PowerPoint PresentationTranscript profiling among different cell typesTranscription Factors Drive the SystemIR FamilySlide 5Founding MembersIdentifying Functional DomainsGeneral Protein Structure for Hormone ReceptorsGlucocorticoid SignalingSteroids & GlucocorticoidsHow Steroids Circulate and Are DistributedInflammation ControlTarget Genes for GCSlide 14Slide 15Small Ligands Provide an Experimental Means to Control Transcriptional ActivityNuclear Run-On AssaySlide 18Means to Achieve Complex Gene RegulationSlide 20Slide 21Slide 22Transcription Factor Cascades Are Commonly Used During DevelopmentSlide 24Slide 25Slide 26Components Build Cooperative ComplexesBenefits of Combinatorial RegulationSlide 29Response ElementsIR Response Elements Typically are Repeated DNA MotifsGR:DNA InteractionsSimple GREDetecting SynergySimple GRE Can Be Used for RepressionComposite Response ElementTethering Response ElementProteins at Tethering REs Physically InteractOne Repression Mechanism at a Tethering GREDNA Sequence is Interpreted by GR at a Tethering GRESome REs Only Work within Chromatin ContextSlide 42Slide 43Slide 44Slide 45Slide 46Slide 47Slide 48Slide 49Slide 50Dimerization at IR Response ElementsDimerization Greatly Increases Control OptionsIndividual IR Variants Peroxisome proliferator - activated receptors (PPARs)Slide 54Cognate LigandsConsequences of Ligand BindingLigand Binding Dictates Coregulator InteractionsAgonists, Antagonists & Partial AgonistsSlide 59Proper Gene Regulation Requires Coactivators1Points for the lecture:Gene regulation complexity increased by combinatorial systemsThree general forms of response elementsInduced conformations can dictate protein interactions-small molecules and/or DNA2•All cells express a common set of genes (~60-70% of the total number of transcripts are found in all cell types)•The mRNA abundances for each transcript varies widely (10-1,000,000 copies per cell)•Cell types vary by only a small change in expressed mRNAs (~5% change)•Disease states (e.g., cancer cell stages) often differ by only small gene expression changes (~2-fold)Transcript profiling among different cell types3Transcription Factors Drive the SystemIntracellular Hormone Receptor Superfamily4IR Family # of receptorsHomo sapiens 48Drosophila melanogaster 19Caenorhabditis elegans 228 (total)homologs 44novel 1845Intracellular Hormone Receptor Signaling small molecules govern large transcriptional programsAporeceptor ComplexHormoneHoloreceptor-Response Element ComplexTranscription Complex6Founding Members7Identifying Functional Domains8General Protein Structure forHormone Receptors9Glucocorticoid Signaling10Steroids & Glucocorticoids•Steroids are small, lipophilic molecules derived from a common precursor, cholesterol.•Steroids regulate a large number of normal and pathologic processes.•There are 4 major types of steroids: progestins, androgens, estrogens and corticoids (mineralocorticoids and glucocorticoids, GC).•Steroids differ in the number of carbon atoms, which differentiates the binding receptor and/or the biological response elicited. Of note, the molecular volume for each is ~300 cubic Å.11How Steroids Circulate and Are Distributed•Steroids are lipophilic, therefore they are transported in the blood in a reversible complex with protein.•The major high capacity/low affinity carrier is albumin, the major low capacity/high affinity is transcortin (a.k.a., corticosteroid binding globulin).•Only 1-10% of circulating GC is free and available to mediate biological functions, thus the effective GC concentration in plasma is much lower than the total.•Importantly, the affinity between GR and cortisol is 30 nM –i.e., in the normal range for plasma concentrations of free hormone.•Stress (exercise, hunger, psychological) may elevate circulating cortisol levels >100-fold. •Synthetic GCs (e.g., dexamethasone) are particularly potent because of their high affinity for GR and relatively low levels of binding to plasma proteins.12Inflammation ControlGlucocorticoids represent one of the most effective clinical treatments for a range of inflammatory conditions including severe acute inflammation. Interleukins and tumour necrosis factor alpha (TNF) are two pivotal proinflammatory cytokines. TNF can induce NFB activity.13Target Genes for GC•Increased gene transcription: tyrosine amino transferase, tryptophan oxygenase, lipocortin-1, -adrenoreceptor, IL-1RA, IBa•Decreased gene transcription: cytokines (IL-1/8, IL-11, IL-13, TNF-, GM-CSF), chemokines (RANTES, eotaxin, MIP-1, MCP-1), enzymes (iNOS, COX-2), adhesion molecules (ICAM-1, VCAM-1), receptors (IL-2R, IL-4R)•Increased mRNA half life: collagenase, fatty acid synthase•Decreased mRNA half life: COX-2, MCP-1, IL-4R, IL-1, IL-3, IL-6, GM-CSF, cyclin D314Protein Expression Is Controlled at Many Steps15Small molecule governs large transcriptional program16Small Ligands Provide an Experimental Means to Control Transcriptional ActivityNorthernBlotWesternBlotRepressed by GR-Hormone Complex17Nuclear Run-On Assay18~2000 transcription factors~350 transcription factorsHow to make a multi-cellular organism with a limited set of transcription factors?19Means to Achieve Complex Gene Regulation•Vary the expression of the transcription factors•Vary basal promoter elements •Combinatorial Regulation–Alter the response element (sequence and configuration)–Monomer, homodimer and heterodimer –Nature of activating signal (e.g., different hormones)•Coregulator expression20Transcription Factor Combinatorial Regulation21Fly Brain Complexity22Temporal Expression of Transcription Factors Dictates Neuron Development23Transcription Factor Cascades Are Commonly Used During Development24Means to Achieve Complex Gene Regulation•Vary the expression of the transcription factors•Vary basal promoter elements •Combinatorial Regulation–Alter the response element (sequence and configuration)–Monomer, homodimer and heterodimer –Nature of activating signal (e.g., different hormones)•Coregulator expression25Basal Elements Have a Significant Impact26Means to Achieve Complex Gene Regulation•Vary the expression of the transcription factors•Vary basal promoter elements •Combinatorial Regulation–Alter the response element (sequence and configuration)–Monomer, homodimer and heterodimer –Nature of activating signal (e.g., different hormones)•Coregulator
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