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Stanford CS 374 - Computational Methods for Studying Chromatin Structure and Remodeling

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Computational Methods for Studying Chromatin Structure and RemodelingHunter Richards8 April 2008ReferencesSegal E, Fondufe-Mittendorf Y, Chen L, Thastrom A, Field Y, Moore I, Wang J, Widom J. 2006. Nature v442 pp772-778Ioshikhes I, Albert I, Zanton S, Pugh B. 2006. Nat. Gene. v38 n10 pp1210-1215Oszolak F, Song J, Liu X, Fisher D. 2007. Nat. Biotech. v25 n2 pp244-248Papers are on class website: cs374.stanford.eduOverviewBackground on chromatinDiscuss three chromatin-related papersWhat determines chromatin structure?How does it regulate genes?Are these papers' findings consistent?Conclusions and QuestionsChromatin: Beyond the Central DogmaDNARNAproteinbiological functionchromatin changes drastically what we know about this stepReview: TranscriptionDNA sequence to complimentary RNA sequence for later translation into protein...Source: en.wikipedia.org/TranscriptionDNA sequence to complimentary RNA sequence for later translation into protein......but if RNA polymerase is blocked, no transcription occurs.Chromatin: Beyond the Central DogmaDNARNAproteinbiological function●chromatin allows another level of regulation of gene expression by blocking transcription●regulation is important in all biological processesWhat is Chromatin?DNA plus specific proteinsGenomic DNA coiled around cylindrical protein complexes called histones~150bp wound around histone; histones linked with ~50bp linker regionsAround histone, blocked; in linker, openChromatin in ContextStructure of a Histone~150 bp of DNA wrapped around histone “core”DNA is fairly rigid; needs AA or TT pairings to give flexibility Source: Segal et al.First, Some TerminologyHistone: a class of cylindrical, DNA-binding proteinsNucleosome: a single histone with its bound ~150bp of DNALinker Region: the ~50bp region between nucleosomesChromatin: many nucleosomes and linkers togetherCompeting Theories: cis and transHow is chromatin structure determined:cis: by the proximal, underlying sequencetrans: by other factors, independent of local DNAProbably a mixture of both (in fact Segal et al. puts it at ~50/50):Sequence control has been demonstrated......but chromatin structure changesCommon Logical Features of Computational PapersOnly then, make predictionsVerification and testing of model against previous dataGeneration of a model based on experimental dataSegal, et al.“A genomic code for nucleosome positioning”Creation of modelTesting of model against other genomesCollect Nucleosome-Bound Sequences“DNA footprint analysis”“DNA footprint analysis”Isolate genomic DNAMicrococcal digestion: digests all it can accessRemove histonesClone and sequence histone-bound DNAAlignment ISource: Segal et al., Sup. Figs.Alignment IISource: Segal et al., Sup. Figs.Probability Distribution●Markov Assumption: A chaining of probabilities.●Takes into account biochemical nature of histones, i.e. AAs and TTs are favoredThermodyanmic Model: Actually Placing HistonesFrom the probabilistic model, derived a thermodynamic model that included spatial exclusionPartition function: for all “legal” configurations –i.e. those that don't overlap -- choose the one that the probability function says is most likelySource: Segal et al.One View of the ModelAA/TT varies in sinusoid with period 10bp:Validate Predictions of the Model:Does it make sense?Assay for nucleosome binding for sequences predicted by model.“Teach” model on random genetic fragments; does your model reappear?Do the histone positions predicted by your model agree with experimental evidence?Agreement with Experiment1.Measure distance between predicted and known nucleosomes: 54% fell within 35bp vs. 39% by chance.2.Re-taught model using chicken histones, no significant difference.3.Outliers: stable histones in the genome were predicted by model; most highly stable predicted histones actually existed.4.~ 50% of all known histone positions predicted solely by sequence factorsFindings and PredictionsPromoter regions are nucleosome poorFindings and PredictionsChromatin structure differentiates between otherwise identical binding sites:Findings and PredictionsExpression activity of different gene classes is somewhat correlated with predicted nucleosome scarcity:Exception proves the rule: ribosomal proteins are “intrinsically” nucleosome-dense, but are kept open under normal conditions by remodeling factors.Ioshikes et al.Only then, make predictionsVerification and testing of model against previous dataGeneration of a model based on experimental dataCorroborationIoshikes et al.Title: “Nucleosome positions predicted through comparative genomics”Looked at promoters and their nucleosome occupation more closelyFound same depletion at start site, but that different types of promoters use nucleosomes in different waysGeneration of ModelIn silico collection of known promoter regions and alignment around TSSAlso used previously measured nucleosome positionsGroup by presence/absence of TATA boxIoshikes et al.: FindingsTATA-dependent and TATA-less promoters use chromatin in similar but distinct ways:Chromatin is Important to Humans, Too●Ozsolak et al.●“High-throughput mapping of the chromatin structure of human promoters”●A chromatin model had been developed in yeast, but did it apply to humans?●What about multicellular organisms?Creating a ModelUsed DNA footprinting to isolate histone DNAHybridized to a microarrayMapped levels for each sequence to the genome, creating a waveform.What is a microarray?Slightly obsolete: high-throughput sequencing allows direct sequencing of massive amounts of DNAKnown gene sequencesGlass slide (chip)Cancer cellNormal cellIsolation RNA Cy3 dyeCy5 dyeValidationChIP-chipinput/input negative controlFindings and PredictionsHow much do different cell lines differ in chromatin structure?Findings and PredictionsIs human chromatin different on expressed vs. unexpressed sequences?Findings and PredictionsCan cell lines with different gene expression be differentiated based on chromatin structure?ConclusionsChromatin is a newly discovered yet integral part of the regulatory network of all eukaryotes.Chromatin structure is partly based on the underlying genetic code and partly on the activity of trans factors.A large part of chromatin structure can be


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Stanford CS 374 - Computational Methods for Studying Chromatin Structure and Remodeling

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