Differential Gene ExpressionDifferential Gene ExpressionChromatin StructureChromatin ConfigurationAnatomy of the GenenRNA ProcessingHuman β-globin SequenceProduction of β-globinPromoters and EnhancersEukaryotic Transcription Initiation ComplexEukaryotic Transcription Initiation ComplexStabilization of Transcriptional Initiation Complex by TAFsEnhancersEnhancersEnhancer GeneralizationsMethod – Reporter GenesTranscription FactorsTranscription Factor DomainsTranscription Factor DomainsTranscription Factor DomainsEnhancer ModulesDNA MethylationDNA MethylationGenomic ImprintingDosage CompensationX Chromosome inactivationa-Tropomyosin alternative splicingDifferential Gene ExpressionBiology 4361 - Developmental BiologySeptember 28, 2006Differential Gene ExpressionChromatin structureGene anatomyGene sequencesControl of gene transcriptionenhancerstranscription factorsRNA processingmethylationimprintingEpigenetic processes(epigenetic - modification of structureof DNA rather than sequence)Chromatin Structure~140 bp~60 bp3. Histone methylation (CH3) – further repressionTranscriptional Regulation:1. “Packing” prevents access2. Acetylation ( ) state of histones controls DNA binding- histone acetyltransferases – allow transcription- histone deacetylases - repressionCH3COchromatin-remains tightly condensed throughout most of the cell cycle - replicates laterJohn H. FrensterChromatin Configurationheterochromatineuchromatin- “active” chromatinAnatomy of the Gene5’ 3’core promoterregiontranscription initiation site+1TATAT upstream downstreamtranscribed regionpromoter exon 1 intron 1 exon 2 intron 2 termination regiongene3’ cleavage,polyadenylationAAAAAAA(n)7-methyl-GpppnRNA Processingsplicingexon 1 exon 27-methyl-Gppp AAAAAAA(n)mature mRNA, ready to be transported to the cytoplasm and translatedAAUAA7-methyl-Gpppprimary transcriptcapcleavage signalpoly(A)polymerasecleavage factortranscription, 5’ cappingHuman β-globin SequenceProduction of β-globinPromoters and EnhancersRNA polymerase II binds to the promoter region at the TATA box- various proteins bind to regulatory sequences upstream and downstream of transcription initiation site- promoter region binds Basal Transcription Factors- basal transcription factors facilitate Pol II binding and activitypromoterregion5’ 3’- however, Pol II cannot initiate transcription aloneEukaryotic Transcription Initiation Complexbasal transcription factors:- sequential binding- binding mediated by small proteins- TBP-associated factors (TAFs)- mediator complex1. TFIID complex binds to the TATA box through its TATA Binding Protein (TBP) subunit 2. TFIID is stabilized by TFIIA3. TFIIB and TFIIH join the complex on the TATA box; TFIIE and TFIIF associate with RNA polymerase IIEukaryotic Transcription Initiation Complex4. RNA polymerase II is positioned by TFIIB, and its carboxy-terminal domain (CTD) is bound by TFIID5. The CTD is phosphorylated by TFIIH and is released by TFIID; RNA polymerase II can now transcribe mRNAStabilization of Transcriptional Initiation Complex by TAFsTBP – TATA binding proteinTAF(s) – TBP-associated factorsEnhancersFunction - bind specific regulatory proteins (i.e. specific transcription factors)Enhancers: DNA sequences that regulate gene expression by affecting the transcription initiation complex on the promoter.Locations - highly variable with respect to the transcribed portion of the gene. - upstream (5’), downstream (3’), within transcribed region - close proximity or as many as 106bp awayEnhancers are modular; e.g. mouse Pax6 gene- expressed in multiple tissues, each under the influence of a different enhancerEnhancers are cis-acting regulatory elementscis – (same or same side); elements that reside on the same DNA strand; e.g. DNA sequencestrans – (other side); elements that originate from another DNA strand, e.g. regulatory proteinsEnhancersEnhancers differ from promoters:3) can work in reverse orientation2) can work at a distance1) need a promoter to workEnhancer Generalizations1. Most gene transcription requires enhancers.2. Enhancers are the major determinants of differential transcription in cell types and through developmental stages.3. There can be multiple signals (e.g. multiple enhancer sites) for a given gene, and each enhancer can be bound by more than one transcription factor (not at the same time).4. Transcription is regulated by the interaction of transcription factors bound to enhancers and the transcription initiation complex assembled at the promoter.5. Enhancers are combinatorial. Various DNA sequences regulate temporal and spatial gene expression; these can be mixed and matched.6. Enhancers are modular. A gene can have several enhancer elements, each of which may turn it on in different sets of cells.7. Enhancers generally activate transcription by remodeling chromatin to expose the promoter, or by facilitating the binding of RNA polymerase to the promoter by stabilizing TAFs.8. Enhancers can also inhibit transcription (aka Silencers).Method – Reporter Genesexons for Pax6Reporter gene construct: enhancer elements for Pax6 attached to structural genes for reporter molecules.add a gene for reporter protein, e.g.β-galactosidase (blue)luciferase (produces light)green fluorescent protein (GFP)LacZ – β-galactosidasemouse – Pax6DrosophilaTranscription FactorsProteins that bind to enhancer or promoter regions- activate or repress transcriptionMost bind to specific DNA sequences (e.g. enhancers)Transcription factors are grouped together in families, based on structural similarities- families share common framework in DNA binding sites- slight differences in binding sites cause differences in recognitionestrogen receptor zinc finger domainTranscription Factor DomainsThree major domains:1. DNA-binding- recognizes particular DNA sequencetranscription factor - engrailedTranscription Factor DomainsThree major domains:1. DNA-binding- recognizes particular DNA sequence2. trans-activation – activates or represses transcription- often involved with proteins involved in binding RNA polymerase II;e.g. TFIIB, TFIIE- often involved with enzymes that modify histones3. protein-protein interaction domain- promotes dimerization- allows it to be modulated by TAFs or other transcription factorsTranscription Factor DomainsTranscription factor MITF- basic helix-loop-helix- homodimer is the functional proteinThe trans-activating domain is contained in the center of the protein.- when