Page 1Page 2Page 3Page 4Page 5Page 6Page 7Page 8Page 9Page 10Page 111Biology 4361Developmental BiologyThe Paradigm of Differential Gene ExpressionSeptember 28, 2006Differential Gene TranscriptionAnatomy of a geneChromatin structureExons and intronsPromoters and enhancers Enhancer: DNA sequence that controls the efficiency and rate of transcription of a specific promoter- instruct where & when a promoter is used; how much gene product to make NOTE - enhancers activate only cis-linked promoterscis - refers to cis-acting or cis-elements; cis = same; i.e. reside on the same strandof DNA, e.g. regulatory sequencestrans - other side; i.e. elements supplied from another DNA strand, e.g.transcription factor proteins- enhancers can be in close proximity or very far away from promoter- also, may be on either side of promoter (5' or 3') or could be in transcribed region- enhancers function by binding specific regulatory proteins = transcription factors- different types of genes have different enhancers- reporter genes - method for identifying enhancer sequences- clone DNA sequences flanking genes of interest- fuse them to reporter genes- reporter gene products identifiable; e.g. luciferase or green fluorescent protein(GFP; both produce light), $-galactosidase (turns blue)- insert constructed genes into cells or embryos; monitor expression of the reportergene (if the enhancer is stimulated by endogenous regulatory proteins, thereporter gene product will also be manufactured)- this method used to monitor temporal- and tissue-specific gene expression- enhancers are modular and are often used in combination with other enhancers (more onthat later) Generalizations about Enhancers and Enhancer Functions:1. Most gene transcription requires enhancers.2. Enhancers are the major determinant of differential transcription in space (cell type) andtime (i.e. developmental stage).3. There can be multiple signals (i.e. multiple enhancer sites) for a given gene, and eachenhancer can be bound by more than one transcription factor (although not at the2same time).4. Transcription is regulated by the interaction of transcription factors bound to enhancersand the transcription initiation complex assembled at the promoter.5. Enhancers are combinatorial: Various DNA sequences regulate temporal an spatial geneexpression; these can be mixed and matched. 6. Enhancers are modular; a gene can have several enhancers elements, each of whichturns it on in a different set of cells7. Enhancers generally activate transcription by remodeling chromatin to expose thepromoter, or by facilitating the binding of RNA polymerase to the promoter bystabilizing TAFs.8. Enhancers can also inhibit transcription (aka Silencers)Transcription factors - proteins that bind to enhancer or promoter regions- activate or repress transcription - most bind to specific DNA sequences - grouped together in families, based on similarities in structure- share common framework in DNA binding sites- slight differences in binding sites cause differences in recognitionTranscription factors have three major domains: 1. DNA-binding domain - recognizes particular DNA sequence 2. trans-activating domain - activates or represses transcription of gene- often involved with proteins involved in binding RNA poly II (e.g. TFIIB, TFIIE)- also involved with enzymes that modify histones 3. protein-protein interaction domain - allows transcription factor to be modulated by TAFsor other transcription factorsExamples of transcription factors: MITF (microphthalmia) protein- transcription factor active in ear and pigment-forming cells of eye and skin- humans heterozygous for a mutation of the MITF gene are deaf, have multicoloredirises, and have a white forelock of hair- MITF protein is a basic helix-loop-helix transcription factor- three functionally important domains;1. protein-protein interaction domain that enables dimerization (with anotherMITF protein)- homodimer is the functional protein- binds to DNA and activates transcription of certain genes2. DNA-binding domain; close to amino-terminal end of proteincontains numerous basic amino acids that make contact with DNA- pigment-cell-specific enzymes of the tyrosinase family have MITF bindingregion in their promoter regions3- without MITF - no protein3. trans-activating domain - long stretch of amino acids in the center of the protein- when bound to promoter or enhancer, trans-activating region able to binda TAF (p300/CBP)- p300/CBP is a histone acetyltransferase; transfers acetyl groups tohistones; destabilizes them; allows transcription- numerous transcription factors operate by recruiting histone acetyltransferases- acetylating histones allows nucleosome dispersal; access for other TFs and RNApolymerase II- some TAFs are histone acetyltransferases Pax 6- needed for mammalian eye, nervous system, pancreas development- contains two potential DNA-binding domains- Pax6-binding sequences found in enhancers of vertebrate lens crystallin genes, and inendocrine cells of the pancreas (secreting insulin, glucagon, somatostatin)- Pax6 binding can either activate of repress the gene- trans-activating domain rich in proline, threonine, serine- mutations in trans-activating regions cause severe nervous system, pancreatic, opticabnormalities in humansCombinatorial regulation of transcriptionThe use of Pax6 by different organs demonstrate combinatorial manner of TF activity e.g.: 1. chick *1 lens crystallin gene- promoter contains site for TBP binding and an upstream promoter elements the bindsSp1 (general transcriptional activator found in all cells)- also, enhancer in 3 intron that controls time and place of crystallin gene expressionrd- enhancer contains two Pax6-binding sites- Pax6 present in early development in CNS and head surface ectoderm of chick- crystallin gene not expressed unless Pax6 is present in the nucleus and bound to theseenhancer sites- also, this enhancer has a binding site for another TF, Sox2- Sox2 not usually found in outer ectoderm, but appears in those outer ectodermalcells that will become lens by virtue of their being induced by the opticvesicle evaginating from the brain (CNS)- thus, only cells containing both Sox2 and Pax6 can express lens crystallin gene- in addition, 3 enhancer site can bind either and activator (*EF3 protein) or repressorrd*EF1 protein)- repressor may be critical in preventing crystallin expression in nervoussystem 2. Pax6 regulation also