Takashi Sera

Zinc-finger-based artificial transcription factors and their applications

Artificial transcription factors (ATFs) are potentially a powerful molecular tool to modulate endogenous target gene expression in living cells and organisms. To date, many DNA-binding molecules have been developed as the DNA-binding domains for ATFs. Among them, ATFs comprising Cys(2)His(2)-type zinc-finger proteins (ZFPs) as the DNA-binding domain have been extensively explored. The zinc-finger-based ATFs specifically recognize targeting sites in chromosomes and effectively up- and downregulate expression of their target genes not only in vitro, but also in vivo. In this review, after briefly introducing Cys(2)His(2)-type ZFPs, I will review the studies of endogenous human gene regulation by zinc-finger-based ATFs and other applications as well.

Inhibition of Virus DNA Replication by Artificial Zinc Finger Proteins

ABSTRACT Prevention of virus infections is a major objective in agriculture and human health. One attractive approach to the prevention is inhibition of virus replication. To demonstrate this concept in vivo, an artificial zinc finger protein (AZP) targeting the replication origin of the Beet severe curly top virus (BSCTV), a model DNA virus, was created. In vitro DNA binding assays indicated that the AZP efficiently blocked binding of the viral replication protein (Rep), which initiates virus replication, to the replication origin. All of the transgenic Arabidopsis plants expressing the AZP showed phenotypes strongly resistant to virus infection, and 84% of the transgenic plants showed no symptom. Southern blot analysis demonstrated that BSCTV replication was completely suppressed in the transgenic plants. Since the mechanism of viral DNA replication is well conserved among plants and mammals, this approach could be applied not only to agricultural crop protection but also to the prevention of virus infections in humans.

Inhibition of DNA replication of human papillomavirus by artificial zinc finger proteins.

ABSTRACT Recently, we demonstrated that plant DNA virus replication was inhibited in planta by using an artificial zinc finger protein (AZP) and created AZP-based transgenic plants resistant to DNA virus infection. Here we apply the AZP technology to the inhibition of replication of a mammalian DNA virus, human papillomavirus type 18 (HPV-18). Two AZPs, designated AZPHPV-1 and AZPHPV-2, were designed by using our nondegenerate recognition code table and were constructed to block binding of the HPV-18 E2 replication protein to the replication origin. Both of the newly designed AZPs had much higher affinities towards the replication origin than did the E2 protein, and they efficiently blocked E2 binding in vitro. In transient replication assays, both AZPs inhibited viral DNA replication, especially AZPHPV-2, which reduced the replication level to approximately 10%. We also demonstrated in transient replication assays, using plasmids with mutant replication origins, that AZPHPV-2 could precisely recognize the replication origin in mammalian cells. Thus, it was demonstrated that the AZP technology could be applied not only to plant DNA viruses but also to mammalian DNA viruses.

Gene- and Protein-Delivered Zinc Finger–Staphylococcal Nuclease Hybrid for Inhibition of DNA Replication of Human Papillomavirus

Previously, we reported that artificial zinc-finger proteins (AZPs) inhibited virus DNA replication in planta and in mammalian cells by blocking binding of a viral replication protein to its replication origin. However, the replication mechanisms of viruses of interest need to be disentangled for the application. To develop more widely applicable methods for antiviral therapy, we explored the feasibility of inhibition of HPV-18 replication as a model system by cleaving its viral genome. To this end, we fused the staphylococcal nuclease cleaving DNA as a monomer to an AZP that binds to the viral genome. The resulting hybrid nuclease (designated AZP–SNase) cleaved its target DNA plasmid efficiently and sequence-specifically in vitro. Then, we confirmed that transfection with a plasmid expressing AZP–SNase inhibited HPV-18 DNA replication in transient replication assays using mammalian cells. Linker-mediated PCR analysis revealed that the AZP–SNase cleaved an HPV-18 ori plasmid around its binding site. Finally, we demonstrated that the protein-delivered AZP–SNase inhibited HPV-18 DNA replication as well and did not show any significant cytotoxicity. Thus, both gene- and protein-delivered hybrid nucleases efficiently inhibited HPV-18 DNA replication, leading to development of a more universal antiviral therapy for human DNA viruses.

Efficient double-stranded DNA cleavage by artificial zinc-finger nucleases composed of one zinc-finger protein and a single-chain FokI dimer

Zinc-finger-FokI nucleases (ZFNs) are useful for manipulating genomic DNA, but two ZFNs are required to cleave one site of double-stranded DNA (dsDNA), which limits the choice of targets. To refine ZFN technology, we constructed artificial zinc-finger nucleases containing an artificial zinc-finger protein (AZP) and a single-chain FokI dimer with nine different peptide linkers between two FokI molecules (designated AZP-scFokI). DNA cleavage assays revealed that the AZP-scFokI variant possessing the longest peptide linker cleaved dsDNA with equal or greater reactivity than the corresponding AZP-FokI dimer. The DNA cleavage pattern of AZP-scFokI suggests that the enhanced dsDNA cleavage was due to increased formation of FokI dimer in AZP-scFokI. Furthermore, we demonstrated that AZP-scFokI site-specifically cleaved its target DNA due to the AZP moiety discriminating one base pair difference. Thus, a single AZP-scFokI molecule is able to cleave dsDNA efficiently and site-specifically, and enhances the usefulness of the ZFN approach.

Enhanced Cleavage of Double-Stranded DNA by Artificial Zinc-Finger Nuclease Sandwiched between Two Zinc-Finger Proteins

To enhance DNA cleavage by zinc-finger nucleases (ZFNs), we sandwiched a DNA cleavage enzyme with two artificial zinc-finger proteins (AZPs). Because the DNA between the two AZP-binding sites is cleaved, the AZP-sandwiched nuclease is expected to bind preferentially to a DNA substrate rather than to cleavage products and thereby cleave it with multiple turnovers. To demonstrate the concept, we sandwiched a staphylococcal nuclease (SNase), which cleaves DNA as a monomer, between two three-finger AZPs. The AZP-sandwiched SNase cleaved large amounts of dsDNA site-specifically. Such multiple-turnover cleavage was not observed with nucleases that possess a single AZP. Thus, AZP-sandwiched nucleases will further refine ZFN technology.

Sandwiched zinc-finger nucleases harboring a single-chain FokI dimer as a DNA-cleavage domain.

Zinc-finger nucleases (ZFNs) are a powerful tool for manipulation of genomic DNA. Recently, we reported a new ZFN composed of one artificial zinc-finger protein (AZP) and a single-chain FokI dimer (scFokI) that refines ZFN technology. While AZP-scFokI cleaved DNA specifically around the AZP-target site, several nucleotide positions were cleaved due to the mobility of the scFokI domain. In the present study, we aimed to improve the DNA-cleavage specificity at the nucleotide level. To this end, we sandwiched a scFokI domain between two AZPs to reduce the mobility of the scFokI moiety when bound to DNA. We demonstrated that the AZP-sandwiched scFokI cleaved DNA at a single nucleotide position of a target plasmid, in which two AZP-binding sites were connected with a 6-bp spacer, with multiple turnovers. Further improvement of AZP-sandwiched scFokI will lead to development of ideal artificial meganucleases.

Deuterium relaxation time of α-d-tryptophan included in cyclodextrin host molecules

Deuterium relaxation times ofd- andl-α-d-tryptophan included in β-cyclodextrin derivatives were directly measured by deuterium NMR spectroscopy. The results showed that the molecular motion of the tryptophan molecule was strongly restricted even in the cavity of unmodified β-cyclodextrin and the additional recognition groupings — ammonium and carboxylate — on β-cyclodextrin did not affect the molecular motion of tryptophan, though the association constants were significantly enhanced.

Construction of plants resistant to TYLCV by using artificial zinc-finger proteins.

Previously, we have demonstrated that plant DNA virus replication could be inhibited in Arabidopsis thaliana by using an artificial zinc-finger protein (AZP) and created AZP-based transgenic A. thaliana resistant to DNA virus infection. Here we apply the AZP technology to tomato yellow leaf curl virus (TYLCV) causing serious damage to an important agricultural crop, tomato. An AZP was designed to block binding of the TYLCV replication protein (Rep) to the replication origin. The designed AZP had much higher affinities towards the replication origin than did the Rep, and efficiently blocked Rep binding in vitro. The AZP gene was then introduced into a plant genome with the help of Agrobacterium tumefaciens to generate the transgenic plants. The current status of the construction of the AZP-expressing transgenic plants will be reported.

Hypoxia-specific upregulation of the endogenous human VEGF-A gene by hypoxia-driven expression of artificial transcription factor

Activation of vascular endothelial growth factor A (VEGF-A) is an attractive approach to treatment of ischemic diseases. Although zinc-finger-based artificial transcription factors (ATFs) were constructed for human VEGF-A and constitutive expression of ATFs upregulated the endogenous VEGF-A gene expression, activation of VEGF-A specifically in ischemic tissues is desirable for therapeutic application of ATF technology. Here, we describe hypoxia-specific activation of human VEGF-A gene by hypoxia-driven expression of the ATF. We constructed a hypoxia-driven promoter for the ATF expression and placed it upstream of the ATF-encoding regions. The resulting hypoxia-driven expression plasmid induced the ATF expression in hypoxia but not in normoxia, and the hypoxia-specific expression of the ATF activated the VEGF-A expression specifically in hypoxia. Thus, the engineered expression system of ATFs may enable activation of VEGF-A expression specifically in ischemic tissues without affecting normal, healthy tissues in vivo.