Chapter 4 - DNA11 methylation-Practice (1)

1、1DNA methylation彭 城Oct 23 2013A few data human genome :about 28 milion CpGs 6080% are generally methylated. Less than 10% of CpGs occur in CG-dense regions that are termed CpG islands; Half of CGI occur at gene body.A few knowledge 1.Three conserved enzymesDNA methyltransferase 1 (DNMT1): DNA methyl

2、ation maintenanceDNMT3A and DNMT3B:de novo methylationDNMT3L:is a catalytically inactive homologue of DNMT3A and DNMT3B2.Demethylation:thymidine DNA glycosylase (TDG)ten eleven translocation methylatosine dioxygenase(TET)activation-induced cytodine deaminase(AID)The many roles of DNA methylationDNMT

3、1DNMT3A/BMarking active regulatory elementsTFStadler et al, Nature, 2011ImprintingBrandeis et al, EMBO J, 1993Alternative promoter regulationMaunakea et al, Nature, 2010Silencing of repeatsAlu/LINE/SINEWalsh et al, Nat Genetics, 1998Gene silencingNaveh-Many and Cedar, PNAS, 1981CytosineMethyl-cytosi

4、neKnown features of DNA methylation in mammalsunknown features of DNA methylation in mammalsFUNCTIONS 转录起始位点的甲基化：有CpG岛的模式，没有CpG岛的模式 Gene body的甲基化 增强子的甲基化 绝缘子的甲基化Patterns at CpG island transcription start sites 大多数的CGIs保持不甲基化的状态。当在活跃的TSS有CGIs时，他们的启动子区域往往有NDRs（核小体贫乏区域），并且NDRs周围存在核小体变体H2A.Z,和H3K4me3。CG

5、I启动子能够被很多的机制所抑制，比如，myogenic differentiation 1 (MYOD1) or paired box 6 (PAX6)被polycomb蛋白，被抑制的启动子TSS区有核小体以及H3K27me3. 许多被抑制的基因同样存在CGIs的甲基化。这些被甲基化的启动子区域的CGIs大多数都是长时期的稳定的抑制状态。（例如，印记基因）Patterns at non-CpG-island TSSs 1.在TSS区域CpG-poor时，表达的基因往往是未甲基化的，而不表达的基因往往是甲基化的。比如OCT4 and NANOG promoters。 2.但是在05年的全基因组层

6、面上，发现甲基化和表达上没有负相关，但是在08年重分析又发现这个负相关的规律是存在。 3.一个问题是究竟甲基化是表达沉默的原因，还是结果。Reason or resultresult：methylation of the Hprt gene on the inactive X chromosome occurred after the chromosome had been inactivatedreason：study showed that the methylase was essential for differentiation of a fairly short-lived cel

7、l type, it seems possible that DNA methylation has a more instructive role in initiating rather than reinforcing the silencingGenome-wide studies in cancer cells have, however,shown that genes with CGI promoters that are alreadysilenced by Polycomb complexes are much more likelythan other genes to b

8、ecome methylated in cancer: thatis, the silent state precedes methylation主流的观点是：DNA甲基化只是起到维持沉默的作用Gene body methylation 1.在外显子区域的DNA甲基化是C-T突变的重要原因。 2.大概一半的CpGIs是在gene body把那些区域称做了orphan promoters，功能还未知，可能与早期发育相关。 3.在人脑细胞中，34%的gene body的CGI甲基化，但是并不阻碍表达。 4.只是转录起始对甲基化敏感。Possible functions of gene body m

9、ethylation 沉默DNA重复元件（retroviruses, LINE1 elements, Alu elements）但是允许管家基因的转录的延伸。 H3K36me3也是与转录的启动相关，但是不影响转录的延伸。 影响剪切. 全基因组分析发现exon高甲基化，intro低甲基化，并且这个数值转变很明显的发生在两者的边界区域。 CTCF的绑定受到DNA甲基化的影响导致了暂停RNA polymerase II的移动，而RNA polymerase II的动态影响剪切。Methylation at enhancers 在老鼠的基因组，增强子不是100%甲基化也不是完全不甲基化，趋向于low-

10、methylated regions (LMRs)。 在T细胞的不同子集，发现在增强子的区域有很大数量的differentially methylated regions (DMRs) 当在增强子区域去甲基化的时候，增强子起作用，但是依然是不知道甲基化是结果还是原因。Methylation at insulators 主要研究CTCF 首先是imprinted IGF2H19，当CTCF绑定的DNA序列被甲基化，影响了增强子与绝缘子之间的关系。 然后是CTCF绑定在表达CD45基因的exon5，对剪切有影响。 但是最新的全基因组的研究表明，不是DNA的甲基化影响CTCF的绑定，反而是CTCF的

11、绑定诱导去甲基化。Methods to detect methylation Bisulfite sequencing (BS-seq) Reduced Represention Bisulfite sequencing (RRBS) TAB-seq/oxBS-seq Methylated DNA Immunoprecipitation assays (MeDIP-seq/MeDIP-chip) HapII tiny fragment Enrichment by Ligation-mediated PCR (HELP) assay MRE-seq, MethylC-seq, MethylCap

12、-seqBS-seq (theory) :BS-seq (procedure) :Genomic DNACGACGACGCGAACGTTTmmBisulfiteconversionTargetedPCRSangersequencingConverted DNAmLibraryamplificationmCGchr20CGACGACGUGAAUGTTTmmmTraditional bisulfite sequencingLister et al, Nature, 2009Ultimate resolutionCCQuantitativeCCC100%50%0%Gary HonBS-seq (as

13、sessment) : Advantages Single base resolution Quantitative Disadvantages Defect: cant distinguish between 5mC and 5hmC Artifact: incomplete conversion (2.21%) Problem: DNA degradation Mapping: low comlexity of converted sequenceRRBS (procedure) :RRBS (assessment) : Advantages Enrichment of CpGs Low sample input Disadvantages Restriction enzyme limitation BS-seq flawsBS-seq, TAB-seq and oxBS-seq-TET oxygenasesBS-seq, TAB-seq and oxBS-seq-schemeBS-seq, TAB-seq and oxBS-seq-resultBisulfite Sequencingmapping schemessequence changes:Problems: Searching space is signif