IPA在DNA甲基化與ncRNA分析中的應用課件

1、Long-lasting alterations to DNA methylation and ncRNAs could underlie the effects of fetal alcohol exposure in mice.PMID: 23580197Ben LauferPI: Shiva M. SinghContributors: Katherine Mantha, Morgan L. Kleiber, Eric J. Diehl, Sean M.F. AddisonInstitution: Western University (formerly University of Wes

2、tern Ontario)Above and Beyond the Genome1Welcome to The Post-Genomics EraWeve sequenced the human genomeIt can now be done for only a few thousand dollars!We can sequence thousands of human genomes!Individual people can now go see their sequence!The benefits weve seen areIs this it?Shouldnt cracking

3、 the code of life give us more breakthroughs than we can dream of?Can 4 base pairs really code for complex life?Whats missing?What treasure is left?, Alper Uzun, PhD.“A mitotically (or meiotically) inheritable change in gene expression, independent of an alteration in DNA sequence”Berger et al. (200

4、9) Genes Dev.The Solution to the Post Genomics Era: EpigeneticsEpigenetic Mechanisms (Macmillan Publishers Ltd: Nature 441: 143-145. 11 May 2006)5Redefining the Central DogmaSaletore et al. Genome Biology 2012, 13:175 Epigenetic MechanismsHistone ModificationsMicroRNADNA Cytosine Methylation8Epigene

5、tic MechanismsHistone ModificationsMicroRNADNA Cytosine Methylation10microRNAsThe second level of the epigenetic landscapeActs at translation, as opposed to Histone modifications and DNA methylation, which act transcriptionAct as fine-tuners, rather than on and off switches like histone modification

6、s and DNA methylation.Typically result in low fold changes in gene expressionHowever, these changes are physiologically relevant.microRNA (miRNA)/chapters/www_microRNA/celmicRfig5.jpg12DNA Methylation (and other modifications)Histones arent the only ones to enjoy modificationsThere arent just four b

7、ase pairs anymoreCytosine has made a few friends/img/articles/epigenetic-base-modifications.pngDNA Cytosine Methylation. Metivier, R. et al. Cyclical DNA methylation of a transcriptionally active promoter. Nature 452, 4550 (2008).15The Epigenetic Landscape/content/m26565/latest/graphics35.jpgIt real

8、ly is a landscapeWaddingtons Epigenetic LandscapeWaddington, Conrad H. 1953. The Epigenetics of birds. Cambridge University PressA metaphor for biological developmentCell fates are established in development by epigenetic marks much like a marble rolls down to the lowest pointIncreasing irreversibil

9、ity of cell type differentiation as ridges rise between the valleys.19Environmental Conditions20Environmental Epigenetics21Environmentally Responsive Genome/content/epigenetics/nutrition/images/pathway.jpg22Fetal Alcohol Exposure Leading cause of preventable birth defects and mental deficits in Nort

10、h AmericaFASDFetal Alcohol Spectrum Disorders2-5% of pregnancies!Umbrella term for a number of physical abnormalities, behavioural and intellectual problemsStrongest manifestation is Fetal Alcohol Syndrome (FAS)Chudley et al. CMAJ 200524Spectrum DisordersWhere does an individual land in the spectrum

11、?Depends on:Genetic BackgroundTiming of ExposureDosage of ExposureOther Epigenetic and Environmental Factors, either:InheritedExperiencedSpectrum Meets LandscapeFetal Alcohol Syndrome (FAS)Partial Fetal Alcohol SyndromeAlcohol-Related Neurodevelopmental Disorder (ARND)Alcohol-Related Birth Defects (

12、ARBD)Fetal Alcohol Effects (FAE)28Mouse ModelsReasons for use:Useful for studies that would be impractical in humansReach sexual maturity early (68 wk)Birth multiple offspringAbbreviated gestational period (1821 days)Many generations can be analyzed within a relatively short timeframe (12 yr)Allows

13、for studying long-term changesAllow for analysis in vivo, which is essential for epigenetic studies.Cell cultures do not exhibit natural epigenetic propertiesSee: Embryo culture and epigenetics. Velker BA, Denomme MM, Mann MR. Methods in Molecular Biology. DisneyFetal Alcohol Exposure in an Animal M

14、odel We have shown that Fetal Alcohol Exposure (FAE) affects behaviour, learning and related genes.We have also recently shown that these changes are maintained for a lifetimeEven after exposure has ceased for weeksAre epigenetic mechanisms responsible?Kleiber et al. Behav. Brain. Res. 2012Kleiber e

15、t al. Brain. Res. 201230Epigenetic Mechanisms of InterestDNA Cytosine MethylationTypically turns expression on or off Metivier, et al 2008. Nature/chapters/www_microRNA/celmicRfig5.jpgNoncoding RNAi.e: miRNAFine tuners of gene expressionLow fold changes31Fetal Alcohol Exposure and MethylationFAE alt

16、ers the methylation and expression of genomically imprinted (uni-parental) genes in cell cultures derived from:Whole embryo PlacentaLiu et al. 2009 Epigenetics.Shukla et al. 2011 Alcohol. Clin. Exp. ResMorison et al. 2005, Trends. Genet. 30% of parentally imprinted transcripts are ncRNA. 32Imprinted

17、 ncRNA Key role in neurodevelopment and memory.Important for early life processesand functionally important for adult brain functions.Many are microRNAsWang et al. 2009 PLoS OneDavies et al. 2008 Adv. Exp. Med. Biolhttp:/4./_Ik_ovkt6ICg/SfjaLNt1ehI/AAAAAAAAAF4/4ZzazV4xx3Y/s320/imprinted+brain.jpg33F

18、etal Alcohol Exposure and miRNAsmiRNAs have been shown to be deregulated by FAE in fetal mouse brain cell cultureCo-incubation with folic acid prevents altered miRNA and target gene expression in mouse embryosFolic Acid is involved in establishing DNA methylation.Association between methylation and

19、expression, but what is the mechanism behind this relationship?Sathyan et al. 2007 J. Neurosci.Wang et al. 2009 Hum. Reprod.34Functional MechanismsAn alteration of methylation in a transcription factor binding site has the potential to affect gene expression.CTCF binding sites in the H19/Igf2 imprin

20、ting control region show differential methylation in FASD placental tissue. CTCF is a highly conserved ubiquitous zinc-finger protein with multiple functions in chromatin organization and gene regulationIt binds in a methylation sensitive manner to target sequencesIs this the functional mechanism fo

21、r the association between gene expression and DNA methylation in FASD?Williams et al 2008. J. Exp. Med. Filippova 2008 Curr. Top. Dev. Biol.Haycock et al. 2009 Biol. Reprod. 35HypothesisAlterations in DNA methylation and ncRNA expression are associated with life-long alterations in gene expression i

22、n the mouse brain after fetal alcohol exposure.36Continuous Preference Drinking (CPD)Free choiceQuantity monitored dailyNo Stress70% preference for 10% EtOHC57BL/6J miceMetabolize alcohol much quicker than humansBlood Alcohol Concentration (BAC)Represents moderate drinking= pregnant human mother who

23、 has a drink or two every now and then.Young & Olney 2006 Neurobiol. Dis.37Experimental DesignEverything done downstream of your workflow is dependent on what has happened upstream.Errors will amplify.For perspective see:Fundamentals of experimental design for cDNA microarrays. Churchill GA. Nature

24、Genetics.Probe set algorithms: is there a rational best bet? Seo J, Hoffman EP. BMC Bioinformatics.Tackling the widespread and critical impact of batch effects in high-throughput data. Leek JT. Nature Reviews Genetics. Whole Brain Methylation Array AnalysisDNA MethylationPND 7039Over 6,600 genes wit

25、h differences in 1 or more promoter regionsMore than half of imprinted genes in genomep 0.01Subjected to Ingenuity Pathway AnalysisTreatedControl40Looking through the noiseWhat is a p-value?Is a p=0.01 stringent enough for data from 2.1 Million probes?Theres going to be some false positives!But do w

26、e want to get rid of all the useful data caught up in those p-values?Particularly relevant for spectrum disorders as they are riddled with heterogeneity since we expect large amounts of variation within the experimental groupSystems BiologyThe antithesis to a reductionist approachReductionism is a p

27、hilosophy that the understanding of a complex system can be achieved in full by understanding its simpler component parts.Systems biology, on the other hand relies on examining the entirety of cellular processes and interactions in concert .Independent Component AnalysisIngenuity Core AnalysisA 1.2

28、fold increase in many genes of a pathway can have a potentially greater physiological impact than a 20-fold increase in a single gene.“Project microarray data into statistically independent components that correspond to putative biological processes, and to cluster genes according to over- or under-

29、expression in each component.”Further Perspective: Application of independent component analysis to microarrays. Lee SI, Batzoglou S. Genome Biology.Enriched Biological FunctionsMolecular and Cellular FunctionsNamep-value# GenesCell Death4.06E-04 - 4.97E-02 224Cellular Development6.24E-04 - 4.52E-02

30、 166Cellular Function and Maintenance9.57E-04 - 4.97E-02 86Cellular Movement4.12E-03 - 4.52E-02 41Cell Signaling8.43E-03 - 4.97E-02 26Physiological System Development and FunctionNamep-value# GenesNervous System Development and Function3.86E-05 - 4.97E-02 273Tissue Morphology1.64E-04 - 4.23E-02 97Be

31、havior1.62E-03 - 1.58E-02 24Embryonic Development1.23E-02 - 4.23E-02 29Organismal Development1.23E-03 - 4.23E-02 25Many have been previously implicated in FASD and all are highly compatible45Using MeDIP and ChIP DataSuper Simple StuffExcel table with column 1 containing gene name from upstream analy

32、sisSubsequent columns for metrics of interest (optional)Top Affected IPA Network“Behaviour, Neurological Disease, and Psychological Disorders” (IPA Score 65)47Network BiologyThe distribution of nodes (i.e: genes) in cellular networks is highly non-uniformMost of the nodes having only a few links to

33、other nodes.However, there are a few nodes with a very large number of links called hubs The importance of the relationship between a system and single gene is highlighted in these cases.While a network can tolerate many disruptions to its lesser-connected nodes, a similar disruption to a single hub

34、 can be catastrophic to the networks it connects. Further Insight:Network biology: understanding the cells functional organization. Barabsi AL, Oltvai ZN. Nature Reviews Genetics.Top Affected IPA Network“Behaviour, Neurological Disease, and Psychological Disorders” (IPA Score 65)40% of hub-genes pro

35、moters investigated had CTCF binding sites49Gene PCTCFGene NameProtein NameH19N/A (ncRNA)Gtl2 (Meg3)N/A (ncRNA)NpyPro-neuropeptide YAkt1RAC-alpha serine/threonine-protein kinaseGhrGrowth hormone receptorNtrk1High affinity nerve growth factor receptorApoeApolipoprotein EGrin2cGlutamate NMDA receptor

36、subunit epsilon-3Gene NameProtein NameAppAmyloid beta A4 proteinMbpMyelin basic proteinAtp1a2Sodium/potassium-transporting ATPase subunit alpha-2Grin1G protein-regulated inducer of neurite outgrowth 1Gene PCTCFCTCFBSDB: a CTCF binding site database for characterization of vertebrate genomic insulato

37、rsBao L et al. Nucleic Acids Research 2008Pten Canonical Signaling PathwaySignificantly affected canonical pathway (p=1.9E-06)54/95 molecules showed significant differential methylation in their promotersControls the tempo of the process of newborn neuron integration during adult neurogenesisincludi

38、ng correct neuron positioning, dendritic development, and synapse formation.Porteous et al. 2009 Neuron51Methylation Array Results SummaryOver 6000 genes with significant differences in their promotersMore than of the molecules involved Pten Signaling affectedNot a random sampleEnriched for relevant

39、 functionsMany CTCF binding sites in important neurodevelopmental genes showed differences in methylationDNA MethylationPND 7052Whole Brain ncRNA Expression Array AnalysisGene ExpressionPND 70microRNA53miRNA Array Heatmapsp 0.05FC 1.2TreatedTreatedTreatedTreatedControlControlControlControlLaufer et

40、al. Disease Models & Mechanisms. 201354ncRNA Venn Diagramp 0.05FC 1.21Laufer et al. Disease Models & Mechanisms. 201355Imprinted Noncoding RNA ClustersLocalized to the brainOnly 3 clusters in mouse genomeSfmbt2 (Chr 2), Snrpn-Ube3a (Chr 7), Dlk1-Dio3 (Chr 12)20% of altered miRNAs in all exposure par

41、adigmsAssociated with FASD related endophenotypes56Cluster of Interest: Snrpn-Ube3a (Chr 7)III5htr2cPre-mRNA IIIIIIIVVaVbVIReceptor with a stronger serotonin responseInclusion of exon Vb without mRNA editing during alternative splicingsnoRNA binds to mRNAH/MBII-52(SNORD115)Showed significant up-regu

42、lation in:all 4 treatment paradigmsCPD and injectionsboth array typesmiRNA and gene Laufer et al. Disease Models & Mechanisms. 201357PND 70microRNAmiRNA and Gene Expression Results SummaryGlobal Expression changesIndividual miRNAs unique to treatment paradigm20% of affected miRNAs belong to imprinte

43、d clustersH/MBII-52 only ncRNA (and gene) affected in all paradigms and arraysGene Expression58Whole Brain Bioinformatic AnalysisGene ExpressionmicroRNADNA MethylationPND 7059Creating a miRNA Target FilterWhat types of Data can be used?Any Gene Expression Data!Most arrays and RNA-Seq technologies as

44、say miRNAsJust create two separate files:microRNA ExpressionGene ExpressionImporting and formatting miRNA ExpressionmicroRNA AnalysisAdding Gene ExpressionExpression PairingHigh Quality Regulatory RelationshipsFilteringEnd Result!Wasnt that Easy?I really enjoyed the user interface of this program. I

45、t makes accessing a large annotated database of genetic information a breeze.Letting you get back to the biology!+ The Power of Ad-HocIPA miRNA Target FiltermiRNA IDmiRNA Fold ChangeGene IDGene Fold ChangeConfidence of Interactionmir-369-5p-1.336Pten1.377Highmir-25-1.224Pten1.377Highmir-495-1.232Pte

46、n1.377Highmir-1521.208Otx2-1.27Highmir-12241.528Nmnat1-1.237Moderatemir-4311.366Nmnat1-1.237Moderatemir-743a1.341Nmnat1-1.237Moderatemir-17*1.451Slitrk2-1.202Highmir-200a*1.178Slitrk2-1.202Moderate34 genes identified with reverse pairwise relationships to predicted miRNAs4 are highly compatible with FASD:71miRNA Target Filter Gene of InterestA novel role for Pten in FASD:Pten is a lipid phosphatase that suppresses Akt activation.Akt:Regulates neuronal developme