Dimerization Systems Transcription

Different AAV serotypes have shown remarkably different expression patterns because of differences in cell entry and intracellular activities (66, 67). Application of the dimerizer-inducible transcriptional regulatory system for AAV has allowed pharmacological regulation of heterologous gene expression in vivo (68). 

The effectiveness of any three-hybrid system depends critically on the CID used to dimerize the transcriptional activator in vivo [23,24], The subject of CIDs has been considered fully in the previous chapter by Clackson, so here we focus on the issues we have found particularly important for the use of CIDs in the... 

Fig. 4.2-11 Use of dimerizer-controlled transcription to achieve long-term regulated expression of a therapeutic gene in a nonhuman primate. At time zero, the animal received a single intramuscular injection of adeno-associated viral vectors encoding primate erythropoietin (Epo) under the control of the rapamycin-regulated dimerization system. Subsequent administrations of rapamycin at the...

A bacterial two-hybrid system has been developed that, similar to the yeast system, functions via activation of transcription (Dove and Hochschild, 1998 Joung et al., 2000). RNA polymerase (RNAP) in E. coli consists of an enzymatic core composed of the a, (3, and (3 subunits in the stoichiometry a2(3(3, and one of several c factors that enable the enzyme to recognize specific promoters (Heilman and Chamberlin, 1988). Many bacterial transcriptional activator proteins bind the promoters they regulate and interact directly with subunits of RNAP. The most commonly observed contact is between activator proteins and the a subunit of RNAP (Ebright and Busby, 1995). The function of the a subunit is to initiate the assembly of RNAP by forming a dimer (Igarashi et al., 1991). 

A variation on the basic theme of receptor Tyr kinases is seen in receptors that have no intrinsic protein kinase activity but, when occupied by their ligand, bind a soluble Tyr kinase. One example is the system that regulates the formation of erythrocytes in mammals. The cytokine (developmental signal) for this system is erythropoietin (EPO), a 165 amino acid protein produced in the kidneys. When EPO binds to its plasma membrane receptor (Fig. 12-9), the receptor dimerizes and can now bind the soluble protein kinase JAK (Janus kinase). This binding activates JAK, which phosphory-lates several Tyr residues in the cytoplasmic domain of the EPO receptor. A family of transcription factors, collectively called STATs (signal transducers and activators of transcription), are also targets of the JAK kinase activity. An SH2 domain in STATS binds (P)-Tyr residues in the EPO receptor, positioning it for this phosphorylation by JAK. When STATS is phosphorylated in re-... 

The leptin signal is transduced by a mechanism also used by receptors for interferon and growth factors, the JAIC-STAT system (Fig. 23-34 see Fig. 12-9). The leptin receptor, which has a single transmembrane segment, dimerizes when leptin binds to the extracellular domain of two monomers. Both monomers are phos-phorylated on a Tyr residue of the intracellular domain by a Janus kinase (JAK). The -Tyr residues become docking sites for three proteins that are signal transducers and activators of transcription (STATs 3, 5, and 6, sometimes called fat-STATS). The docked STATs are then phosphorylated on Tyr residues by the... 

In yeast model systems it has been observed that myc is a transcription activator but only when present in a heterodimer with max. Max appears to be essential for DNA binding. Max dimer can bind to DNA on its own but it does not activate transcription on its own. Mammalian genes that are normally activated by myc are still not known. 

As outlined in Fig. 7.1, the Y2H system consists of two protein chimeras, and a reporter gene downstream from the binding site for the transcriptional activator. If the two proteins of interest (X and Y) interact, they effectively dimerize the DNA-binding protein chimera (DBD-X) and the transcription activation protein chimera (AD-Y). Dimerization of the DBD and the transcription AD helps to recruit the transcription machinery to a promoter adjacent to the binding site for the transcriptional activator, thereby activating transcription of the reporter gene. 

More recently Michnick and co-workers have introduced a dihydrofolate reductase complementation system, which seems to be particularly robust [61 - 65], They attribute the success of this system to the fact that the N-terminal (1 - 105) and C-terminal (106 - 186) DHFR fragments do not fold until they are dimerized. In addition to the obvious selection for essential metabolites dependent on the reduction of dihydrofolate to tetrahydrofolate, protein-protein interactions are detected based on the retention of a fluorescein-methotrexate conjugate. Several other enzymes have been employed for the design of complementation assays, including green fluorescent protein, which allows screens based on fluorescence or FRET [66 - 68]. As with the bacterial transcription assays, these complementation systems are new. It will be interesting to see if, as the selections are optimized, these systems prove competitive with the Y2H assay. 

Copper ion homeostasis in prokaryotes involves Cu ion efflux and sequestration. The proteins involved in these processes are regulated in their biosynthesis by the cellular Cu ion status. The best studied bacterial Cu metalloregulation system is found in the gram-positive bacterium Enterococcus hirae. Cellular Cu levels in this bacterium control the expression of two P-type ATPases critical for Cu homeostasis (Odermatt and Solioz, 1995). The CopA ATPase functions in Cu ion uptake, whereas the CopB ATPase is a Cu(I) efflux pump (Solioz and Odermatt, 1995). The biosynthesis of both ATPases is regulated by a Cu-responsive transcription factor, CopY (Harrison et al., 2000). In low ambient Cu levels Cop Y represses transcription of the two ATPase genes. On exposure to Cu(I), CopY dissociates from promoter/operator sites on DNA with a for Cu of 20 jlM (Strausak and Solioz, 1997). Transcription of copA and copB proceeds after dissociation of CuCopY. The only other metal ions that induce CopY dissociation from DNA in vitro are Ag(I) and Cd(II), although the in vivo activation of copA and copB is specihc to Cu salts. The CuCopY complex is dimeric with two Cu(I) ions binding per monomer (C. T. Dameron, personal communication). The structural basis for the Cu-induced dissociation of CopY is unknown. Curiously, CopY is also activated in Cu-dehcient cells, but the mechanism is distinct from the described Cu-induced dissociation from DNA (Wunderh-Ye and Solioz, 1999). 

Chemical inducers of dimerization (CID) are bivalent small molecules that bind t vo proteins simultaneously. The purpose of these molecules is to bring the proteins together to induce signal transduction [27]. For brevity, we will limit our discussion to CIDs that directly control transcription. The basic architecture of these systems consists of two chimeric proteins. The first contains a DNA-binding domain (DBD) fused to a ligand-binding domain (LBD) and the second chimera contains an LBD and an activation domain (AD). The small molecule that binds both of these proteins simultaneously induces proximity of the two proteins, resulting in transcription activation (Fig. 8.11). 

Fig.8.n Chemical inducers of dimerization (CID) to control transcription. This system requires two chimeric proteins, one comprising a DNA-binding domain (DBD) fused to a ligand-binding domain (LBD), and the other comprising an LBD fused to a transcriptional... 

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