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Download Control of gene expression in eukaryotes Transcriptional regulation
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2/11/11 Control of gene expression in eukaryotes Transcriptional regulation Developmental mutants in Drosophila Master genes Effector genes Relevance to disease, evolution General principles from study of lac operon: Gene expression controlled by binding of proteins to upstream regulatory elements (promoter / operator) Each gene controlled by both activators and repressors Separate response elements for different proteins Mutations in regulatory proteins or in the DNA elements can alter expression Do these principles apply to eukaryotes? Important differences in gene expression in eukaryotes: Prokaryotes: Eukaryotes: Transcription and mRNA processing happen in nucleus Translation happens in cytosol 1 2/11/11 Eukaryotic DNA packaged into chromatin Structure of the nucleosome Histone proteins 2 2/11/11 Chromatin can be in an “open” state, allowing transcription or in a “closed” transcriptionally silent state Eukaryotic transcription involves a complex of “basal” transcription factors that help recruit RNA polymerase to the promoter (defined by TATA box) RNA polymerase can access open but not closed chromatin Once bound, RNA polymerase can transcribe through chromatin 3 2/11/11 Chromatin and gene regulation Chromatin can be open or closed (active or silent) RNA polymerase can access and transcribe through open chromatin Regulation of chromatin structure is one level of transcriptional regulation in eukaryotes Also have specific activators and repressors as in prokaryotes that regulate transcription These can influence chromatin structure and/or affect binding of basal transcriptional machinery While molecular mechanisms of transcriptional regulation in eukaryotes differ somewhat from prokaryotes due to presence of chromatin, the basic concepts are conserved: Activators and repressors bind regulatory elements in the DNA (enhancers and silencers) to control access or activity of basal transcriptional machinery Binding of these factors is: Specific Modular - elements function independently and can be combined to give different patterns of expression in different genes Activators and repressors bind enhancers and silencers to regulate transcription 4 2/11/11 Investigating the control of gene expression in eukaryotes Homeotic mutations in Drosophila Embryogenesis in Drosophila melanogaster 5 2/11/11 Different segments in embryo give rise to different structures in adult Homeotic mutants Isolated by Ed Lewis in 1970’s Wanted to understand development and differentiation - isolated mutants with transformations of body structures, e.g., Antp: antennae -> legs Bithorax mutants - third thoracic segment transformed to fate of second thoracic segment (wings instead of halteres) 6 2/11/11 Many different mutants isolated affecting segment or structure identity Mapped to two regions of the genome - the antennapedia (ANTP) complex - the bithorax (BX) complex Genes are arranged on chromosomes in same order as segments they specify Hox genes define segment identity Hox genes are arranged in linear order on chromosome that corresponds to order of segments they specify ANTP-C BX-C Some homeotic mutations in protein-coding genes and others in regulatory elements 7 2/11/11 Hox gene expression defines segment identity Hox genes are arranged in linear order on chromosome that corresponds to expression domain in embryo ANTP-C BX-C Visualising Hox gene expression in the embryo Visualising mRNA expression In situ RNA hybridisation Tagged Antisense RNA probe binds mRNA Antibody used to bind to tag Enzyme fused to antibody - makes coloured substrate 8 2/11/11 Visualising protein expression Primary antibody binds protein X Secondary antibody binds primary Enzymatic reaction or fluorescent tag Hox gene expression established by cascade of developmental genes Hox proteins Homeotic genes encode Hox proteins - when cloned, found to look like… lac repressor! => bind DNA , act as transcription factors Control expression of many other genes - they are called “master” regulatory genes - control other Hox genes - and downstream “effector” genes All contain homeodomain - conserved DNA-binding domain 9 2/11/11 Homeodomain proteins control expression of many other genes parasegment number Hox genes specify identity of segments in which they are expressed Removal of Hox genes leads to transformation of segments to more anterior fates Why? Posteriorly expressed Hox genes repress more anteriorly expressed ones 10 2/11/11 Explanation of bithorax mutant Bithorax: - Ubx gene deleted - Antp expression expands posteriorly - T3 -> T2 - halteres -> wings Target genes: e.g., wingless - necessary for wing formation (mutant has no wings) - activated by Antp, repressed by Ubx Variation in targets of Ubx could be associated with evolution of diverse wing types Explanation of antennapedia mutant: Antennapedia: - deletion of silencer element - Antp gene now expressed in antennal segment (antennae -> legs) - Antp represses homothorax gene (necessary for antennal formation) - Antp turns on distal-less (necessary for limb formation) 11 2/11/11 Hox genes are remarkably highly conserved Mammals have four copies of whole cluster Highly conserved at protein sequence level Some can substitute across species (i.e., mouse gene can rescue fly mutant) Mammals have four copies of Hox gene cluster, arranged on different chromosomes Order on chromosome also corresponds to expression domain in embryo Mutations in several human HOX genes associated with congenital defects e.g., HOXD13 mutations result in synpolydactyly (multiple fused digits) 12 2/11/11 Another example of a “master” regulatory gene: Eyeless: necessary for eye formation in flies Also encodes homeodomain protein Also conserved - called Pax6 in mammals Eyeless gene sufficient to drive eye formation in flies if expressed elsewhere Even expression of Pax6 can drive formation of ectopic eyes in fly! eyeless mutants in Drosophila have reduced or totally absent eyes Wild-type eyeless mutants Pax6 mutants in mouse also lack eyes Wild-type Pax6 mutant 13 2/11/11 Pax6 mutations in humans affect eye development, leading to aniridia, lens defects and cataracts Driving eyeless expression in other tissues leads to ectopic eyes - Achieved by fusing ey protein-coding sequence to regulatory elements of gene normally expressed in limbs Driving mouse Pax6 expression also leads to ectopic eyes - can still activate correct downstream targets in fly 14 2/11/11 Eyeless (or Pax6) directly activates several target genes: Optix Eyes absent Sine oculis Also transcription factors Mutations in any of these genes lead to loss of eyes => All those genes necessary for proper eye formation Only eyeless is sufficient to drive eye formation (it is “master” regulator) Implies eyes did NOT evolve multiple times but just once eyes no eyes eyes Hypothetical common ancestor of insects and mammals must have had some photoreceptive organ specified by eyeless/Pax6 homologue 15 2/11/11 Summary Transcription in eukaryotes involves combined effects of activators and repressors These bind specific regulatory elements Mutations in proteins or in regulatory elements can alter expression Master genes specify segments or body structures by regulating many downstream genes Remarkable conservation of these genes across species 16