PowerPoint PresentationFundamental StatesSlide 3Complexity is RequiredSlide 5Determining Gene Regulatory PatternsOrdered Nuclear Entry of Proteins Following Cell DivisionImmediately Following Cell Division Transcription Proteins are in the CytoplasmRNAP II Entry Precedes Splicing FactorsPhosphorylated RNAP II Enters After Splicing FactorsHigh Global Transcription Correlates with Ser-2 PhosphorylationSlide 12Slide 13Slide 14Invading Condensed ChromatinIn vitro Heterochromatin SystemHnf3 and GATA4 Promote Access In vitroTissue-Specific Access In vivoHnf3 & GATA4 Insert Between OctamersStructural Similarities with HistonesNucleosome Depleted Region and the +1 NucleosomeSlide 22IFNb Enhanceosome is a Cooperative RE UnitProper IFNb Regulation Involves a Positioned NucleosomeCombinatorial Regulation May Involve NucleosomesPositioned Nucleosome Slides Upon Gene ActivationChange in Acetylation PatternHistone Code Cascade Occurs Prior to Activation of IFNb GeneReconstituting Recombinant Histones at IFNb PromoterIn vitro Acetylation is Important for SWI/SNF and TFIID Promoter BindingFunctional Interpretation of the CodeSwi/Snf and PIC Association Requires CBPWho Uses What at IFNbOrdered Recruitment In vivoSequential Recruitment at IFNb PromoterDifferent Promoter, Different OrderEstradiol-Regulated TranscriptionEstradiol Responsive Genes are Transcribed CyclicallyRegulation of pS2Double ChIPDouble ChIP May be Used for Complex DeterminationEarly Promoter EventsMid Promoter EventsLate Promoter EventsIn Between EventsRapid Interactions During Each CycleFirst ER Binding PeakSecond ER Binding PeakFinal Cyclical ModelIs Improper Order a Dead End?1Points for the lecture:Biological processes are typically multi-step pathwaysOrder of transcription pathways depend on cell and promoter contextsFundamental States23Complexity is Required45Numerous Cofactors Feed into Transcription EventsDetermining Gene Regulatory Patterns67Ordered Nuclear Entry of Proteins Following Cell Division8Immediately Following Cell Division Transcription Proteins are in the CytoplasmDNATranscriptionProteinsNuclearMembranePhos-Ser5Total9RNAP II Entry Precedes Splicing FactorsDNA RNAP II Splicing Factors10Phosphorylated RNAP II Enters After Splicing FactorsDNA Phos. RNAP II Splicing FactorsPhos-Ser211High Global Transcription Correlates with Ser-2 Phosphorylation12Flow of MaterialMesodermEndodermCell Fate Changes Require Major Shifts in Chromatin States EctodermPluripotentCellOCT4SOX2KLF4cMYCPluripotentCell14Cell Differences Can be Subtle15Invading Condensed Chromatin16In vitro Heterochromatin System17Hnf3 and GATA4 Promote Access In vitroDNase I Restriction Enzyme18Tissue-Specific Access In vivo19Hnf3 & GATA4 Insert Between Octamers20Structural Similarities with HistonesNucleosome Depleted Region and the +1 Nucleosome2122HAT first or Chromatin Remodeler?IFN Gene Activation23IFN Enhanceosome is a Cooperative RE Unit24Proper IFN Regulation Involves a Positioned NucleosomeFactor Recruitment by ChIPPosition of Nucleosome Influences Activity In vitro Transcription25Combinatorial Regulation May Involve Nucleosomes26Positioned Nucleosome Slides Upon Gene ActivationPosition is Important for Proper Timing27Change in Acetylation PatternChromatin Immunoprecipitation results:28Histone Code Cascade Occurs Prior to Activation of IFNGene29Reconstituting Recombinant Histones at IFN Promoter30In vitro Acetylation is Important for SWI/SNF and TFIID Promoter Binding31Functional Interpretation of the CodeMutation of certain lysines can alter in vitro transcriptionExcessive SWI/SNF can compensate for certain mutationsMay require both SWI/SNF and TFIID32Swi/Snf and PIC Association Requires CBP33Who Uses What at IFN34Ordered Recruitment In vivo35Sequential Recruitment at IFNPromoter36Different Promoter, Different Order37Estradiol-Regulated Transcription38Estradiol Responsive Genes are Transcribed CyclicallyNuclearRun-OnChIP39Regulation of pS2Transcript Production Basic ChIPComplete Single ChIP40Double ChIP41Double ChIP May be Used for Complex Determination42Early Promoter Events43Mid Promoter Events44Late Promoter Events45In Between Events46Rapid Interactions During Each Cycle47First ER Binding Peak48Second ER Binding Peak49Final Cyclical Model50Is Improper Order a Dead
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