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Neutralizing the Function of a Β-Globin–Associated Cis-Regulatory DNA Element Using an Artificial Zinc finger DNA-Binding Domain

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Neutralizing the Function of a Β-Globin–Associated Cis-Regulatory DNA Element Using an Artificial Zinc finger DNA-Binding Domain Neutralizing the function of a β-globin–associated cis-regulatory DNA element using an artificial zinc finger DNA-binding domain Joeva J. Barrow, Jude Masannat, and Jörg Bungert1 Department of Biochemistry and Molecular Biology, College of Medicine, Center for Epigenetics, Genetics Institute, Shands Cancer Center, Powell-Gene Therapy Center, University of Florida, Gainesville, FL 32610 Edited by Mark Groudine, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved September 19, 2012 (received for review May 6, 2012) Gene expression is primarily regulated by cis-regulatory DNA ele- The purpose of our study was to examine the applicability of ments and trans-interacting proteins. Transcription factors bind in using a ZF-DBD to assess the contribution of a cis-regulatory DNA a DNA sequence–specific manner and recruit activities that modu- element to gene expression and transcription factor binding in vivo. late the association and activity of transcription complexes at spe- We designed and expressed a ZF-DBD, harboring six ZFs, that cific genes. Often, transcription factors belong to families of related specifically interacts with a critical cis-regulatory element associated proteins that interact with similar DNA sequences. Furthermore, with adult βmin-globin gene expression—the −90 CACCC box genes are regulated by multiple, sometimes redundant, cis-regula- located about 90 bp upstream of the βmin-globin transcription start tory elements. Thus, the analysis of the role of a specific DNA regu- site (15). We termed this artificial binding protein −90 β-ZF-DBD. latory sequence and the interacting proteins in the context of intact The CACCC site interacts with transcription factor KLF1, a ZF cells is challenging. In this study, we designed and functionally char- protein important for red blood cell function (16–18). Mutation of acterized an artificial DNA-binding domain that neutralizes the func- the β-promoter–associated CACCC site in the human population tion of a cis-regulatory DNA element associated with adult β-globin has been associated with β-thalassemia (19). The −90 β-ZF-DBD, gene expression. The zinc finger DNA-binding domain (ZF-DBD), when expressed in erythroid cells, displaced KLF1 from the βmin- comprising six ZFs, interacted specifically with a CACCC site located globin promoter and reduced expression of the βmin-globin gene. 90 bp upstream of the transcription start site (–90 β-ZF-DBD), which Other KLF1-regulated genes, such as the dematin and β-spectrin is normally occupied by KLF1, a major regulator of adult β-globin genes, were not affected by the −90 β-ZF-DBD. Synthetic ZF- gene expression. Stable expression of the –90 β-ZF-DBD in mouse DBDs may provide a tool for the treatment of sickle cell anemia erythroleukemia cells reduced the binding of KLF1 with the β-globin and other hemoglobinopathies. Specifically, a combination of ZF- gene, but not with locus control region element HS2, and led to DBDs could be designed that reduce expression of mutant β-globin reduced transcription. Transient transgenic embryos expressing and enhance expression of therapeutic γ-globin genes. the –90 β-ZF-DBD developed normally but revealed reduced expres- sion of the adult β-globin gene. These results demonstrate that ar- Results tificial DNA-binding proteins lacking effector domains are useful We designed a ZF-DBD comprising six ZFs that would bind to 18 tools for studying and modulating the function of cis-regulatory bp of DNA containing the βmin-globin promoter–associated −90 DNA elements. CACCC box (−90 β-ZF-DBD, Fig. 1A). Statistically, an 18-bp sequence occurs once per mammalian genome. There are two artificial transcription factor | red cell | gene regulation CACCC sites in the βmin-globin gene promoter, and the −90 β-ZF-DBD is predicted to block both of them. Specific amino he modulation and functional analysis of gene regulation in acid–nucleic acid recognition sequences were derived using a web- Tvivo is challenging because of redundancies in transcription based program developed by Barbas and colleagues (20, 21). The factors and cis-regulatory DNA elements (1–4). Transgenic or ZF-DBD protein was generated using a modified protocol from reporter gene assays are powerful but limited by potential position Cathomen et al. (22), as outlined in Fig. 1B. The −90 β-ZF-DBD effects (5). Genetic manipulation of cis-regulatory DNA elements coding segment was cloned into the pT7-Flag2 vector and is technically demanding and time-consuming (6). The zinc finger expressed in and purified from Escherichia coli (Fig. 1 C and D). (ZF) domain, the most commonly found DNA-binding domain in The −90 β-ZF-DBD protein migrated at an expected size of 24 eukaryotic transcription factors (7), is characterized by a DNA- kDa. Electrophoretic mobility shift assays (EMSAs) demonstrated binding α-helix, known as the α-helix reading head, which is sta- that the −90 β-ZF-DBD specifically interacted with an oligonu- bilized by an adjacent finger-like structure in which histidine or cleotide containing the 18-bp target WT sequence harboring the cysteine residues coordinate a zinc atom (8–10). The mode of −90 CACCC site, but not with a mutant oligonucleotide (Fig. 1E). DNA binding by ZF proteins is very well understood, and this An excess of unlabeled WT oligonucleotides efficiently competed knowledge led to the development of artificial proteins containing for the binding, whereas mutant oligonucleotides did not perturb adefined ZF DNA-binding domain (ZF-DBD) that interacts with binding. The addition of a Flag antibody to the binding reaction a specific sequence of interest (10, 11). Each α-helix reading head eliminated formation of the protein-DNA complex, indicating that of a ZF-DBD recognizes three to four specific DNA base pairs, this interaction was specific. These data demonstrate that the −90 and reading heads can be designed to essentially recognize any possible triplet of DNA base pairs (11, 12). Furthermore, the interactions are modular in nature; therefore, arranging these ZFs Author contributions: J.J.B. and J.B. designed research; J.J.B., J.M., and J.B. performed in tandem provides the recognition of extended asymmetrical research; J.J.B. and J.B. analyzed data; and J.J.B. and J.B. wrote the paper. DNA sequences. Previous studies linked artificial ZF-DBDs to The authors declare no conflict of interest. nucleases or to effector domains that enhance or repress tran- This article is a PNAS Direct Submission. scription (11–13). Most of the artificial ZF proteins studied to date Freely available online through the PNAS open access option. appear to interact with desired target sequences with high speci- 1To whom correspondence should be addressed. E-mail: jbungert@ufl.edu. fi city; however, off-target binding has been detected, which is This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. a concern if ZF-DBDs are expressed with effector domains (14). 1073/pnas.1207677109/-/DCSupplemental. 17948–17953 | PNAS | October 30, 2012 | vol. 109 | no. 44 www.pnas.org/cgi/doi/10.1073/pnas.1207677109 Downloaded by guest on September 24, 2021 Fig. 1. In vitro characterization of the −90 β-ZF- DBD. (A) Representation of the adult murine βmin- globin gene with the −90 β-ZF-DBD (oval) binding to an 18-bp sequence encompassing the −90 KLF1 binding site (CACCC box). (B) Overall design sche- matic of the −90 β-ZF-DBD. The coding DNA frag- ment of the −90 β-ZF-DBD containing six ZF domains was generated using two PCR reactions encoding for ZF 1–3 and ZF 4–6, respectively. The two DNA fragments were assembled separately using a series of overlapping oligonucleotides coding for the constant ZF backbone (blue) or the variable α-helix reading heads (black). The DNA fragments were annealed and gaps were sealed by PCR. Ligation of the two fragments and a canonical linker yielded the complete six-fingered ZF-DBD, which was then amplified by PCR using flanking forward and re- verse primers and cloned into prokaryotic or eukaryotic expression vectors. (C) Immunoblot with aFlag-specific antibody. The −90 β-ZF-DBD mi- grated with an apparent molecular weight of 24 kDa. (D) SDS/PAGE and subsequent Coomassie stain of proteins from induced and uninduced E. coli (lanes 1 and 2), respectively. The −90 β-ZF-DBD was immunopurified from crude protein lysates using anti-Flag magnetic beads (lane 4). (E) EMSA of the purified recombinant −90 β-ZF-DBD using oligonucleotides representing the −90 KLF1 site (WT) or a mutant oligonucleotide (Left, lanes 1 and 3). Binding of the −90 β-ZF-DBD to the labeled WT oligonucleotide was abolished in the presence of 500 molar excess of unlabeled WT competitor but was unaffected by excess of unlabeled mutant oligonucleotides (Right). The band labeled with # is nonspecific. The lane labeled WT (*) included a Flag-specific antibody during the binding reaction (Left). β-ZF-DBD interacts with the target 18-bp sequence encompassing (23). Immunofluorescence microscopic analysis in induced MEL the −90 CACCC box in a sequence-specific manner. cells using an antibody against the backbone of the ZF-DBD For studies in eukaryotic cells, the coding sequence for the −90 revealed that the −90 β-ZF-DBD localized to the nucleus (Fig. 2 β-ZF-DBD was cloned into the retroviral pMSCV (murine stem A-F). We next fractionated the MEL cells into cytosolic and nuclear cell virus)-neo plasmid and modified to include a nuclear localiza- compartments and performed an immunoblot analysis (Fig. 2G). tion sequence (NLS). After packaging, viruses either harboring the Brg1, a nuclear chromatin regulatory protein, and α-tubulin, a pre- vector encoding the −90 β-ZF-DBD or an empty vector were used dominantly cytoplasmic protein, were used as controls to indicate to transduce mouse erythroleukemia (MEL) cells and single-cell complete cellular fractionation.
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