基因组甲基化1课件

1、基因组甲基化Liu Qingyou 20080318主要内容基本概念基因组甲基化的原理基因组甲基化的研究方法基因组甲基化在胚胎发育中的模式与作用表观遗传学在基因组中除了DNA和RNA序列以外，还有许多调控基因的信息，它们虽然本身不改变基因的序列，但是可以通过基因修饰，蛋白质与蛋白质、DNA和其它分子的相互作用，而影响和调节遗传的基因的功能和特性，并且通过细胞分裂和增殖周期影响遗传。这就是表观遗传学（epigenetics），表观遗传学又称为实验遗传学、化学遗传学、特异性遗传学、后遗传学、表遗传学和基因外调节系统，它是生命科学中一个普遍而又十分重要的 epigenetics是一种关于区别于DNA

2、序列改变的基因表达可遗传变化基因组含有两类遗传信息,一类是传统意义上的遗传信息,即DNA 序列所提供的遗传信息,另一类是表观遗传学信息,它提供了何时、何地、以何种方式去应用遗传信息的指令。 组织细胞时空特异性的表达Nature 441, 143-145 (11 May 2006) CpG isolandCpG岛位于基因的启动子和第一个外显子区,富含CpG二核苷酸,故称为CpG岛,一般作为启动子和基因复制的起始区.在哺乳动物中,几乎所有的甲基化位点均在CpG二核苷酸,并通过对CpG的甲基化来调节基因的表达,因为DNA的甲基化可改变染色质的结构,从而引起不同DNA结合蛋白的结合 。所有管家基因和4

3、%组织特异性基因的5端调控区存在CpG高频区 CpG島（CpG islands）是指DNA上一個區域，此區域含有大量相聯的胞嘧啶（C）、鳥嘌呤（G），以及使兩者相連的磷酸酯鍵（p）。哺乳類基因中的啟動子上，含有約40%的CpG島（人類約70%）。一般CpG島的長度約300到3000個鹼基對（bp）。 定義是指一個至少含有200bp的區域，其中GC所佔比例超過50%，且CpG的觀察值/預測值比例必須高於0.6。此部份的CpG島與基因相連，可用來作為限制酶的辨識位置。 鉑複合物需要電荷和順式位置的平衡才能表現其抗腫瘤效果 前已知鉑複合物可以廣泛地抑制動物和人類的腫瘤 DNA甲基化DNA甲基化是指真

4、核生物以S-腺苷甲硫氨酸(SAM)作为甲基供体,在DNA甲基转移酶(DNAmethyltransferases,DNMTs)的作用下,将甲基转移到特定碱基C上,形成5甲基胞嘧啶(5mC) 。Functions of DNA methylation in mammals Transcriptional gene silencingChromatin compactionGenome stabilitySuppression of homologous recombination between repeatsGenome defense（抵御）X chromosome inactivation

5、(females)Methyl groups(red) in DNA(map byDr. Craig Cooney) Calculations of binding affinities for MGMT/inhibitor complexes with computer simulation techniques DNA甲基化转移酶今后可能象限制性内切酶一样成为实验室常用的工具酶。来自立陶宛的HHMI研究人员SauliusKlimasauskas领导的小组利用DNA甲基化转移酶把一些化学分子转移到DNA上，为基因诊断和纳米生物技术的应用奠定了基础，有关结果发表在NatureChemicalB

6、iology。 DNMT1the most abundant and catalytically active enzyme in most cell types, which associates with S-phase replication foci via interaction with PCNA (Yokochi et al. 2002). Its primary role is believed to be that of a maintenance methyltransferase ( Bestor 2000), copying DNA methylation patter

7、ns following DNA replication. Murine knockouts of Dnmt1 are embryonic lethal at day E8.5. knockout of Dnmt3L, which is not a functional enzyme due to lack of critical catalytic site motifs, results in maternal DNA methylation imprint failure and male sterility in mice (Hata et al. 2002). There are c

8、urrently five members of the DNA methyltransferase family in mammalian cells. All of these proteins have their catalytic domain in the C-terminal region, and (with the exception of DNMT2) a regulatory domain in the N-terminal region. Most of the protein-protein interactions are mediated by the N-ter

9、minal region. DNA methyltransferases interact with many other proteins. Proteins involved in modulating chromatin structure include pRb, HDACs, HP1, SIN3A, hSNF2H, SUV39H1, and hCAP-C/E.Transcriptionally active chromatin regions tend to be hyperacetylated and hypomethylated. If a region of DNA or a

10、gene is destined for silencing, chromatin remodeling enzymes such as histone deacetylases and ATP-dependent chromatin remodelers likely begin the gene silencing process. One or more of these activities may recruit DNA methyltransferase resulting in DNA methylation, followed finally by recruitment of

11、 the methyl-CpG binding proteins. The region of DNA will then be heritably maintained in an inactive state. DNM 与 染色质remodelDNA Methylation as a Biomarker for CancerLoss of normal DNA methylation patterns in somatic cells results in loss of cell growth control. In normal cells, DNA methylation is fo

12、und predominantly in the repetitive fraction of the genome, including satellite DNA and parasitic elements (LINES, SINES, endogenous retroviruses) (Yoder et al. 1997).Tumor cells exhibit global hypomethylation of the genome accompanied by region-specific hypermethylation events (Baylin et al. 2001).

13、 Most of the hypomethylation occurs in repetitive DNA that is normally heavily methylated (Yoder et al. 1997).In normal cells (top) DNA methylation is concentrated in repetitive regions of the genome and most CpG island promoters are unmethylated. In tumor cells, the compartmentalization breaks down

14、 and repetitive DNA loses methylation while CpG island promoters acquire it, resulting in silencing of the associated gene. The DNMTs are likely targeted to particular regions via protein-protein interactions within chromatin.DNA Methylation and Chromatin Remodeling Modifications to the chromatin an

15、d the core histone tails impact gene expression and the ability of proteins that act on DNA to access their target sequences. Recent studies indicate that these epigenetic processes, as well as chromatin remodeling, cooperate to control chromatin structure and ultimately gene expression and DNA meth

16、ylation itself. What comes first, histone deacetylation, histone methylation, or DNA methylation? Epigenetics and the Regulation of Pluripotency and Differentiation of Stem Cells 基因组甲基化的研究方法一种比较传统的方法，能够定量测定基因组整体水平DNA甲基化水平。它由Kuo等1980年首次报道。过程是将DNA样品先经盐酸或氢氟酸水解成碱基，水解产物通过色谱柱，结果与标准品比较，用紫外光测定吸收峰值及其量，计算5mC/

17、(5mC+5C)的积分面积就得到基因组整体的甲基化水平。HPLC免疫化学法这种方法是基于单克隆抗体能够与5mC发生特异性反应。应用荧光素标记抗体使之与预先已固定在DEAE膜上的样品DNA特异性结合，对DEAE膜上的荧光素进行扫描得到5mC的水平，其荧光素强度与5mC水平成正比。直接测序法重亚硫酸盐使DNA中未发生甲基化的胞嘧啶脱氨基C转变成尿嘧啶U,而甲基化的胞嘧啶保持不变，进行PCR扩增所需片段,则尿嘧啶U全部转化成胸腺嘧啶T，最后,对PCR产物进行测序并且与未经处理的序列比较,判断是否CpG位点发生甲基化重亚硫酸盐处理The examples of bisulphite treated s

18、equences The example of a non-methylated clone is shown above (cytosines are converted to thymines). 甲基化特异性的PCR（methylation-specific PCR, MS-PCR）设计两对引物：1.引物末端均设计至检测位点结束；2.两对引物分别只能与重亚硫酸盐处理后的序列互补配对，即一对结合处理后的甲基化DNA链，另一对结合处理后的非甲基化DNA链。甲基化敏感性单核苷酸引物延伸（methylation-sensitive single nucleotide primer extensi

19、on，Ms-SnuPE）结合重亚硫酸盐的限制性内切酶法（combined bisulfite restriction analysis，COBRA）荧光法（Methylight）设计一个能与待测位点区互补的探针，探针的5端连接报告荧光，3端连接淬灭荧光，随后行实时定量PCR。如果探针能够与DNA杂交，则在PCR用引物延伸时，TaqDNA聚合酶5到3端的外切酶活性会将探针序列上5端的报告荧光切下，淬灭荧光不再能对报告荧光进行抑制，这样报告荧光发光，测定每个循环报告荧光的强度即可得到该位点的甲基化情况及水平基因芯片全基因组扫描Yan等2001年40将以分子杂交为基础的微阵列技术应用于DNA甲基化检测中，这种方法是基于杂交的寡核苷酸微阵列，是一种在基因组中寻找新位点的方法。包括用于整个基因组范围内扫描的差异甲基化（D