PowerPoint PresentationSlide 2Slide 3Slide 4General Coactivation ComplexesCommon Coactivators of IRsSlide 7Slide 8Two-Hybrid in YeastSlide 10GRIP1 Interacts with Many IRsValidation of Results from First Assay with a Second TestGST-Pull DownLigand Dependent Activity and InteractionsGRIP1 is a Hormone-Dependent IR CoactivatorIR:p160 Interaction Relies on a Small MotifNR-Boxes Display SpecificityLXXLL Motif ProteinsCoactivator Peptide Stabilizes the Receptor:Ligand ComplexSo Far…How Does GRIP1 Coactivate?GRIP1 Has Two Activation DomainsWhat Interacts with GRIP1 ADs?GRIP1 Activation Domains Interact with Distinct 2° CoactivatorsCARM1 Binds p160sSlide 26Synergy is Apparent at Physiological (Low) Protein LevelsNucleating Enzymatic ActivitiesFunctional Domains of CARM1CARM1 Preferentially Methylates H3Slide 311° and 2° CoactivatorsGRIP1 AD1 Interacts with CBPCBP Mediates RNAPII Recruitment at IFN EnhanceosomeCoactivators Provide Multiple Interaction SurfacesCBP and p300Multiple KAT complexesPCAF Increases Transcriptional Output of IRsPCAF Stimulates Transcription Better then a p160PCAF/SAGA ComplexesMediator/TRAP/DRIP/ARCMediator Copurifies with Affinity Tagged TRMediator & RNAP IIMediator has an LXXLL Motif used for Receptor InteractionsMediator’s Conformational State Fluctuates with the ActivatorMediator InteractionsMediator Does not Contain p160s or p300sp160 & Mediator Recruitment is Mutually ExclusiveDistinct ComplexesIR Cofactors Use the Common Interaction Motif LXXLLIs Improper Order a Dead End?1Reading for Final Week: Review:Protein dynamics: implications for nuclear architecture and gene expression. Misteli T. Science. 2001 Feb 2;291(5505):843-7.Transcription dynamics.Hager GL, McNally JG, Misteli T.Mol Cell. 2009 Sep 24;35(6):741-53.2Points for the lecture:Biological complexes are built with multiple componentsSubunits form cooperative structuresCoregulators often have enzymatic activitiesCoregulatory complexes can be mutually exclusive3How to Build Diversity with a Set Number of Transcription Factors?GR is present in most tissues yet gene activation varies greatly4Coactivators Permit Cell- and Promoter-Dependent Activation Potentials5General Coactivation Complexes6Common Coactivators of IRsChromatinRemodelersLysine Acetylases (KATs) Mediator7Identifying Coactivators1. Classic biochemistry2. Affinity-tag pull-down3. Two-hybrid4. Proteome Microarrays5. High-throughput MS8How do Intracellular Hormone Receptors Interact with Coactivators? DNA binding ligand bindingtranscriptionalregulationActivation Function 1 (AF1)transcriptionalregulation(AF2)functional domains:9Two-Hybrid in YeastReporter GenesHIS3-galactosidaseTetRZeocinetc.10GRIP1 the First Coactivator11GRIP1 Interacts with Many IRs12Validation of Results from First Assay with a Second Test13GST-Pull Down14Ligand Dependent Activity and Interactions15GRIP1 is a Hormone-Dependent IR Coactivator16IR:p160 Interaction Relies on a Small Motif17NR-Boxes Display Specificity18LXXLL Motif ProteinsGRIP1/TIF2 (p160)SRC1CBP (HAT)p300Ada3Gcn5TRAP220 (Mediator)TRAP100DRIP205 BAF250 (Chromatin Remodeling)Hsp90 (Chaperone)TRAM1RAP250RIP140Dax1PGC1STAT1STAT6TReP132NCoAJak2 (tyrosine kinase)R-MGMT (DNA methyltransferase)19Coactivator Peptide Stabilizes the Receptor:Ligand Complex20So Far…21How Does GRIP1 Coactivate?bHLH PAS RID22GRIP1 Has Two Activation Domains23What Interacts with GRIP1 ADs?bHLHAD1AD2PAS RID24GRIP1 Activation Domains Interact with Distinct 2° Coactivators25CARM1 Binds p160s26p160s Form Multi-Subunit Cooperative Complexes27Synergy is Apparent at Physiological (Low) Protein Levels28Nucleating Enzymatic ActivitiesbHLHAD1CBP/p300AD2CARM1PAS RID29Functional Domains of CARM130CARM1 Preferentially Methylates H331CARM1 Methylates p300321° and 2° Coactivators33GRIP1 AD1 Interacts with CBP34CBP Mediates RNAPII Recruitment at IFN Enhanceosome35Coactivators Provide Multiple Interaction SurfacesBromo-domainTFIIB36CBP and p30037Multiple KAT complexesLysine Acetylases (KATs)38PCAF Increases Transcriptional Output of IRs39PCAF Stimulates Transcription Better then a p16040PCAF/SAGA Complexes41Mediator/TRAP/DRIP/ARCMediator42Mediator Copurifies with Affinity Tagged TR43Mediator & RNAP IIMediator has an LXXLL Motif used for Receptor Interactions44Mediator’s Conformational State Fluctuates with the Activator4546Mediator Interactions47Mediator Does not Contain p160s or p300s48p160 & Mediator Recruitment is Mutually Exclusive49Distinct Complexes50IR Cofactors Use the Common Interaction Motif LXXLLCoregulatorsHistone Acetyl Transferase p300/CBP p160s (GRIP1, SRC1)Histone Methyl Transferase Asc-2Chromatin Remodeling BAF250Mediator TRAP220/DRIP20551Is Improper Order a Dead
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