催化剂的特性，酶的化学本质，辅酶2、酶的分类和命名3、酶的活力测定和分离纯化4、核酶、抗体酶、寡聚酶、同工酶及诱导酶（九）酶促反应动力学1、酶促反应的动力学（米氏学说，米氏常数，双倒数作图法，多种底物反应的不同机理，抑制剂对酶反应的影响）2、酶的抑制作用3、酶反应的影响因素（十）酶的作用机制和酶的调节1、酶的活性中心及其作用原理（酶的专一性，酶的活性中心，影响酶催化效率的因素）2、酶活性的调节控制和调节酶（别构效应、序变模型、齐变模型、胰蛋白酶）（十一）维生素与辅酶1、维生素的概念，和辅酶的关系2、脂溶性维生素和水溶性维生素（维生素A在视觉中的作用，维生素D与固醇，维生素C与坏血病，维生素B2与FMN、FAD，泛酸，叶酸，生物素，维生素B6，维生素B族与辅酶。
结直肠癌患者DNA甲基转移酶3A、3B基因单核苷酸多态性观察
山东 医药 ２ ０ １ ３年第 ５ ３卷 第 １ １期
结直肠癌患者 Ｄ Ｎ Ａ 甲基 转 移 酶 ３ Ａ、 ３ Ｂ 基 因单 核 苷 酸多 态 性 观 察
徐 峰。 ， 张 国梁 。 邹 江
（ １天 津 医科 大学 第一 中心 临床 学院 ， 天津３ ０ ０ １ ９ ２ ； ２天 津 市第一 中心 医院）
析， 判 断各位点与 Ｃ Ｒ Ｃ的关联 。结果 Ｄ Ｎ ＭＴ ３ Ａ的 ４个位 点之间不存 在紧密 连锁 ， Ｄ Ｎ ＭＴ ３ Ｂ的 ６个位 点相互 间均 呈强连锁不平衡 （ ｒ ２ ＞０ ． ８ ） ， 形成 的 ４种单体 型中以 Ｔ Ｇ Ｔ Ａ Ｇ Ｇ和 Ｃ Ａ Ｔ Ａ Ｇ Ｇ为主 ， ｒ ｓ ６ １ １ ９ ９ ５ ４和 ｒ ｓ ２ ４ ２ ４ ９ ０ ８的等位 基因 频率在观察组和对照组 间差异有 统计 学意义 （ Ｐ＜０ ． ０ ５ ） 。结 论 Ｃ Ｒ Ｃ患者 Ｄ Ｎ Ｍ Ｔ ３ Ｂ ｒ ｓ ６ １ １ ９ ９ ５ ４和 ｒ ｓ ２ ４ ２ ４ ９ ０ ８的等
（ ｏ ｂ ｓ ｅ ｒ ｖ ａ ｔ ｉ ｏ ｎ ｇ ｒ ｏ ｕ ｐ ） ．Ｔ ｈ ｅ ｓ ａ ｍｅ ｓ ａ ｍｐ ｌ ｅ ｓ ｗ ｅ ｒ ｅ ｃ ｏ ｌ ｌ ｅ ｃ ｔ ｅ ｄ ｒ ｆ ｏ ｍ ｔ ｈ ｅ ｏ ｔ ｈ ｅ ｒ ９ ４ ｃ ｏ ｎ ｔ ｒ ｏ ｌ ｓ ｕ ｂ ｊ ｅ ｃ ｔ ｓ（ ｃ ｏ ｎ ｔ ｒ ｏ ｌ ｇ ｒ ｏ ｕ ｐ ） ．Ｓ ｅ ｑ ｕ ｅ ｎ ｏ ｍ Ｍａ ｓ — ｓ Ａ ｒ ｒ ａ ｙ ｓ ｙ ｓ ｔ ｅ ｍ ｗ ａ ｓ ｕ ｓ ｅ ｄ ｔ ｏ ａ ｎ ａ ｌ ｙ ｚ ｅ ｔ ｅ ｎ Ｓ Ｎ Ｐ ｓ ｏ ｆ Ｄ Ｎ ＭＴ ３ Ａ（ ｒ ｓ １ ５ ５ ０ １ １ ７ ，ｒ ｓ １ ３ ４ ２ ０ ８ ２ ７ ，ｒ ｓ ｌ １ ８ ８ ７ １ ２ ０， ｒ ｓ １ ３ ４ ２ ８ ８ １ ２ ）ａ ｎ ｄ Ｄ Ｎ —
2010医学分子生物学考博
英语
VOA News、BBC、时事新闻、新闻场景
作文是环境保护与经济发展，大路边的题目
研究方法、熟悉内容、composition on demand
医学分子生物学
1.酶原和酶原激活
2.操纵子operon
3.原癌基因
4.分子杂交
5.密码子和反密码子
简答
1.什么是逆转录，复制和转录的异同
2.PKC、Ca-CaM-PKC的途径
3.DNA损伤与修复的4种机制及其中酶的作用
4.PCR反应组分、过程、用途（体外扩增、突变分析、定量、测序）
5.紫外线引起凋亡的途径及P53在其中起的作用
6.真核调控的层次有哪些？举三例说明其中3个层次（X染色质、乳糖操纵子、胰酶）
7.Waston、Crick DNA 双螺旋模型
论述
1、DNA合成的三种方法及所用的酶
2、细胞周期的影响因素
医学遗传学
1、遗传异质性
2、不等交换
3、Consanguity
4、剂量补偿
5、开米拉
问答
1、问什么染色体病常引起综合征？
2、何谓RNA加工？RNA加工的过程、意义
3、21-三体的主要成因、如何分析？
4、线粒体基因组的特点
5、杜氏肌营养不良患儿的母亲、表型正常的姨妈再发风险估计
6、RFLP定义、用途、举例
论述
给出A型短指症的系谱，如何进行基因定位？
1、英语口语
2、乳腺上皮细胞培养
3、荧光定量、FISH、免疫组化、基因芯片
4、单体型、lod值连锁分析、GW A、SNP基因组扫描、perl、python处理信息的能力。
DNA甲基转移酶：DNAmethyltransferase
DNA甲基转移酶July2011Molecule of the Month by David Goodsell译者：吕育松（农科院水稻所）关键词：表观遗传学，DNA甲基转移酶，DNMT1,DNMT3,限制性修饰系统引言：人体由表皮细胞、神经细胞、骨细胞以及其他各类细胞构成。
这些细胞具有不同的形态和大小，每种细胞根据其功能需要表达不同特异性的蛋白质。
然而身体内的每个细胞都包含相同的遗传信息，编码DNA序列。
那么细胞是如何决定哪些基因参与表达哪些基因要被舍弃呢？遗传学与表观遗传学：科学家已经发现DNA信息并不是简单的根据碱基序列表达而终止的。
细胞层控制顶端遗传密码的额外结构产生了修饰特定基因的“表观遗传”信息。
在一些情况下，这种修饰由核小体定位执行的。
在其他情况下，DNA被甲基化导致在蛋白质合成中修饰了编码框的阅读。
“清白历史”：在生命起始阶段当我们还是由单细胞组成时，这种表观信息就已经被清除掉了。
如在已经受精的鸡蛋中，这些甲基化基团已经被清除干净了，因而每个基因都一样。
当这些细胞发育成胚时，它们必须“决定”怎么发育——是成为表皮细胞亦或神经细胞还是取决于“命运”的安排呢？在这个时候甲基化转移酶就起到作用了。
使基因上的DNA被甲基化，导致一些基因失活、一些基因活性加强。
如DNA甲基化转移酶DNMT3（图1），通过基因组产生合适的表观遗传编码的甲基化基团。
图1DNA甲基转移酶3（DNMT3）和DNA甲基转移酶1(DNMT1)的结构甲基的保护：一旦细胞决定好了自己的命运，这些表观遗传编码必须被维持稳定。
当细胞分化时这种信息必须同时转移新的细胞中去。
DNA甲基化转移酶DNMT1（图1）就是起这样的作用：当DNA复制时，DNMT1将适合的甲基基团加到新形成DNA链上。
甲基基团一般都加在---CG---序列中胞嘧啶碱基上。
由于两条链中都含有胞嘧啶碱基。
由于两条链中都含有胞嘧啶碱基，因此在DNA甲基化区域中，每条链都含有一个甲基基团。
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ChromatinmethylationactivityofDnmt3aandDnmt3a/3LisguidedbyinteractionoftheADDdomainwiththehistoneH3tail
YingyingZhang1,RenataJurkowska1,SzabolcsSoeroes2,ArumugamRajavelu1,ArunkumarDhayalan1,InaBock1,PhilippRathert3,OleBrandt4,RichardReinhardt5,WolfgangFischle2andAlbertJeltsch1,*
1BiochemistryLaboratory,SchoolofEngineeringandScience,JacobsUniversityBremen,CampusRing1,
28759Bremen,2LaboratoryofChromatinBiochemistry,Max-PlanckInstituteforBiophysicalChemistry,D-37077Go¨ttingen,Germany,3ActiveMotifEurope,AvF.D.Roosevelt,104,BE-1330Rixensart,Belgium,
4INTAVISBioanalyticalInstrumentsAG,Nattermannallee1,50829Ko¨lnand5MaxPlanckInstitutefor
MolecularGenetics,Ihnestrasse63-73,D-14195Berlin-Dahlem,Germany
ReceivedDecember4,2009;RevisedFebruary11,2010;AcceptedFebruary18,2010
ABSTRACTUsingpeptidearraysandbindingtonativehistoneproteins,weshowthattheADDdomainofDnmt3aspecificallyinteractswiththeH3histone1–19tail.Bindingisdisruptedbydi-andtrimethylationofK4,phosphorylationofT3,S10orT11andacetylationofK4.WedidnotobservebindingtotheH41–19tail.TheADDdomainofDnmt3bshowsthesamebindingspecificity,suggestingthatthedistinctbio-logicalfunctionsofbothenzymesarenotrelatedtotheirADDdomains.ToestablishafunctionalroleoftheADDdomainbindingtounmodifiedH3tails,weanalyzedtheDNAmethylationofinvitroreconstitutedchromatinwithDnmt3a2,theDnmt3a2/Dnmt3Lcomplex,andthecatalyticdomainofDnmt3a.AllDnmt3acomplexespreferentiallymethylatedlinkerDNAregions.ChromatinsubstrateswithunmodifiedH3tailorwithH3K9me3modificationweremethylatedmoreefficientlybyfull-lengthDnmt3aandfull-lengthDnmt3a/3LcomplexesthanchromatintrimethylatedatH3K4.Incontrast,thecatalyticdomainofDnmt3awasnotaffectedbytheH3K4me3modification.TheseresultsdemonstratethatthebindingoftheADDdomaintoH3tailsunmethylatedatK4leadstothepreferentialmethylationofDNAboundtochromatinwiththismodificationstate.OurinvitroresultsrecapitulateDNAmethylationpatternsobservedingenome-wideDNAmethylationstudies.
INTRODUCTIONDNAmethylationisamajorformofepigeneticmodifica-tionandplaysessentialrolesingeneexpressionregulationandchromatinstructureremodeling(1–3).Themethyla-tionstateofDNAiscloselyconnectedtootherepigeneticsignalsincludinghistonemodifications,suchasmethyla-tionoracetylation,whichareknowntoactivateorsilencegeneexpression(4).ThemethylationofCpGdinucleo-tides(CpG)inmammaliancellsiscatalyzedbyDNAmethyltransferases(Dnmts),comprisingDnmt3aand3b,whichestablishDNAmethylationpatternsduringembry-onicdevelopmentandDnmt1,whichmaintainsthemethylationpatternafterDNAreplication(1,2,5).Dnmt3aand3bcontainlargeN-terminalpartsincludingaPWWPdomainandaPHD-likeADDdomain,whichinteractwithotherproteins,andaC-terminaldomainhar-boringthecatalyticcenter.TheisolatedcatalyticdomainsofDnmt3aand3bareenzymaticallyactive(6).AnothermemberoftheDnmt3family,Dnmt3L(Dnmt3-like),ishomologoustotheDnmt3enzymes,butlackscatalyticactivity.ItactsasaregulatoryfactorandcanstimulatethecatalyticactivityofDnmt3aand3b(7–10).TheADDdomainofDnmt3LwasshowntointeractspecificallywithhistoneH3tailsthatareunmethylatedatlysine4(11).TheDnmt3a/3Lcomplexformsaheterotetramer(12),suggestingthattheinteractionoftheDnmt3LADDdomainwiththeH3tailcoulddirectDNAmethylationbyDnmt3a(11).TheADDdomainsofDnmt3aand3bshareconsiderablehomologywithDnmt3LandrecentlybindingoftheDnmt3aADDdomaintoH3tailsunmodifiedatK4hasbeenshownandthestructureofthiscomplexsolved(13).Inaddition,an
*Towhomcorrespondenceshouldbeaddressed.Tel:+494212003247;Fax:+494212003249;Email:a.jeltsch@jacobs-university.de
4246–4253NucleicAcidsResearch,2010,Vol.38,No.13Publishedonline11March2010doi:10.1093/nar/gkq147
ßTheAuthor(s)2010.PublishedbyOxfordUniversityPress.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense(http://creativecommons.org/licenses/by-nc/2.5),whichpermitsunrestrictednon-commercialuse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.interactionoftheDnmt3aADDwithH4R3me2speptideshasbeenreportedaswell(14).Here,westudiedtheinter-actionoftheDnmt3aand3bADDdomainswithmodifiedhistonetailsbyapplyingahypothesisfreepeptidearraybindingapproach.Independentexperimentalevidencesuggestinganinflu-enceofhistonetailmodificationonDNAmethylationhasbeenprovidedthroughseveralepigenomicstudies.Genome-wideDNAmethylationandhistonemodificationstudiesrevealedastronganti-correlationofDNAmethylationandhistoneH3lysine4trimethylation(H3K4me3)(15–18),andacorrelationofH3K9me3withDNAmethylation(16).Afunctionalconnectionofthesetwosilencingmarks(DNAmethylationandH3K9methylation)hasbeenobservedbeforeinNeurosporacrassa(19,20),plants(21)andmammaliancells(22),wheredisruptionoftheH3K9me3signalledtoalossofDNAmethylation.Asdescribedabove,biochemicalandstructuraldatasuggestadirectroleoftheADDdomainofDnmt3LinthetargetingofDnmt3atochromatinunmethylatedatH3K4(11).However,sofarexperimentalevidenceforpreferentialmethylationofDNAboundtochromatin,whichcarriesaparticularmodificationpattern,hasnotbeenprovided.InordertoestablishthemolecularmechanismofDNAmethylationguidancebyhistonemodificationstates,wesetupacompleteinvitrosystemthatallowedustostudytheinfluenceofchromatinmodificationsontheactivityofpurifiedDnmt3aorDnmt3a/3L.Tothisend,histonesweregeneratedbynativepeptideligationtocontainspecificallyH3K4andH3K9methylation.Themodifiedhistoneswereassembledintooctamers,boundtoDNAandthereconstitutedoligonucleosomeswereusedassubstratesformethylationwithDnmt3aandDnmt3a/3L.