1、BioSci 145A lecture 13 1 copyright Bruce Blumberg 2000. All rights reserved,BioSci 145A Lecture 13 - 2/18/2003 Principles of Gene regulation,Topics we will cover today Example of targeted gene replacement/transgenic Regulated gene expression Ecdysone Tetracycline and reverse tetracycline Hybrid tetr
2、acycline regulated system implications of transgenic technology Principles of gene regulation Identification of regulatory elementstranscription factor resources http:/transfac.gbf-braunschweig.de/TRANSFAC/ http:/bioinformatics.weizmann.ac.il/transfac/ detailed transcription factor database http:/co
3、pan.bioz.unibas.ch/homeo.html collected information about homeobox genes http:/biochem1.basic-ci.georgetown.edu/nrr/nrr.html nuclear receptor resourcereferences nuclear transport Nakielny and Dreyfuss (1999) Cell 99, 677-690. Nuclear pore structure Daneholt (1997) Cell 88, 585-588.,BioSci 145A lectu
4、re 13 2 copyright Bruce Blumberg 2000. All rights reserved,Example of targeted gene replacement,Goal: Confirm the function of a novel orphan nuclear receptor (SXR) Is it a xenobiotic receptor? Is it involved in steroid metabolism Can characteristics of human receptor be transferred to mouse? Pharmac
5、ology of human and mouse receptors is very different Designing a targeting constructSouthern analysis should be able to distinguish 8.5 kb wild type allele and 5.1 kb knockout allele,BioSci 145A lecture 13 3 copyright Bruce Blumberg 2000. All rights reserved,Example of targeted gene replacement - co
6、ntd,Southern Analysis Stoichiometry is correct Knockout fragment changes predictablyNorthern analysis Is the mRNA expressed? Are target genes affected?Is the induction of target genes lost in KO? Induction by PCN lostHow about by other activators? DEX - PXR 3MC - AhR Conclusion PXR regulates xenobio
7、tic response, in vivo,BioSci 145A lecture 13 4 copyright Bruce Blumberg 2000. All rights reserved,Example of targeted gene replacement - contd,Can rodent be humanized Rat target gene/luc reporter Activated by rat-specific but not human ligands Addition of human receptor confers human responseHuman t
8、arget gene/luc reporter Addition of human receptor confers human response but rat response still present ? Does human receptor work on rodent gene?,BioSci 145A lecture 13 5 copyright Bruce Blumberg 2000. All rights reserved,Example of targeted gene replacement - contd,Rat target gene response elemen
9、t is activated by human receptor Conclusion elements are functionally interchangeable Humanized mouse will activate mouse xenobiotic response,BioSci 145A lecture 13 6 copyright Bruce Blumberg 2000. All rights reserved,Example of targeted gene replacement - contd,How to make a humanized mouse Best -
10、replace mouse gene with human gene Time consuming, difficult fierce competition (Glaxo) Made a human transgenic line and crossed into knockout background Either human gene or Constitutively active human gene (gain-of-function)Make transgenics and test expression/function SXR confers induction of tar
11、gets in appropriate tissues,BioSci 145A lecture 13 7 copyright Bruce Blumberg 2000. All rights reserved,Example of targeted gene replacement - contd,SXR transgene directed partially “humanized response” in mouse liver - wt mouse + SXR transgenic Get appropriate responseEffect of adding human- specif
12、ic activator is time-dependent and reversible in “humanized mice”Effects are dose- dependent,BioSci 145A lecture 13 8 copyright Bruce Blumberg 2000. All rights reserved,Example of targeted gene replacement - contd,Fully humanized response in PXR -/-, Alb-SXR mice RIF activation depends on SXR PCN do
13、es not activate in ko SXR is expressed as predicted (only in “humanized”)VP16-SXR mice rapidly overcome anesthetics SXR induces metabolism of these anesthetics VP16-SXR confers increased protection to toxic chemicals,BioSci 145A lecture 13 9 copyright Bruce Blumberg 2000. All rights reserved,Example
14、 of targeted gene replacement - contd,Conclusions from this paper SXR(PXR) is a critical regulator of xenobiotic response in vivo Knockout proves necessity Transgenic proves sufficiency Proved our original model that SXR functions as a xenobiotic sensor in vivo Changing a single transcription factor
15、 alters species specificity of response Few examples of this “humanized” mouse can serve as a better model for toxicology and pharmacologyCriticisms? Not fully humanized would have been better to “knock” human receptor into mouse locus Pharmacologists and toxicologists dont want to be bothered with
16、mice use rats exclusively,BioSci 145A lecture 13 10 copyright Bruce Blumberg 2000. All rights reserved,How is gene expression regulated?,BioSci 145A lecture 13 11 copyright Bruce Blumberg 2000. All rights reserved,Regulated expression of introduced genes,Several such systems are available Inherently
17、 regulated promoters Metallothionin Heat shock proteins All are leaky Ligand regulated promoters Metallothionin Lac Combination systems (receptor/ligand) Estrogen receptor/tamoxifen Glucocorticoid receptor fusions GAL4 2 part system Ecdysone tetracycline,BioSci 145A lecture 13 12 copyright Bruce Blu
18、mberg 2000. All rights reserved,Regulated expression - ecdysone,Background No et al (1996) PNAS 93, 3346-3351 20-OH ecdysone is a steroid hormone that controls metamorphosis in invertebrates family of hormones called ecdysteroids regulates transcription by interacting with a specific cellular recept
19、or, the ecdysone receptor functional ecdysone receptor is a heterodimer of two different but related proteins, ecr and usp (ultraspiracle) both partners of the heterodimer are required for ligand binding and transcriptional activation properties of the system ecdysone is not present in vertebrates a
20、nd has no detectable effects in rodents human effects? Activators are lipophilic molecules that can penetrate most tissues, including brain muristerone A ponasterone A rapidly metabolized by cytochrome p450s not stored requires multiple components, RXR, EcR, EcRE target gene construct.,BioSci 145A l
21、ecture 13 13 copyright Bruce Blumberg 2000. All rights reserved,Regulated expression - ecdysone (contd),BioSci 145A lecture 13 14 copyright Bruce Blumberg 2000. All rights reserved,Regulated expression - ecdysone (contd),applications in vitro regulation of transfected genes muristerone A is not read
22、ily available in quantity other inducers are not as good Also expensive, must be purified regulating targeted gene disruption in ES cells and embryos advantages commercially available (InVitrogen, Stratagene) may have no deleterious effects in mammalian cells could work in transgenic animals if acti
23、vators were affordable and widely available disadvantages requires multiple constructs/cell expense and unavailability of ligands little literature or experience questionable utility for gene therapy requires high concentration of ligand (M) caveats works fairly well in cell culture figures in paper
24、 are misleading, doesnt work as well as claimed vs tetracycline system nuances of reporter construction.,BioSci 145A lecture 13 15 copyright Bruce Blumberg 2000. All rights reserved,Regulated expression of introduced genes - tetracycline,Background Gossen and Bujard (1992) PNAS 89, 5547-5551 is the
25、original publication based on the E. coli tetracycline (tc) resistance operon derived from Tn10. tetO - tetracycline operator tetR - tetracycline repressor protein. In the absence of tc, the wild-type protein binds to tetO and represses transcription in the presence of tc, the repressor is dissociat
26、ed and repression is abolished many fusion proteins and other mutations have been engineered into the system to obtain desirable transcriptional effects properties of the original system (called std tet) Clontech - Tet-OFF is commercial product tetR is fused to VP16, strong transcriptional activator
27、 from herpes simplex virus under the control of a strong promoter tetO is placed adjacent to a minimal promoter, eg CMV. Choice of minimal promoter has profound effects on basal activity! Main difference between ecdysone system and tet from the No et al paper is the use of different minimal promoter
28、s, tk vs MTV,BioSci 145A lecture 13 16 copyright Bruce Blumberg 2000. All rights reserved,Regulated expression - tetracycline (contd),Properties (contd) the VP16-TetR fusion protein constitutively activates transcription from promoters containing tetO in the absence of tc or doxycycline (dox) in the
29、 presence of tc or dox, the repressor dissociates from tetO and activation is lost. Typical amount of dox required for full activity is in the ng/ml range, this is 2 nM Applications primarily used in cell culture, difficult to ensure a continuous supply of tc or dox in embryos some literature on the
30、 use of this system in embryos,BioSci 145A lecture 13 17 copyright Bruce Blumberg 2000. All rights reserved,Regulated expression - tetracycline (contd),Caveats and pitfalls for best results, stable cell lines should be used. Viral vectors have recently simplified process effector plasmid must be in
31、large excess to response plasmid in transient transfections bovine serum may contain tetracycline or its relatives advantages target gene expression in the absence of inducer may work better for some experiments, occasionally turning a gene off disadvantages may be difficult to completely abrogate e
32、xpression of target gene in transient transfections unpredictable inheritance of plasmids influences high intracellular concentrations of VP16-tetR are required to ensure full promoter occupancy.may need to use small amounts of dox to titrate toxic effects considerable optimization is required for s
33、uccess cell type specific differences in behavior are not uncommon time lag for effects of tc or dox addition or removal 1/2 life of mRNA or protein clearance of drug,BioSci 145A lecture 13 18 copyright Bruce Blumberg 2000. All rights reserved,Regulated expression - reverse tetracycline,Background G
34、ossen et al (1995) Science 268, 1766-1769 designed to behave like a more standard inducible system to comfort some molecular biologists addition of inducer activates transcription properties of the system mutated tetR such that binding of dox induces DNA binding rather than abrogating it, rtetR. VP1
35、6-rtetR fusion is then an activator only in the presence of dox (tc doesnt work well) applications appears to be more amenable to precise regulation than std tet commonly used in transgenic mice Caveats and pitfalls for best results, stable cell lines should be used. Viral vectors have recently simp
36、lified process minimal promoter selection CRITICAL for success bovine serum may contain tetracycline or its relatives,BioSci 145A lecture 13 19 copyright Bruce Blumberg 2000. All rights reserved,Regulated expression - reverse tetracycline (contd),advantages inducer only required to activate gene exp
37、ression conceptually and practically easier no requirement for high levels of VP16-rtetR protein as with std tet. Better for transient transfection than std tet disadvantages somewhat leaky, basal expression can be problematic choice of minimal promoter much higher levels of dox required than for st
38、d tet - toxicity is problematic,BioSci 145A lecture 13 20 copyright Bruce Blumberg 2000. All rights reserved,Regulated expression - hybrid tetracycline systems,Background references Kringstein et al (1998) PNAS 95, 13670-75 Baron et al (1999) PNAS 96, 1013-1018 Blau and Rossi (1999) PNAS 96, 797-799
39、 utilizes highly engineered tet and reverse tet proteins to get specific effects properties of the system what happens when one puts proteins into the cell that respond differently to the same effector compound? If they can dimerize with each other can not dimerize with each other or if they do not
40、dimerize and bind to different and non-overlapping operator sequences different function, can heterodimerize in this case, a fair number will make unproductive heterodimers and interfere with desired effect this will also be problematic even if the two dimers have been engineered to bind different t
41、argets so for maximum effect, we must prevent heterodimerization between effectors that can bind to different sequences have different functions (activator vs repressor,BioSci 145A lecture 13 21 copyright Bruce Blumberg 2000. All rights reserved,Regulated expression - hybrid tetracycline (contd),Opp
42、osite function - same DNA target use pure tetR and VP16-rtetR at low dox, the repressor will dominate as dox increases, the repressor will dissociate and VP16-rtetR will activate increases dynamic range of activation 105 fold increases sensitivity as well,Heterodimers possiblesame DNA targetsdiffere
43、nt DNA targets,Heterodimers not possiblefunctional discrimination works,BioSci 145A lecture 13 22 copyright Bruce Blumberg 2000. All rights reserved,Regulated expression - hybrid tetracycline (contd),Opposite function - different DNA target use std tet with one type of tetO to regulate gene A and re
44、v tet with another type of tetO to regulate gene B in the absence of dox, gene A will be activated while B will be silent in the presence of high dox, gene A will be inactivated and gene B will be activated,BioSci 145A lecture 13 23 copyright Bruce Blumberg 2000. All rights reserved,Regulated expres
45、sion - hybrid tetracycline (contd),Applications activator and repressor very sensitive regulation of responsive gene expression tightly regulated expression over 5 logs can readily measure effects of subtle changes in gene expression how much change in expression is required to get effects? Do effec
46、ts differ at different levels of expression? Activator/repressor two targets can create “conditional mutants” that have one activity at low levels of effector substance and another at high levels can regulate two different genes or two alleles of a single gene mutually exclusive expression or expres
47、sion of neither can repeatedly switch between two states and observe effects at high resolution possible to perturb intracellular equilibria in small increments and follow the effects on phenotype,BioSci 145A lecture 13 24 copyright Bruce Blumberg 2000. All rights reserved,Regulated expression - hyb
48、rid tetracycline (contd),Caveats and pitfalls best done with stable cell lines advantages much more versatile than ecdysone possible to fine tune expression of a single gene, or two genes with unprecendented resolution can make conditional mutants without genetics eg in model systems not amenable to
49、 genetics such as Xenopus or chicken can make conditional replacements in vivo knock repressible tetO into an endogenous gene e.g., mouse PXR introduce a transgene under the control of the activatable tetO e.g., human SXRbreed these mice with a line expressing the dual tet repressors increasing dox
50、will inactivate the endogenous gene and induce expression of the transgene disadvantages technically demanding multiple steps required may not work as well as predicted due to complexity virus-based systems may not work in ES cells Clontechs TRE-effector plasmids have high background - need to be remade,
