UtroUp is a novel six zinc finger artificial transcription factor that recognises 18 base pairs of the utrophin promoter and efficiently drives utrophin upregulation
© Onori et al.; licensee BioMed Central Ltd. 2013
Received: 30 March 2012
Accepted: 12 December 2012
Published: 30 January 2013
Duchenne muscular dystrophy (DMD) is the most common X-linked muscle degenerative disease and it is due to the absence of the cytoskeletal protein dystrophin. Currently there is no effective treatment for DMD. Among the different strategies for achieving a functional recovery of the dystrophic muscle, the upregulation of the dystrophin-related gene utrophin is becoming more and more feasible.
We have previously shown that the zinc finger-based artificial transcriptional factor “Jazz” corrects the dystrophic pathology in mdx mice by upregulating utrophin gene expression. Here we describe a novel artificial transcription factor, named “UtroUp”, engineered to further improve the DNA-binding specificity. UtroUp has been designed to recognise an extended DNA target sequence on both the human and mouse utrophin gene promoters. The UtroUp DNA-binding domain contains six zinc finger motifs in tandem, which is able to recognise an 18-base-pair DNA target sequence that statistically is present only once in the human genome. To achieve a higher transcriptional activation, we coupled the UtroUp DNA-binding domain with the innovative transcriptional activation domain, which was derived from the multivalent adaptor protein Che-1/AATF. We show that the artificial transcription factor UtroUp, due to its six zinc finger tandem motif, possesses a low dissociation constant that is consistent with a strong affinity/specificity toward its DNA-binding site. When expressed in mammalian cell lines, UtroUp promotes utrophin transcription and efficiently accesses active chromatin promoting accumulation of the acetylated form of histone H3 in the utrophin promoter locus.
This novel artificial molecule may represent an improved platform for the development of future applications in DMD treatment.
KeywordsDMD Dystrophin Utrophin Zinc finger Artificial transcription factor Activation domain Che-1/AATF
Duchenne Muscular Dystrophy (DMD) is the most common X-linked degenerative muscle disease. The diagnostic marker for DMD is the absence of the cytoskeletal protein dystrophin, which plays a major structural role in muscle by providing stability to the sarcolemma during muscle contractions. DMD still lacks an effective cure; although different therapeutic strategies for DMD are currently being explored[2–6], a variety of drawbacks has significantly delayed their clinical translation. A promising approach for DMD therapy is based on increasing the levels of utrophin, a cytoskeletal protein that is similar to dystrophin and is able to compensate for its absence. Dystrophin and utrophin display a high degree of homology, and both bind members of the dystrophin-associated protein complex (DAPC). In adult muscle, utrophin is localised preferentially at the neuromuscular junction (NMJ) and myotendinous junctions, while dystrophin is localised along the entire length of the sarcolemma. However, utrophin is also found along the sarcolemma in developing muscle, in regenerating muscle after injury and in mdx (dystrophin-deficient) skeletal muscle. In DMD patients, utrophin is often upregulated, but this upregulation is not sufficient to prevent the progression of muscular dystrophy. Although the adenoviral delivery of utrophin in the mouse model of DMD (mdx) and in the dystrophin-deficient dog ameliorates the pathology, the huge size of the utrophin gene is a critical disadvantage. Therefore, studies developing natural or synthetic small molecules that upregulate utrophin could accelerate the clinical translation process[11–17]. To obtain upregulation of utrophin, we have engineered artificial zinc finger-based transcription factors that are capable of binding and activating transcription from promoter “A” of both the human and mouse utrophin genes[18–23]. Zinc finger domains have been shown to be optimal building blocks for generating artificial transcription factors due to their versatility and modularity[24, 25]. In particular, a “recognition code” that relates the amino acids of a single zinc finger to its associated DNA target has been utilised as a guide for the DNA binding design[24–30]. Changes in the key amino acid positions (−1, +3 and +6) of the zinc finger alpha-helix alter the DNA-binding specificity of a zinc finger and enable it to bind the programmed DNA-binding site. We generated transgenic mice that specifically over-express an artificial three zinc finger protein in the muscle, named “Jazz”, which is able to specifically upregulate the utrophin gene. Crossing the Jazz transgenic mice with the mouse Duchenne muscular dystrophy mouse model “mdx” results in a strong amelioration of the dystrophic phenotype[22, 23]. In a continued attempt to improve the artificial transcription factor’s DNA-binding affinity/specificity, we engineered “UtroUp” that recognises a longer DNA target sequence than its prototype gene Jazz. UtroUp has been designed to target the eighteen-base-pair DNA sequence present in both human and mouse utrophin gene promoters “A”. This target sequence is unique and conserved in both genomes. Here, we show that the artificial transcription factor UtroUp, due to its six zinc finger tandem motif, and its low dissociation constant (Kd) possesses a strong affinity/specificity toward its DNA-binding site. UtroUp, coupled with its activation domain that was derived from the adaptor protein Che-1/AATF, efficiently accesses the active chromatin in the utrophin promoter locus and strongly activates utrophin transcription. This novel artificial molecule represents an improved platform for the development of future applications in the DMD gene therapy field.
The synthesis of the UtroUp gene was performed by GenScript (New Jersey, USA). The company provided us with the gene cloned into the pUC57 vector at the EcoRI and XhoI restriction sites. We sub-cloned the synthetic gene into pGEX-4T1 bacterial expression vector (GST-UtroUp) and into the pRK5/myc mammalian expression vector, under the control of cytomegalovirus regulatory regions, in fusion with the CJ7 trans-activation domain (CJ7-UtroUp). CJ7-Jazz molecule was cloned in the same pRK5/myc mammalian expression vector. Note that here, we simply referred to CJ7 as the activation domain, which is one hundred amino acids long and strictly derived from the human Che-1/AATF protein, and not as the entire fusion protein that was described in our previous publication.
Expression of the bacterial recombinant fusion proteins
The recombinant GST-UtroUp protein and the recombinant GST-Jazz protein were expressed in BL21(DE3) host bacteria by an IPTG induction and were purified using glutathione Sepharose 4B beads as previously described. The GST fusion proteins were separated by SDS-PAGE electrophoresis, visualized by Coomassie blue staining and quantified.
Electrophoretic mobility shift assay (EMSA) and dissociation constant
The EMSA were performed as previously described. The oligonucleotide probes containing one copy of the WT UtroUp DNA target (underlined) were the following: forward primer 5′-A TTA AGC CGG GCT GCT GCG GGC TGG GAG TAT GAT CC-3′ and reverse primer 5′-GG ATC ATA CTC CCA GCC CGC AGC AGC CCG GCT TAA T-3′. The mutagenized oligonucleotide probes were: “Mutated” UtroUp forward primer 5′-A TTA AGC CGG GCT GCT taa taa TGG GAG TAT GAT CC-3′ and reverse primer 5′-GG ATC ATA CTC CCA tta tta AGC AGC CCG GCT TAA T-3′; “Scrambled” UtroUp forward primer 5′-A TTA AGC CGG ACA ACC ATC GAT GTC CGT TAT GAT CC-3′ and reverse primer 5′-GG ATC ATA ACG GAC ATC GAT GGT TGT CCG GCT TAA T-3′. The oligos used in the EMSA were labelled by a T4 polynucleotide kinase. The binding buffer that was used contained 20 mM Hepes pH 7.5, 0.5 mM DTT, 100 mM NaCl, 50 μM ZnCl2, 50 μg/ml BSA, 100 ng of poly (dI-dC), 0.05% NP40 and 5% glycerol. Percentage of both inputs: WT and the “6 base pair” mutated DNA target probes shifted at each protein concentration were quantified using ImageJ analysis software and plotted in a graph. To measure and compare the apparent dissociation constant (Kd) of the GST-UtroUp and GST-Jazz complexed with their DNA-target, we performed a series of EMSA with increasing amount of GST-UtroUp (1, 2, 3, 4, 8, 12, 30, 60 nM and 90 nM) or increasing amount of GST-Jazz (5, 8, 16, 32, 64 and 96 nM) incubated with a constant amount of labelled UtroUp-Jazz DNA target, following the procedure previously described. Briefly, the radioactive signals were visualised by autoradiography and quantified by the ImageQuant software (Molecular Dynamics, Sunnyvale, CA) and the data were analysed with the KaleidaGraph programme (Abelbeck Software, Reading, PA). The Kd values were determined as an average of data as mean ± S.D. of five separate experiments.
Cell lines, transient transfections and reporter gene assay
The human HeLa cell line was grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco Corporation, Grand Island, NY, USA) supplemented with 10% foetal calf serum. Transient transfection experiments in the HeLa cell line were carried out using Lipofectin and PLUS reagents (Life Technologies Corporation, Carlsbad, Ca.) according to the manufacturer’s instructions. Cell extracts were prepared and assayed for luciferase (LUC) activity according to the manufacturer’s instructions (Promega, Madison, WI, USA) using a Berthold LB9506 luminometer. The total protein in the extracts was quantified using a Bradford assay, and the LUC activity from equal amounts of protein was determined and normalised for beta-galactosidase activity.
Chromatin immunoprecipitation assay (ChIP)
Chromatin Immunoprecipitation was performed using a ChIP assay kit according to the manufacturer’s instructions (Upstate Biotechnology, Charlottesville, VA, USA). Approximately 8 million cells were cross-linked with 1% formaldehyde for 10 min at 37°C and then lysed. The cell lysate was sonicated on ice, which resulted in DNA fragments of approximately 500 bp in length. Equal amounts of chromatin from each sample were immunoprecipitated over night with either an anti-acetylated-Histone H3 rabbit polyclonal antibody (Upstate Biotechnology) or anti-myc 9E10 monoclonal antibody. DNA representing 0.005% of the sonicated chromatin solution (input) and 10% of the immunoprecipitated sonicated chromatin solution were amplified using the following primers: human utrophin promoter region primers forward 5′-CGGCACGCACGGTTCACTCTGGAGCGC-3′ and reverse 5′-CAGCAACTTTGTTCCGGAAGATCAGCC-3′; human thymidine kinase promoter region–specific primers forward 5′-GCCCCTTTAAACTTGGTGGGCGG-3′ and reverse 5′-TTGCGCCTCCGGGAAGTTCACG-3′; human dystrophin promoter region–specific primers forward 5′-GTGTTTTAAGAATTGGCACCAG-3′ and reverse 5′-AGTCTGAATAAGAGAAGCAGCA -3′; human chromosome 16 region–specific primers forward 5′-AGGACCACTCGCTGGGTAAGCA-3′ and reverse 5′-CGCGGAGGGTGACATGGGGT-3′; human chromosome 17 region–specific primers forward 5′-ACCTGTGTGTGGGTGGTGAGA-3′ and reverse 5′-CTGTAGGGCCCCAGGCACCAT-3′. The DNA sequence of the potential off-target site present in chromosome 16 is: 5′-GCTGCTGgGGGCTGGGgc-3′, with 15/18 matches with UtroUp target sequence. The DNA sequence of the potential off-target present in chromosome 17 is: 5′-GCTGCTGCGGGCTGGGga-3′, with 16/18 matches with UtroUp Target sequence. The PCR conditions were the following: 30 cycles at 95°C for 45 s, 62°C for 30 s, 72°C for 30 s and a final extension at 72°C for 5 min. Quantification of each chromatin immunoprecipitation (ChIP) experiment was performed using ImageJ software and plotted in a graph.
RNA extraction, reverse transcriptase reaction and real-time PCR
Total RNA was extracted from HeLa cells expressing either CJ7-UtroUp or the negative control pRK5 using the TRIzol reagent according to the manufacturer’s instructions (Life Technologies). Two μg of RNA was reverse transcribed using oligo (dT)12–18 primers and Superscript II (Life Technologies) in a final volume of 20 μl at 42°C for 50 min. A real-time PCR assay was performed in a 96-well format using the ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). Primers and probes for utrophin (target gene) and for GAPDH (housekeeping gene) were purchased as TaqMan Gene Expression Assays (Applied Biosystems). PCR mixtures containing the cDNA template, the TaqMan Universal PCR master mix (Applied Biosystems) and the primers/probes in a final volume of 25 μl were analysed in triplicate using the following conditions: an incubation at 50°C for 2 min, denaturing at 95°C for 10 min and then 40 cycles of the amplification step at 95°C for 15 s and 60°C for 1 min. For each gene amplification, a standard curve was generated using serial dilutions (200, 40, 8, 1.6 and 0.32 ng) of cDNA from the HeLa cells (negative control). The results were analysed using the Applied Biosystems analysis software. The data are expressed as the ratio between utrophin and GAPDH mRNA expression.
Proteins were extracted from HeLa cells using the following lysis buffer (50 mM Tris pH 7.4, 50 mM EDTA, 250 mM NaCl, 50 mM NaF, 0.1% Triton X-100, 0.1 mM NaVO4 and 0.5% glycerol) containing protease inhibitors. Equal amounts of total protein (30–40 μg) were separated by SDS-PAGE electrophoresis, transferred to membranes and immunoblotted with the following antibodies: mouse monoclonal anti-myc 9E10, mouse monoclonal anti-alpha tubulin (Sigma Corporation, St. Louis, Missouri, USA), mouse monoclonal anti-utrophin (NOVOCASTRA, Newcastle, UK), rabbit polyclonal anti-laminin (Sigma Corporation, St. Louis, Missouri, USA), mouse monoclonal anti-beta-Actin (Sigma Corporation, St. Louis, Missouri, USA), rabbit polyclonal anti-Sp1 (Santa Cruz, Santa Cruz, CA). Densitometric analysis of utrophin protein level was performed using ImageJ analysis software.
Results and discussion
The synthetic transcription factor Jazz, with its three zinc finger domains, binds a 9-base-pair DNA sequence. Bagly, the first Jazz variation, has four zinc finger domains and binds a 12-base-pair DNA sequence. As described here, UtroUp, with its six zinc finger domains, binds an 18-base-pair DNA sequence. Notably, a 9-base-pair DNA sequence is statistically present in the human genome (≅ 3.5 × 109 bp) approximately 1.3 × 104 times, a 12-base-pair sequence is present 210 times and an 18-base-pair sequence is present only once. Moreover, when fused with our strong activation domain CJ7, UtroUp specifically and efficiently upregulates the utrophin gene expression.
In an attempt to improve the efficacy of our artificial transcription factors, engineered to reprogram the expression of the utrophin gene, we focused on tuning both DNA-binding affinity/specificity and transcriptional performances. Our novel artificial transcription factor, CJ7-UtroUp, represents a good compromise between small size, high DNA target affinity/specificity and efficient transcriptional activation. The small size makes UtroUp particular suitable for viral vector delivery and the possibility of targeting a unique site in the genome extremely reduces off-target effects on global transcription. UtroUp and its upcoming variations may represent an important platform for the development of future applications in the DMD gene therapy field. Moreover, human myogenic cell lines and mouse models expressing UtroUp can offer an unique model system for screening novel compounds that are able to upregulate the utrophin gene for DMD treatment.
Importantly, our results contribute to demonstrate that artificial zinc finger transcription factors may be a class of therapeutic reagents for treatment of crucial inherited diseases.
Duchenne Muscular Dystrophy
Dystrophin-associated protein complex
Electrophoretic mobility shift assays
Basic Local Alignment Search Tool.
This manuscript was edited for proper English language by American Journal Experts; Certificate Verification Key: C219-9C5A-B8C2-8EAF-792C. We thank Dr. R. G. Ruscitti for her valuable assistance. This work was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC) project IG 2009, by the Telethon project GGP10094, by the AFM project grant N° 14353 and PRIN-2008-81213. The UtroUp gene is the object of the following patents: CNR European patent n° 2193200 and CNR Italian patent n° 1868 RM 2007 A00523.
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