a protein homodimerizer is utilized mainly for ligand-induced receptor dimerization and activation of intracellular signaling, thanks to the use of chimeric receptors that contain the ligand-binding domain of FKBP12. These synthetic receptors have been engineered to undergo dimerization in the presence of FKBP binders but not of endogenous ligands (88,89,93).
The modular nature of eukaryotic transcription factors enabled the construction of a dimerizer-induced regulatory system (94). DNA Binding Domains (DBDs) and Activation Domains (ADs) can be expressed independently of each other. Each domain maintains its proper folding: a DBD bound to its target promoter is unable to activate transcription unless the AD is placed in close proximity. This simple concept has been extensively exploited in the setting of two-hybrid assays in yeast (95). By fusing a DBD and an AD with a drug-binding domain, assembling an active transcription factor thus depends on adding of a dimerizer-ligand capable of bridging the DBD and AD by simultaneously interacting with the drug-binding domains of the fusion proteins. In this context, however, fusion of the DBD and the AD with the same ligand-binding domain and the use of homodimerizing drugs are not appropriate, as it would also lead to the generation of inactive DBD-DBD and AD-AD homodimers. To prevent homodi-merization, this system has been evolved to incorporate chemical heterodimerizers. Since no chemical entity is known with sufficient affinity and selectivity to bring together a DBD and an AD, attention has been directed to utilizing the natural targets of known chemical heterodimerizers. One such molecule is another immunosuppressant drug, rapamycin, a natural product macrolide (Fig. 11).
Rapamycin binds the immunophilin FKBP12 with a nano-molar affinity (96), and this complex interacts with FRAP (FKBP rapamycin-associated protein) (97) also called mTOR (mammalian target of rapamycin), a PI3-kinase homolog involved in control of cell growth and division (98). FKBP and an 89-aa fragment of FRAP, called FRB, sufficient to bind the FKBP-rapamycin complex (99), can be linked to a DBD and to an AD, respectively. These two components, namely DBD-FKBP and FRB-AD, associate noncovalently in the presence of rapamycin. A gene cloned downstream of a promoter containing sites recognized by the DBD-FKBP fusion is thus only transcribed in the presence of the dimerizer drug, according to the scheme shown in Fig. 12.
The dimerizer-regulated system was constructed by using protein domains of human origin in order to minimize its potential immunogenicity. DBD-FKBP and FRB-AD were thus generated by using human protein domains. The two heterodimeriz-ing components FKBP and FRB are human proteins. The selected DBD, ZFHD1, is a composite DNA-binding domain with novel DNA recognition specificity (100). It is a 122 poly-peptide constituted by a 58 aa zinc-finger domain from the natural human Zif268 zinc-finger protein linked via two glycine residues to the 62 aa POU domain of the human transcrip tion factor Oct-1. The AD is derived from the carboxy-termi-nal region of the human transcription factor NF-kB p65 (78). The two transcription components have been called ZFD1-FKBP (the version that carries three tandem copies of FKBP, named ZF3, was initially selected because of its excellent drug-dependent activity in transfected cells) and FRB-p65, respectively (also called S1) (101). They can be expressed either from separate transcription units or from a single bicis-tronic transcript by means of the ECMV (encephalomyocardi-tis virus) internal ribosome entry site (IRES).
Further modifications/improvements of these transcription factors have been obtained, through the generation of more potent FRB-carrying components. A widely accepted concept is that enhancing the efficiency with which activators are delivered to a promoter strongly potentiates transcription efficiency (102). Linking two ADs, namely those of human p65 and of human heat shock factor 1, to FRB, generated a more potent factor (103). Use of this more potent factor may be appropriate when higher sensitivity is required, for example when low gene copies/cell are transduced by retroviral vectors (103). A second elegant example was the generation of FRB-p65 fusions that carry a portion of the E. coli lactose repressor between the two domains that form tetrameric bundles and therefore multimers of the AD (104). These improved activators induce higher maximum expression levels of the regulated reporter genes and are more responsive to rapamycin. However, while the first solution is entirely based on human proteins, the second suffers from the potential immunogenicity of its E. coli-derived component.
More recently, a system similar to that induced by rapa-mycin has been described that responds to nonimmunosup-pressive analogs of FK506. FK506 heterodimerizes FKBP and calcineurin, a protein phosphatase that mediates the mitogenic stimuli from the T cell receptor (105). The FK506-FKBP binding interface of calcineurin is composed of aminoacid residues that belong to its component polypeptides, called can and CnB. Based on the X-ray structure of the FKBP-FK506-can-CnB complex, protein design was used to generate a synthetic calcineurin A-B fusion, called mCAB, which binds FKBP-FK506. A yeast three-hybrid system was then used to identify mCAB mutants that interact with FK506 analogs unable to bind calcineurin and therefore not immunosuppressive. A regulatory switch based on DBD-FKBP and mCAB-AD components was significantly activated by these FK506 analogs in transiently transfected cells (106). The potential of this system for gene therapy applications remains to be verified (see below).
As with other ligand-dependent regulatory systems, dimer-izer-responsive promoters are essentially composed of a downstream element, the minimal promoter, and an upstream element with a variable number of binding site(s) for the indu-cible transcription factor. The downstream elements used are
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