Papers that describe the use of three-hybrid technology are grouped in a separate list at the end of this section.
2006
- Indraccolo, S., Moserle, L., Tisato, V., Gola, E., Minuzzo, S., Roni, V., Persano, L., Chieco-Bianchi, L., and Amadori, A. (2006) Gene therapy of ovarian cancer with IFN-alpha-producing fibroblasts: comparison of constitutive and inducible vectors. Gene Ther 13:953-965.
Describes the use of AP21967 to control expression of interferon-alpha in mice, following infusion into the animals of fibroblasts transduced with the retroviral ARGENT gene expression system. - Sanftner, L.M., Rivera, V.M., Suzuki, B.M., Feng, L., Berk, L., Zhou, S., Forsayeth, J.R., Clackson, T., and Cunningham, J. (2006) Dimerizer regulation of AADC expression and behavioral response in AAV-transduced 6-OHDA lesioned rats. Mol Ther 13:167-174.
AAV vectors containing rapamycin-regulated AADC gene casettes were delivered to the striata of rats modeling Parkinson disease. Rapamycin-regulated expression of AADC and a robust behavioral response were demonstrated. - Wang, J., Voutetakis, A., Papa, M., Rivera, V.M., Clackson, T., Lodde, B.M., Mineshiba, F., and Baum, B.J. (2006) Rapamycin control of transgene expression from a single AAV vector in mouse salivary glands. Gene Ther 13:187-190.
A single AAV vector expressing rapamycin-inducible erythropoietin (Epo) was transduced into the salivary glands of mice. Administration of rapamycin led to the dose-dependent, reversible production of Epo that could be detected in serum.
2005
- Lebherz, C., Auricchio, A., Maguire, A.M., Rivera, V.M., Tang, W., Grant, R.L., Clackson, T., Bennett, J., and Wilson, J.M. (2005) Long-term inducible gene expression in the eye via adeno-associated virus gene transfer in nonhuman primates. Hum Gene Ther 16:178-186.
Demonstrates long-term rapamycin and AP22594-regulated expression of erythropoietin in the eyes of nonhuman primates transduced with AAV vectors. - Mukherjee, S., and Conrad, S.E. (2005) c-Myc suppresses p21WAF1/CIP1 expression during estrogen signaling and antiestrogen resistance in human breast cancer cells. J Biol Chem 280:17617-17625.
Uses the AP1510 homodimerization system to regulate transcription of c-Myc in stably transduced MCF-7 cells. - Rivera, V.M., Gao, G.P., Grant, R.L., Schnell, M.A., Zoltick, P.W., Rozamus, L.W., Clackson, T., and Wilson, J.M. (2005) Long-term pharmacologically regulated expression of erythropoietin in primates following AAV-mediated gene transfer. Blood 105:1424-1430.
Demonstrates long-term (greater than 6 years) rapamycin and AP22594-regulated expression of erythropoietin in nonhuman primates transduced intramuscularly with AAV vectors. In addition, for the first time, the regulated transcription factor and the target gene cassettes are combined in a single AAV vector with long-term, tightly controlled expression maintained.
2004
- Horswill, A.R., Savinov, S.N., and Benkovic, S.J. (2004) A systematic method for identifying small-molecule modulators of protein-protein interactions. Proc Natl Acad Sci U S A 101:15591-15596.
Uses rapamycin as a heterodimerizer to demonstrate the feasibility of a new selection system for identifying inhibitors of protein-protein interactions. Rapamycin is used to control transcription in E.coli cells. - Sudomoina, M., Latypova, E., Favorova, O.O., Golemis, E.A., and Serebriiskii, I.G. (2004) A gene expression system offering multiple levels of regulation: the Dual Drug Control (DDC) system. BMC Biotechnol 4:9.
Describes a gene regulation system that uses AP21967-mediated transcription of a tetracycline regulated transcription factor to control expression of a target gene. - Wang, J., Voutetakis, A., Zheng, C., and Baum, B.J. (2004) Rapamycin control of exocrine protein levels in saliva after adenoviral vector-mediated gene transfer. Gene Ther 11:729-733.
Provides the first demonstration of regulated gene expression (using rapamycin) after adenoviral-mediated gene transfer to salivary glands in rats. - Zhang, H., Wu, W., Du, Y., Santos, S.J., Conrad, S.E., Watson, J.T., Grammatikakis, N., and Gallo, K.A. (2004) Hsp90/p50cdc37 is required for mixed-lineage kinase (MLK) 3 signaling. J Biol Chem 279:19457-19463.
AP21967 used to inducibly express mixed-lineage kinase 3 in stable cell lines to study its function.
2003
- Crittenden, M., Gough, M., Chester, J., Kottke, T., Thompson, J., Ruchatz, A., Clackson, T., Cosset, F.L., Chong, H., Diaz, R.M., Harrington, K., Alvarez Vallina, L., and Vile, R. (2003) Pharmacologically regulated production of targeted retrovirus from T cells for systemic antitumor gene therapy. Cancer Res 63:3173-80.
Describes a system which allows T cells to be engineered to produce a retrovirus in vivo following transcriptional induction by AP21967. - Johnston, J., Tazelaar, J., Rivera, V.M., Clackson, T., Gao, G.P., and Wilson, J.M. (2003) Regulated expression of erythropoietin from an AAV vector safely improves the anemia of beta-thalassemia in a mouse model. Mol Ther 7:493-7.
Demonstrates rapamycin-mediated control of Epo levels and hematocrit upon delivery of AAV vectors to the muscle of beta-thalassemic mice. - Wang, S., Petravicz, J., and Breakefield, X.O. (2003) Single HSV-amplicon vector mediates drug-induced gene expression via dimerizer system. Mol Ther 7:790-800.
Demonstrates rapamycin-dependent expression of a reporter gene in culture and in rat brain delivered in a single HSV-amplicon vector. - Xu, Z.L., Mizuguchi, H., Mayumi, T., and Hayakawa, T. (2003) Regulated gene expression from adenovirus vectors: a systematic comparison of various inducible systems. Gene 309:145-51.
In a direct comparison of 5 different gene regulation systems delivered to cells using adenoviral vectors, the AP21967-based dimerizer system is shown to have the lowest basal expression and highest induction ratio. - Yang, W., Keenan, T.P., Rozamus, L.W., Wang, X., Rivera, V.M., Rollins, C.T., Clackson, T., and Holt, D.A. (2003) Regulation of gene expression by synthetic dimerizers with novel specificity. Bioorg Med Chem Lett 13:3181-4.
Explores the structure-activity relationship of synthetic homodimerizers applied toward the regulation of gene transcription. Demonstrates that simple, "bumped" homodimerizers such as AP1966 and AP1889 can potently activate gene expression in cells expressing transcription factors fused to the specific FKBP mutant F36V.
2002
- Auricchio, A., Gao, G.P., Yu, Q.C., Raper, S., Rivera, V.M., Clackson, T., and Wilson, J.M. (2002) Constitutive and regulated expression of processed insulin following in vivo hepatic gene transfer. Gene Ther 9:963-71.
Demonstrates tight, rapamycin-dependent transcription of insulin, and control of glucose levels, following transduction of mouse liver by adenoviral vectors. - Auricchio, A., Rivera, V.M., Clackson, T., O'Connor, E.E., Maguire, A.M., Tolentino, M.J., Bennett, J., and Wilson, J.M. (2002) Pharmacological regulation of protein expression from adeno-associated viral vectors in the eye. Mol Ther 6:238-42.
Demonstrates tight, dose-dependent regulation of gene expression upon delivery of AAV vectors to the eyes of rats and a non-human primate following systemic administration of the inducer rapamycin. - Chong, H., Ruchatz, A., Clackson, T., Rivera, V.M., and Vile, R.G. (2002) A system for small-molecule control of conditionally replication- competent adenoviral vectors. Mol Ther 5:195-203.
Uses the rapamycin analog AP21967 to control E1 gene expression and thereby control replication of adenoviral vectors in vitro and in vivo. - Go, W.Y., and Ho, S.N. (2002) Optimization and direct comparison of the dimerizer and reverse tet transcriptional control systems. J Gene Med 4:258-70.
Demonstrates tight, rapamycin-induced expression of a reporter gene in transiently transfected cells. - Pollock, R., and Clackson, T. (2002) Dimerizer-regulated gene expression. Curr Opin Biotechnol 13:459-67.
Reviews recent progress made using various dimerizer-based systems for regulating gene expression. - Pollock, R., Giel, M., Linher, K., and Clackson, T. (2002) Regulation of endogenous gene expression with a small-molecule dimerizer. Nat Biotechnol 20:729-33.
The dimerizer-regulated gene expression system was combined with targeted zinc finger DNA binding domain technology to permit small molecule control of endogenous gene expression. AP21967-dependent expression of the chromosomal human VEGF gene was rapid, tight and dose-dependent and exceeded levels produced by the natural hypoxic response.
2001
- Senner, V., Sotoodeh, A., and Paulus, W. (2001) Regulated gene expression in glioma cells: a comparison of three inducible systems. Neurochem Res 26:521-4.
Demonstrates tight, highly-inducible expression of a reporter gene in glioma cells using the AP1510-based transcriptional regulation system.
2000
- Clackson, T. (2000) Regulated gene expression systems. Gene Ther 7:120-5.
Reviews the various systems being developed for regulating gene expression in the context of gene therapy. - Pollock, R., Issner, R., Zoller, K., Natesan, S., Rivera, V.M., and Clackson, T. (2000) Delivery of a stringent dimerizer-regulated gene expression system in a single retroviral vector. Proc Natl Acad Sci USA 97:13221-6.
Describes several improvements in the rapamycin regulated transcription system, including incorporation of more potent transcriptional activation domains to improve the level of target gene transcription and the incorporation of the entire system into a single retroviral vector. A variety of cell lines transduced with the all-in-one retroviral vector display negligible basal expression and give induction ratios of at least three orders of magnitude in the presence of rapamycin or a nonimmunosuppressive analog.
1999
- Molinari, E., Gilman, M., and Natesan, S. (1999) Proteasome-mediated degradation of transcriptional activators correlates with activation domain potency in vivo. Embo J 18:6439-47.
Uses rapamycin-regulated recruitment of an activation domain to DNA to explore proteasome-mediated degradation of transcription factors. - Natesan, S., Molinari, E., Rivera, V.M., Rickles, R.J., and Gilman, M. (1999) A general strategy to enhance the potency of chimeric transcriptional activators. Proc Natl Acad Sci USA 96:13898-903.
Demonstrates that the effectiveness of chimeric transcriptional activators can be dramatically improved by expressing them as noncovalent tetrameric bundles. Bundling is shown to improve the performance of both the rapamycin-regulated gene expression system and a mammalian two-hybrid system. - Pollock, R., and Rivera, V.M. (1999) Regulation of gene expression with synthetic dimerizers. Methods Enzymol 306:263-81.
Describes the development of a transcription switch based on the use of a "third generation" synthetic homodimerizer, AP1889, which binds specifically to a mutated FKBP over the wild type protein. - Rivera, V.M., Ye, X., Courage, N.L., Sachar, J., Cerasoli, F., Jr., Wilson, J.M., and Gilman, M. (1999) Long-term regulated expression of growth hormone in mice after intramuscular gene transfer. Proc Natl Acad Sci USA 96:8657-62.
AAV-mediated delivery of the rapamycin regulated transcription system to the muscle of mice allows long-term, tightly regulated control over the production of growth hormone. - Ye, X., Rivera, V.M., Zoltick, P., Cerasoli, F., Jr., Schnell, M.A., Gao, G., Hughes, J.V., Gilman, M., and Wilson, J.M. (1999) Regulated delivery of therapeutic proteins after in vivo somatic cell gene transfer. Science 283:88-91.
The first demonstration of regulated gene expression in a primate. AAV-mediated delivery of the rapamycin regulated transcription system into the muscle of mice and a non-human primate allows tightly regulated expression of erythropoietin.
1998
- Rivera, V.M. (1998) Controlling gene expression using synthetic ligands. Methods 14:421-9.
A detailed description of the methods involved in employing the AP1510-based dimerizer system to control transcription.
1997
- Amara, J.F., Clackson, T., Rivera, V.M., Guo, T., Keenan, T., Natesan, S., Pollock, R., Yang, W., Courage, N.L., Holt, D.A., and Gilman, M. (1997) A versatile synthetic dimerizer for the regulation of protein-protein interactions. Proc Natl Acad Sci USA 94:10618-23.
Describes the development of the second generation, wholly synthetic FKBP dimerizer, AP1510. The synthetic nature of AP1510 makes it more amenable to modification and more readily synthesized in larger quantities. AP1510-mediated oligomerization of a Fas receptor and an engineered transcription factor are shown to induce apoptosis and transcription of a target gene, respectively. - Clackson, T. (1997) Controlling mammalian gene expression with small molecules. Curr Opin Chem Biol 1:210-8.
Reviews the regulatory systems that have been developed for controlling gene transcription, including the rapamycin-based system. - Freiberg, R.A., Ho, S.N., and Khavari, P.A. (1997) Transcriptional control in keratinocytes and fibroblasts using synthetic ligands. J Clin Invest 99:2610-5.
Uses FK1012-mediated dimerization of a transcription factor to control transcription in keratinocytes and fibroblasts. - Liberles, S.D., Diver, S.T., Austin, D.J., and Schreiber, S.L. (1997) Inducible gene expression and protein translocation using nontoxic ligands identified by a mammalian three-hybrid screen. Proc Natl Acad Sci USA 94:7825-30.
Describes the modification of the rapamycin heterodimerizer system to function with one class of non-immunosuppressive analogs. A mutant FRAP domain is identified that binds to an analog called Ma-rap that has greatly reduced affinity for wild type FRAP. The utility of the modified system for controlling the subcellular localization of a protein and the activity of a transcription factor is demonstrated. - Magari, S.R., Rivera, V.M., Iuliucci, J.D., Gilman, M., and Cerasoli, F., Jr. (1997) Pharmacologic control of a humanized gene therapy system implanted into nude mice. J Clin Invest 100:2865-72.
Demonstrates precise, in vivo, control of protein expression from cells engineered to express human growth hormone in response to rapamycin.
1996
- Belshaw, P.J., Ho, S.N., Crabtree, G.R., and Schreiber, S.L. (1996) Controlling protein association and subcellular localization with a synthetic ligand that induces heterodimerization of proteins. Proc Natl Acad Sci USA 93:4604-7.
Uses an FKBP-cyclophilin heterodimerizer (FKCsA) to alter the subcellular localization of a fusion protein or control the activity of a transcription factor. - Ho, S.N., Biggar, S.R., Spencer, D.M., Schreiber, S.L., and Crabtree, G.R. (1996) Dimeric ligands define a role for transcriptional activation domains in reinitiation. Nature 382:822-6.
Uses FK1012, FK506 and rapamycin to control the activity of engineered transcription factors in order to explore the role of transcriptional activation domains in the reinitiation of transcription. - Rivera, V.M., Clackson, T., Natesan, S., Pollock, R., Amara, J.F., Keenan, T., Magari, S.R., Phillips, T., Courage, N.L., Cerasoli, F., Jr., Holt, D.A., and Gilman, M. (1996) A humanized system for pharmacologic control of gene expression. Nat Med 2:1028-32.
Describes the development of a humanized system for controlling gene transcription based on the use of the heterodimerizer rapamycin to control the activity of a transcription factor. Inducible growth hormone expression is demonstrated in vitro and in vivo in implanted mice.
Three-Hybrid techology
A particular use of dimerizer-controlled transcription is in three-hybrid assays and screens. In these applications the "third hybrid" is the dimerizer, and gene activation serves merely as an assay to report on the interaction between a dimerizer and the two fusion proteins, rather than as the end in itself. Three-hybrid can be used to identify target proteins for a given small molecule, or vice versa. Most of the three-hybrid papers below use the transcriptional system although a few use alternative readouts.
2006
- Caligiuri, M., Molz, L., Liu, Q., Kaplan, F., Xu, J.P., Majeti, J.Z., Ramos-Kelsey, R., Murthi, K., Lievens, S., Tavernier, J., and Kley, N. (2006) MASPIT: three-hybrid trap for quantitative proteome fingerprinting of small molecule-protein interactions in mammalian cells. Chem Biol 13:711-722.
Describes a three-hybrid system in mammalian cells, in which novel small molecules are fused to methotrexate, and the readout based on ligand-dependent activation of JAK/STAT signaling. The system is used to identify new targets for a previously known Src kinase inhibitor.
2005
- Caligiuri, M., Becker, F., Murthi, K., Kaplan, F., Dedier, S., Kaufmann, C., Machl, A., Zybarth, G., Richard, J., Bockovich, N., Kluge, A., and Kley, N. (2005) A proteome-wide CDK/CRK-specific kinase inhibitor promotes tumor cell death in the absence of cell cycle progression. Chem Biol 12:1103-1115.
Describes the use of a yeast three-hybrid screen to identify multiple cyclin-dependent kinases (CDKs) and other related kinases as targets of a novel small-molecule kinase inhibitor.
2004
- Becker, F., Murthi, K., Smith, C., Come, J., Costa-Roldan, N., Kaufmann, C., Hanke, U., Degenhart, C., Baumann, S., Wallner, W., Huber, A., Dedier, S., Dill, S., Kinsman, D., Hediger, M., Bockovich, N., Meier-Ewert, S., Kluge, A.F., and Kley, N. (2004) A three-hybrid approach to scanning the proteome for targets of small molecule kinase inhibitors. Chem Biol 11:211-223.
Describes use of a yeast three-hybrid system, based on heterodimerizers that fuse methotrexate to a small molecule of interest, to identify both known and novel targets of cyclin-dependent kinase (CDK) inhibitors from cDNA libraries. - de Felipe, K.S., Carter, B.T., Althoff, E.A., and Cornish, V.W. (2004) Correlation between ligand-receptor affinity and the transcription readout in a yeast three-hybrid system. Biochemistry 43:10353-10363.
Carefully quantitates the sensitivity and dose-response of a yeast three-hybrid system based on use of dexamethasone-methotrexate heterodimerizers. - Kley, N. (2004) Chemical dimerizers and three-hybrid systems: scanning the proteome for targets of organic small molecules. Chem Biol 11:599-608.
A review of three-hybrid approaches to identifying the targets for small molecules, in both yeast and mammalian cells. - Lefurgy, S., and Cornish, V. (2004) Finding Cinderella after the ball: a three-hybrid approach to drug target identification. Chem Biol 11:151-153.
Briefly reviews the yeast three-hybrid approach to identifying targets for small molecules, in particular the paper by Becker et al (2004). -
Muddana, S.S., and Peterson, B.R. (2004) Facile synthesis of cids: biotinylated estrone oximes efficiently heterodimerize estrogen receptor and streptavidin proteins in yeast three hybrid systems. Org Lett 6:1409-1412.
Describes a simple chemical synthesis for heterodimerizers based on estrogen-type steroids and biotin, and their use in a yeast-three hybrid system.
2003
- Athavankar, S., and Peterson, B.R. (2003) Control of gene expression with small molecules: biotin-mediated acylation of targeted lysine residues in recombinant yeast. Chem Biol 10:1245-1253.
Describes a novel variation on the principle of chemically-induced dimerization, in which one target protein is fused to streptavidin and the other to an "Avitag" domain that is biotinylated in vivo. Addition of biotin to yeast cells results in modification of the Avitag and tight interaction of the fusion proteins. The system was validated in a yeast three-hybrid assay. - Baker, K., Sengupta, D., Salazar-Jimenez, G., and Cornish, V.W. (2003) An optimized dexamethasone-methotrexate yeast 3-hybrid system for high-throughput screening of small molecule-protein interactions. Anal Biochem 315:134-137.
Describes an optimized yeast three-hybrid system for use with dexamethasone-methotrexate heterodimerizers, using stably transfected yeast strains. - Hussey, S.L., Muddana, S.S., and Peterson, B.R. (2003) Synthesis of a beta-estradiol-biotin chimera that potently heterodimerizes estrogen receptor and streptavidin proteins in a yeast three-hybrid system. J Am Chem Soc 125:3692-3693.
Describes a new class of heterodimerizers based on estrogen-type steroids and biotin, and their use in a yeast-three hybrid system.
2002
- Abida, W.M., Carter, B.T., Althoff, E.A., Lin, H., and Cornish, V.W. (2002) Receptor-dependence of the transcription read-out in a small-molecule three-hybrid system. Chembiochem 3:887-95.
Further characterizes the use of a dexamethasone-methotrexate (Dex-Mtx) heterodimerizer in yeast. - Althoff, E.A., and Cornish, V.W. (2002) A bacterial small-molecule three-hybrid system. Angew. Chemie 41:2327-30.
Describes the development of a novel alternative heterodimerizer, Mtx-SLF, which can heterodimerize dihydrofolate reductase- and FKBP- containing fusion proteins. SLF is a monomeric FKBP ligand based on the early-generation homodimerizer AP1510. These reagents are used to construct a three-hybrid system that functions in bacteria. - Baker, K., Bleczinski, C., Lin, H., Salazar-Jimenez, G., Sengupta, D., Krane, S., and Cornish, V.W. (2002) Chemical complementation: a reaction-independent genetic assay for enzyme catalysis. Proc Natl Acad Sci USA 99:16537-42.
Uses a dexamethasone-methotrexate (Dex-Mtx) heterodimerizer system to develop a general yeast-based screen for enzymatic activity.
2000
- Griffith, E.C., Licitra, E.J., and Liu, J.O. (2000) Yeast three-hybrid system for detecting ligand-receptor interactions. Methods Enzymol 328:89-103.
Provides detailed protocols for the use of the three hybrid system for identifying or characterizing novel small molecule protein ligands.
1996
- Licitra, E.J., and Liu, J.O. (1996) A three-hybrid system for detecting small ligand-protein receptor interactions. Proc Natl Acad Sci USA 93:12817-21.
Describes the use of the chemical dimerizer concept to develop a "three-hybrid" system for identifying or characterizing novel small molecule-protein interactions. In this proof-of-concept, a heterodimerizer comprising FK506 and dexamethasone is used to bring together transcription factor fragments fused to a glucocorticoid receptor and FKBP, activating reporter gene expression in yeast. By substituting dexamethasone (or FK506) for a novel ligand, and the glucocorticoid receptor for a cDNA library, this system could be used to discover receptors for novel ligands; or conversely to screen for new ligands to known receptors.
