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 tetracycline regulated system –implications of transgenic technology –Principles of gene regulation –Identification of regulatory elements transcription factor resources –http://transfac.gbf-braunschweig.de/TRANSFAC/ –http://bioinformatics.weizmann.ac.il/transfac/ detailed transcription factor database –http://copan.bioz.unibas.ch/homeo.html collected information about homeobox genes –http://biochem1.basic-ci.georgetown.edu/nrr/nrr.html nuclear receptor resource references –nuclear transport Nakielny and Dreyfuss (1999) Cell 99, 677-690. –Nuclear pore structure Daneholt (1997) Cell 88, 585- 588.

2. BioSci 145A lecture 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? Pharmacology of human and mouse receptors is very different Designing a targeting construct –Southern analysis should be able to distinguish 8.5 kb wild type allele and 5.1 kb knockout allele

3. BioSci 145A lecture 13 3 © copyright Bruce Blumberg 2000. All rights reserved Example of targeted gene replacement - contd Southern Analysis –Stoichiometry is correct –Knockout fragment changes predictably Northern analysis –Is the mRNA expressed? –Are target genes affected? Is the induction of target genes lost in KO? –Induction by PCN lost –How about by other activators? –DEX -> PXR –3MC -> AhR Conclusion – PXR regulates xenobiotic response, in vivo

4. 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 response –Human target gene/luc reporter Addition of human receptor confers human response but rat response still present ? Does human receptor work on rodent gene?

5. BioSci 145A lecture 13 5 © copyright Bruce Blumberg 2000. All rights reserved Example of targeted gene replacement - contd Rat target gene response element is activated by human receptor –Conclusion – elements are functionally interchangeable –Humanized mouse will activate mouse xenobiotic response

6. 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 -> 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 targets in appropriate tissues

7. 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 response –Effect of adding human- specific activator is time-dependent and reversible in “humanized mice” –Effects are dose- dependent

8. 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 does 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

9. BioSci 145A lecture 13 9 © copyright Bruce Blumberg 2000. All rights reserved Example 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 alters species specificity of response Few examples of this –“humanized” mouse can serve as a better model for toxicology and pharmacology Criticisms? –Not fully humanized – would have been better to “knock” human receptor into mouse locus –Pharmacologists and toxicologists don’t want to be bothered with mice – use rats exclusively

10. BioSci 145A lecture 13 10 © copyright Bruce Blumberg 2000. All rights reserved How is gene expression regulated?

11. BioSci 145A lecture 13 11 © copyright Bruce Blumberg 2000. All rights reserved Regulated expression of introduced genes Several such systems are available –Inherently 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

12. BioSci 145A lecture 13 12 © copyright Bruce Blumberg 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 receptor, 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 and 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.

13. BioSci 145A lecture 13 13 © copyright Bruce Blumberg 2000. All rights reserved Regulated expression - ecdysone (contd)

14. 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 readily 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 activators 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 are misleading, doesn’t work as well as claimed vs tetracycline system nuances of reporter construction.

15. 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 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 dissociated 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 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 promoters, tk vs  MTV

16. 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 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 use of this system in embryos

17. 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 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 expression 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 success –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

18. BioSci 145A lecture 13 18 © copyright Bruce Blumberg 2000. All rights reserved Regulated expression - reverse tetracycline Background –Gossen 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. –VP16-rtetR fusion is then an activator only in the presence of dox (tc doesn’t 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 simplified process –minimal promoter selection CRITICAL for success –bovine serum may contain tetracycline or its relatives

19. BioSci 145A lecture 13 19 © copyright Bruce Blumberg 2000. All rights reserved Regulated expression - reverse tetracycline (contd) advantages –inducer only required to activate gene expression 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 std tet - toxicity is problematic

20. 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 –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 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 targets so for maximum effect, we must prevent heterodimerization between effectors that –can bind to different sequences –have different functions (activator vs repressor

21. BioSci 145A lecture 13 21 © copyright Bruce Blumberg 2000. All rights reserved Regulated expression - hybrid tetracycline (contd) –Opposite 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 ~10 5 fold –increases sensitivity as well Heterodimers possible same DNA targets different DNA targets Heterodimers not possible functional discrimination works

22. 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 rev 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

23. BioSci 145A lecture 13 23 © copyright Bruce Blumberg 2000. All rights reserved Regulated expression - 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 effects 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 expression 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

24. BioSci 145A lecture 13 24 © copyright Bruce Blumberg 2000. All rights reserved Regulated expression - hybrid 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 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 SXR breed these mice with a line expressing the dual tet repressors increasing dox 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 –Clontech’s TRE-effector plasmids have high background - need to be remade

25. BioSci 145A lecture 13 25 © copyright Bruce Blumberg 2000. All rights reserved Regulated gene expression - summary what system do you need to use? –Transgenic animals? –Gene therapy? –Cell culture? How much tolerance is there for modulation of non target genes? –Eg glucocorticoids, estrogens, progestins and thyroid hormones are very important physiologically and cannot be modulated without collateral effects in adults however, these are very good in the early embryo of model organisms How stringent must the regulation be? –The more stringent the requirements for control the greater the likelihood that complex techniques will be required eg hybrid tetracycline Is there a need for regulating multiple genes? –Hybrid tet is the only way to go What are the commercial implications? –Licenses may be required for various technologies tet is controlled by BASF ecdysone by the Salk Institute –depending on the license agreement, one technique might be preferred (eg ecdysone)

26. BioSci 145A lecture 13 26 © copyright Bruce Blumberg 2000. All rights reserved Gene transfer technology - implications Genetics and reverse genetics –gene transfer and selection technology speeds up genetic analysis by orders of magnitude –virtually all conceivable experiments are now possible all questions are askable BUT should all questions be asked? –much more straightforward to understand gene function using knockouts and transgenics gene sequences are coming at an unprecedented rate from the genome projects Knockouts and transgenics remain very expensive to practice –other yet undiscovered technologies will be required to understand gene function. Clinical genetics –Molecular diagnostics are becoming very widespread as genes are matched with diseases huge growth area for the future big pharma is dumping billions into diagnostics –room for great benefit and widespread abuse diagnostics will enable early identification and treatment of diseases but insurance companies will want access to these data to maximize profits

27. BioSci 145A lecture 13 27 © copyright Bruce Blumberg 2000. All rights reserved Gene transfer technology - implications (contd) gene therapy –new viral vector technology is making this a reality efficient transfer and reasonable regulation possible –long lag time from laboratory to clinic, still working with old technology in many cases –The Biotech Death of Jesse Gelsinger. Sheryl Gay Stolberg, NY Times, Sunday Magazine, 28 Nov 99 http://www.frenchanderson.org/history/biotech.pdf protein engineering –not as widely appreciated as more glamorous techniques such as gene therapy and transgenic crops –better drugs, e.g., more stable insulin, TPA for heart attacks and strokes, etc. –more efficient enzymes (e.g. subtilisin in detergents) –safe and effective vaccines just produce antigenic proteins rather than using inactivated or attenuated organisms to reduce undesirable side effects metabolite engineering –enhanced microbial synthesis of valuable products eg indigo (jeans) vitamin C –generation of entirely new small molecules transfer of antibiotic producing genes to related species yields new antibiotics (badly needed) –reduction of undesirable side reactions faster more efficient production of beer

28. BioSci 145A lecture 13 28 © copyright Bruce Blumberg 2000. All rights reserved Gene transfer technology - implications (contd) transgenic food –gene transfer techniques have allowed the creation of desirable mutations into animals and crops of commercial value disease resistance (various viruses) pest resistance (Bt cotton) pesticide resistance herbicide and fungicide resistance growth hormone and milk production –effective but necessary? –negative implications pesticide and herbicide resistance lead to much higher use of toxic compounds results are not predictable due to small datasets at least one herbicide (bromoxynil) for which resistance was engineered has since been banned plants as producers of specialty chemicals –still very underutilized since plant technology yet lags behind techniques in animals –great interest in using plants as factories to produce materials more cheaply and efficiently especially replacements for petrochemicals –plants and herbs are the original source of many pharmaceutical products hence it remains possible to engineer them to overproduce desirable substances