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; Jörg Bungert

doi:10.1073/pnas.1207677109

Edited by Mark Groudine, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved September 19, 2012 (received for review May 6, 2012)

Abstract

Gene expression is primarily regulated by cis-regulatory DNA elements and trans-interacting proteins. Transcription factors bind in a DNA sequence–specific manner and recruit activities that modulate the association and activity of transcription complexes at specific genes. Often, transcription factors belong to families of related proteins that interact with similar DNA sequences. Furthermore, genes are regulated by multiple, sometimes redundant, cis-regulatory elements. Thus, the analysis of the role of a specific DNA regulatory sequence and the interacting proteins in the context of intact cells is challenging. In this study, we designed and functionally characterized an artificial DNA-binding domain that neutralizes the function of a cis-regulatory DNA element associated with adult β-globin gene expression. The zinc finger DNA-binding domain (ZF-DBD), comprising six ZFs, interacted specifically with a CACCC site located 90 bp upstream of the transcription start site (–90 β-ZF-DBD), which is normally occupied by KLF1, a major regulator of adult β-globin gene expression. Stable expression of the –90 β-ZF-DBD in mouse erythroleukemia cells reduced the binding of KLF1 with the β-globin gene, but not with locus control region element HS2, and led to reduced transcription. Transient transgenic embryos expressing the –90 β-ZF-DBD developed normally but revealed reduced expression of the adult β-globin gene. These results demonstrate that artificial DNA-binding proteins lacking effector domains are useful tools for studying and modulating the function of cis-regulatory DNA elements.

The modulation and functional analysis of gene regulation in vivo is challenging because of redundancies in transcription factors and cis-regulatory DNA elements (1 ⇓⇓–4). Transgenic or reporter gene assays are powerful but limited by potential position effects (5). Genetic manipulation of cis-regulatory DNA elements is technically demanding and time-consuming (6). The zinc finger (ZF) domain, the most commonly found DNA-binding domain in eukaryotic transcription factors (7), is characterized by a DNA-binding α-helix, known as the α-helix reading head, which is stabilized by an adjacent finger-like structure in which histidine or cysteine residues coordinate a zinc atom (8 ⇓–10). The mode of DNA binding by ZF proteins is very well understood, and this knowledge led to the development of artificial proteins containing a defined ZF DNA-binding domain (ZF-DBD) that interacts with a specific sequence of interest (10, 11). Each α-helix reading head of a ZF-DBD recognizes three to four specific DNA base pairs, 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 in tandem provides the recognition of extended asymmetrical DNA sequences. Previous studies linked artificial ZF-DBDs to nucleases or to effector domains that enhance or repress transcription (11 ⇓–13). Most of the artificial ZF proteins studied to date appear to interact with desired target sequences with high specificity; however, off-target binding has been detected, which is a concern if ZF-DBDs are expressed with effector domains (14).

The purpose of our study was to examine the applicability of using a ZF-DBD to assess the contribution of a cis-regulatory DNA element to gene expression and transcription factor binding in vivo. We designed and expressed a ZF-DBD, harboring six ZFs, that specifically interacts with a critical cis-regulatory element associated with adult βmin-globin gene expression—the −90 CACCC box located about 90 bp upstream of the βmin-globin transcription start site (15). We termed this artificial binding protein −90 β-ZF-DBD. The CACCC site interacts with transcription factor KLF1, a ZF protein important for red blood cell function (16 ⇓–18). Mutation of the β-promoter–associated CACCC site in the human population has been associated with β-thalassemia (19). The −90 β-ZF-DBD, when expressed in erythroid cells, displaced KLF1 from the βmin-globin promoter and reduced expression of the βmin-globin gene. Other KLF1-regulated genes, such as the dematin and β-spectrin genes, were not affected by the −90 β-ZF-DBD. Synthetic ZF-DBDs may provide a tool for the treatment of sickle cell anemia and other hemoglobinopathies. Specifically, a combination of ZF-DBDs could be designed that reduce expression of mutant β-globin and enhance expression of therapeutic γ-globin genes.

Results

We designed a ZF-DBD comprising six ZFs that would bind to 18 bp of DNA containing the βmin-globin promoter–associated −90 CACCC box (−90 β-ZF-DBD, Fig. 1A). Statistically, an 18-bp sequence occurs once per mammalian genome. There are two CACCC sites in the βmin-globin gene promoter, and the −90 β-ZF-DBD is predicted to block both of them. Specific amino acid–nucleic acid recognition sequences were derived using a web-based program developed by Barbas and colleagues (20, 21). The ZF-DBD protein was generated using a modified protocol from Cathomen et al. (22), as outlined in Fig. 1B. The −90 β-ZF-DBD coding segment was cloned into the pT7-Flag2 vector and expressed in and purified from Escherichia coli (Fig. 1 C and D). The −90 β-ZF-DBD protein migrated at an expected size of 24 kDa. Electrophoretic mobility shift assays (EMSAs) demonstrated that the −90 β-ZF-DBD specifically interacted with an oligonucleotide containing the 18-bp target WT sequence harboring the −90 CACCC site, but not with a mutant oligonucleotide (Fig. 1E). An excess of unlabeled WT oligonucleotides efficiently competed for the binding, whereas mutant oligonucleotides did not perturb binding. The addition of a Flag antibody to the binding reaction eliminated formation of the protein-DNA complex, indicating that this interaction was specific. These data demonstrate that the −90 β-ZF-DBD interacts with the target 18-bp sequence encompassing the −90 CACCC box in a sequence-specific manner.

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 schematic of the −90 β-ZF-DBD. The coding DNA fragment 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 reverse primers and cloned into prokaryotic or eukaryotic expression vectors. (C) Immunoblot with a Flag-specific antibody. The −90 β-ZF-DBD migrated 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).

For studies in eukaryotic cells, the coding sequence for the −90 β-ZF-DBD was cloned into the retroviral pMSCV (murine stem cell virus)-neo plasmid and modified to include a nuclear localization sequence (NLS). After packaging, viruses either harboring the vector encoding the −90 β-ZF-DBD or an empty vector were used to transduce mouse erythroleukemia (MEL) cells and single-cell clones were generated from transduced MEL population pools. MEL cells are erythroid progenitor cells that express low levels of the adult α- and β-globin genes and can be chemically induced to differentiate by DMSO, leading to high-level globin gene expression (23). Immunofluorescence microscopic analysis in induced MEL cells using an antibody against the backbone of the ZF-DBD revealed that the −90 β-ZF-DBD localized to the nucleus (Fig. 2 A-F). We next fractionated the MEL cells into cytosolic and nuclear compartments and performed an immunoblot analysis (Fig. 2G). Brg1, a nuclear chromatin regulatory protein, and α-tubulin, a predominantly cytoplasmic protein, were used as controls to indicate complete cellular fractionation. The −90 β-ZF-DBD was only present in the nuclear fraction in induced MEL cells.

Fig. 2.

The −90 β-ZF-DBD localizes to the nucleus in DMSO-induced MEL cells. (A–F) MEL cells were stably transduced, fixed, stained, and subjected to immunofluorescence microscopy. DAPI stains chromatin in the nucleus (blue) and the −90 β-ZF-DBD was visualized using antisera against the ZF backbone (green). The bottom panel represents the merge of the two previous panels. (G) Induced stable MEL cells were fractionated into cytoplasmic (C) and nuclear (N) fractions that were subjected to immunoblot analyses using antibodies specific for Brg1 and α-tubulin as indicated.

We next analyzed expression of specific genes in single-cell clones from MEL cells that stably expressed either the −90 β-ZF-DBD or contained empty vector. First, we stained induced MEL cells with benzidine, which stains hemoglobin. There was a marked reduction in benzidine-positive cells that stably expressed the −90 β-ZF-DBD compared with control cells (Fig. 3 A and B). Consistent with these results, MEL cells expressing the −90 β-ZF-DBD revealed a reduction of βmin-globin gene expression compared with control cells harboring the vector, suggesting that the ZF-DBD was targeted specifically to the promoter of this gene (Fig. 3C). We evaluated the relative expression levels of the βmin- and βmaj-globin genes in MEL cells and found that both genes are expressed at equivalent levels and that expression of both genes is reduced in cells expressing the −90 β-ZF-DBD (Figs. S1 and S2). The reduction in βmaj-globin gene expression could be due to cooperative interactions and coregulation between the adult promoters, as has recently been observed for many coregulated genes in mammalian cells (24).

Fig. 3.

Reduction in βmin-globin gene transcription in MEL cells expressing the −90 β-ZF-DBD. (A) Benzidine staining of MEL cells expressing the −90 β-ZF-DBD or harboring the empty vector. (B) Percent benzidine-positive MEL cells expressing the −90 β-ZF-DBD or harboring the empty vector. RT-qPCR analysis of βmin-globin (C), β-spectrin (E), and dematin (E) expression in MEL cells expressing the −90 β-ZF-DBD or harboring the empty vector. Data presented is the average of three independent experiments ± SEM. IN, induced Mel cells; UN, uninduced Mel cells.

Dematin (also known as band 4.9) and β-spectrin are genes both induced on erythroid differentiation and positively regulated by KLF1 (25). Importantly, the KLF1-binding sites in the β-spectrin and dematin promoter regions are almost identical to the −90 CACCC site in the βmin-globin gene promoter, but the flanking sequences are unique for each of the three genes (Fig. 4D). RT-quantitative (q)PCR analysis revealed that expression of β-spectrin (Fig. 3D) and dematin (Fig. 3E) was increased on DMSO-induced differentiation of MEL cells harboring the empty vector. However, there were no differences in expression levels of β-spectrin and dematin in induced and uninduced MEL cells expressing the −90 β-ZF-DBD compared with vehicle-control cells. This demonstrates that the −90 β-ZF-DBD specifically reduced expression of the adult β-globin genes but did not affect expression of other KLF1-regulated genes in erythroid cells.

Fig. 4.

Displacement of KLF1 and RNA polymerase II from the βmin-globin gene promoter by the −90 β-ZF-DBD in MEL cells. MEL cells expressing the −90 β-ZF-DBD or harboring the empty vector were induced to differentiate by 2% DMSO for 3 d. The cells were cross-linked and subjected to ChIP analysis using antibodies specific for the ZF-DBD, KLF1, or RNA polymerase II (Pol II). ChIP analysis of the interaction of the −90 β-ZF-DBD (A) or KLF1 (B) with LCR element HS2, the βmin-globin gene promoter, and the dematin promoter, as indicated. (C) ChIP analysis of the interaction of RNA polymerase II (Pol II) with LCR element HS2 and the βmin-globin gene promoter. Data presented are the average of two independent ChIP experiments with PCRs run in triplicate ± the SEM. (D) Four sequences derived from either the dematin or beta-spectrin promoter harboring potential CAC boxes (underlined) were aligned with the −90 β-ZF-DBD target site (18-bp sequence shown above the line). Sequence homology is indicated by the number in parentheses to the right of the sequence. A (+10) therefore denotes a 10 of 18 bp match to the target sequence of the −90 β-ZF-DBD target site.

Next, using ChIP, we analyzed the occupancy of the −90 β-ZF-DBD at the βmin-globin gene promoter, at the promoter of the dematin gene, and at locus control region (LCR) element HS2, all of which contain KLF1-binding sites (Fig. 4A). The −90 β-ZF-DBD was specifically associated with the βmin-globin promoter but not with LCR element HS2. Furthermore, expression of the −90 β-ZF-DBD drastically reduced the binding of KLF1 at the βmin-globin promoter but not at LCR HS2 (Fig. 4B). We also observed binding of the −90 β-ZF-DBD at the dematin promoter, but it was not as pronounced as the interaction with the βmin-globin promoter region. Moreover, KLF1 binding to this region was not affected by expression of the −90 β-ZF-DBD.

Previous studies have shown that Pol II interacts with LCR HS2 and with the adult β-globin gene promoter and that the binding increases on induction of MEL cell differentiation (26 ⇓⇓–29). As shown in Fig. 4C, Pol II bound efficiently to the βmin-globin promoter and to a lesser extent to LCR element HS2 in induced MEL cells harboring the empty vector. The interaction of Pol II was reduced at the βmin-globin promoter in MEL cells expressing the −90 β-ZF-DBD. We also observed a reduction in Pol II binding at LCR element HS2, which could indicate a reduction in interaction frequencies between the LCR and the β-globin promoter in cells expressing the −90 β-ZF-DBD. The data demonstrate that occupancy of the −90 β-ZF-DBD at the β-min promoter prevents KLF1 from binding to the −90 CACCC site, thereby inhibiting the recruitment of transcription complexes.

To test the efficacy of the −90 β-ZF-DBD in vivo, we generated and analyzed transient transgenic embryos (Fig. 5). The switch from the embryonic to the fetal/adult globin expression program in mice occurs around day 11.5 dpc and embryos were thus analyzed at 13.5 days post coitum (dpc). Fig. 5A shows a representative transgenic −90 β-ZF-DBD-expressing embryo and a nontransgenic littermate. Transgenic embryos were more pale compared with nontransgenic littermates but did not reveal gross abnormalities, indicating that expression of the −90 β-ZF-DBD did not affect development and differentiation. Two transient transgenic embryos expressing the −90 β-ZF-DBD revealed reduced expression of adult βmin-globin gene expression (Fig. 5B). Porcu et al. (30) previously demonstrated that βmin-globin gene expression predominates in the early fetal liver and that it is preferentially affected by a lack of KLF1 (30). The data illustrate that synthetic ZF-DBDs can be used to analyze and modulate the function of cis-regulatory elements in vivo.

Fig. 5.

Transient transgenic mouse embryos expressing the −90 β-ZF-DBD exhibit reduced expression of the βmin-globin gene. A plasmid harboring the −90 β-ZF-DBD coding region under control of the CMV promoter/enhancer was linearized and used to generate transgenic embryos that were harvested at 13.5 dpc. Transgenic embryos were identified by PCR. (A) Images of 13.5 dpc WT and −90 β-ZF-DBD transgenic embryos. (B) RT-qPCR analysis of βmin-globin gene expression in two WT (4 and 16) and two −90 β-ZF-DBD (1 and 17) transgenic embryos. Expression levels in WT were set at 1. Data represent two independent RNA isolations with RT-qPCR performed in triplicate ± SEM. (C) Model illustrating ZF-DBD–mediated disruption of KLF1 binding and Pol II recruitment at the adult βmin-globin gene promoter.

Discussion

Artificial DNA-binding domains represent promising tools for studying and modulating gene regulation in vivo. As shown in this study, ZF-DBDs without effector domains can be used to alter expression of genes by preventing transcription factors from accessing DNA at specific sites. These synthetic proteins can be used to assess the function of a cis-regulatory DNA element and the identity of interacting proteins in vivo. Several different strategies have been developed to generate artificial binding domains; the majority are based on DNA-binding domains found in either ZF or transcription activator-like effector (TALE) proteins (11, 31, 32). TALE proteins, identified in prokaryotic plant pathogens, are modular in nature and like ZF proteins can be combined to recognize extended DNA sequences. The advantage of ZF proteins over TALE proteins is that they are relatively small, minimizing effects resulting from potential protein interactions and perhaps in the long term allowing delivery by protein transduction (33, 34).

Much is known about the configuration of amino acids in the ZF α-helix reading head that interact with specific DNA base pair triplets, and this led to the development of algorithms that have been used to design artificial ZF proteins that bind to specific DNA sequences (20). Klug and colleagues first described the use of a synthetic 3-ZF protein that blocked the activating sequence that arose from a chromosomal translocation (10).

In this study, we used the Zinc Finger Tools web site (21) to design a ZF-DBD targeting the −90 CACCC site in the murine adult βmin-globin gene promoter. The −90 β-ZF-DBD interacted with the βmin-globin CACCC box and efficiently blocked association of KLF1 with this site. As a consequence, βmin-globin gene expression was reduced. In previous studies, Bieker and colleagues overexpressed the KLF1 DNA-binding domain in erythroid cells, which led to reduced βmaj-globin expression levels (35). However, in contrast to the −90 β-ZF-DBD, the KLF1-binding domain interacts with many gene regulatory elements in erythroid cells. The −90 β-ZF-DBD did not interact with LCR element HS2, which also contains a KLF1-binding site. We detected a weak interaction of the −90 β-ZF-DBD with the dematin promoter in induced MEL cells. However, this binding event did not affect binding of KLF1 or dematin expression, strongly suggesting that the −90 β-ZF-DBD failed to interfere with KLF1 function at this particular site. We also observed a reduction in expression of the βmaj-globin gene in cells expressing the −90 β-ZF-DBD (Figs. S1 and S2). The reason for this is not known but could indicate that the adult β-globin promoters cooperatively regulate each other. This would be consistent with a recent study demonstrating extensive promoter–promoter interactions and cross-regulation in mammalian cells (24). Alternatively, the artificial zinc finger protein could bind to both adult globin gene promoters due to close proximity.

A recent study by Lam et al. (36) demonstrated that the majority of modular artificial C2H2 ZF proteins bind DNA with high sequence specificity and preferentially associate with sites to which they were targeted. Although we did not perform a global analysis of the interaction of the −90 β-ZF-DBD in MEL cells, the analysis of selected sites that resemble the targeted site suggests that the −90 β-ZF-DBD preferentially associates with the βmin-globin gene promoter. This is further supported by our observation that transient transgenic embryos expressing the −90 β-ZF-DBD ubiquitously had reduced β-globin gene expression but did not reveal any other developmental abnormalities.

The study presented here demonstrates that ZF-DBDs without effector domains can be used to alter expression patterns of genes in vivo. These artificial proteins thus provide a tool for modulating and analyzing cis-regulatory DNA elements and may also impact therapeutic approaches aimed at altering expression of specific genes.

Methods

ZF Design and Plasmid Construction.

The six-fingered −90 β-ZF-DBD was designed using the Zinc Finger Tools website (20, 21). Detailed recognition helices and oligonucleotides used to assemble the ZF-DBD are listed in Tables S1 and S2. The ZF-DBD was constructed as described previously (22), with some modifications including introducing a Flag tag and using a viral approach for eukaryotic studies. The six-fingered ZF-DBD was split into two reactions generating DNA fragments that encode three-fingered ZF domains termed ZF 1–3 and ZF 4–6. Oligonucleotides coding for either the ZF constant backbone (37) or the variable oligonucleotides tailored to recognize the target sequence were hybridized, and gaps annealed in a phase I modular PCR assembly using a high-fidelity Pfu polymerase (Agilent). PCR was performed according to the manufacturer’s protocol (Agilent). The ZF fragments and a canonical linker were gel purified (Qiagen), digested with appropriate restriction enzymes (Table S2), and ligated in frame to generate the coding sequence of the six-fingered ZF protein. The ZF fragment was then PCR-amplified with Pfu polymerase and subcloned into the pT7-Flag2 vector (Sigma) using EcoRI/KpnI (NEB) restriction sites. For expression in MEL cells, the coding region of the −90 β-ZF-DBD was generated by PCR from pT7-Flag with modified oligonucleotides (Table S2) to incorporate an NLS and introduced into the pMSCV-neo vector (Clontech) at the HpaI site. The sequence of the −90 β-ZF-DBD coding region in the context of the expression vectors was verified by Sanger sequencing.

Expression and Purification of the ZF-DBD.

The pT7-Flag2 vector containing the −90 β-ZF-DBD was transformed into BL21-DE3 cells (Invitrogen). Cells were cultured in Luria broth medium supplemented with 100 μg/mL ampicillin, and expression of the −90 β-ZF-DBD was induced on addition of 1 mM IPTG (Sigma) and 100 μM ZnCl₂ (Sigma) following a 4-h incubation at 37 °C. Cells were then centrifuged at 1,900 × g at 4 °C for 20 min and pellets were resuspended in storage buffer as described elsewhere (22). Cells were lysed twice by French press, treated with 200 μg DNase I (Sigma), and centrifuged at 44,000 × g for 30 min at 4 °C. The supernatant was passed through a 0.2-μM filter and immunopurified using anti-Flag M2 magnetic beads (Sigma) according to the manufacturer’s protocol. Flag-tagged −90 β-ZF-DBD was eluted using 3× Flag peptide (Sigma) in 10 mM Tris (pH 8.0), 90 mM KCl, 100 μM ZnCl₂, 5 mM DTT, 0.1% (vol/vol) Triton X-100, and 30% (vol/vol) glycerol and stored at −80 °C. Protein concentrations were determined by the Bradford method, and protein purity was assessed by separating the eluted proteins on a 4–15% (wt/vol) SDS/PAGE (Bio-Rad) and staining with Coomassie Blue (Bio-Rad).

EMSA.

EMSA was performed using the Lightshift Chemiluminescent Kit (Thermo Scientific) according to the manufacturer’s protocol. Double-stranded oligonucleotides representing either the murine −90 CACCC box (5′-GGATCCGAATTCCTGCAGGGTAACACCCTGGCATTGGCCAA-3′) or a mutant sequence (5′-GGATCCGAATTCAGTACTTTGCCTGTTTCAATGCCTTAACC-3′) were annealed in 250 mM Tris-HCl, pH 7.7, to their complement antisense sequences by heating to 95 °C and cooling in 0.5 °C increments to 4 °C for several hours. Oligonucleotides were digested with BamHI (NEB), purified, and biotinylated using bio-dATP and bio-dCTP (Invitrogen). A total of 1 μg of purified recombinant −90 β-ZF-DBD protein was incubated with 2 ng of either biotinylated WT or mutant oligonucleotides. Binding was challenged with 1 μg of excess unlabeled WT or mutant double-stranded oligonucleotides or with 1 μg of mouse M2 anti-Flag antibody (Sigma) as indicated.

Immunoblot Analysis.

Proteins were isolated from MEL cells as described by Leach et al. (38), with modifications to include a mechanical lysis step with a microgrinder (Radnoti) before centrifugation at 20,800 × g for 15 min at 4 °C. A total of 10–20 μg protein was loaded onto a 4–15% (wt/vol) TGX Tris-HCl gel (Bio-Rad), separated by SDS/PAGE, and transferred to PVDF membranes. Proteins were probed with the following antibodies: mouse anti-Flag (Sigma, F3565), rabbit anti-ZF sera (gift from Carlos Barbas, Scripps Research Institute, La Jolla, CA), mouse anti-BRG1 (gift from David Reisman, University of Florida, Gainesville, FL), and mouse anti-Tubulin (Santa Cruz). Anti-mouse and anti-rabbit secondary antibodies were purchased from Santa Cruz. Proteins were detected by ECL reagent (Millipore) and visualized on X-ray film (Kodak). Compartmentalization immunoblot was performed using the NE-PER kit (Thermo Scientific) according to the manufacturer’s instruction, and isolated proteins were analyzed as described previously.

Immunofluorescence Microscopy.

A total of 1.0 × 10⁶ induced MEL cells were plated on poly-lysine (Sigma) coated plates and cultured overnight at 37 °C with 5% CO₂ before being fixed with 4% (wt/vol) paraformaldehyde (Sigma) for 10 min. Cells were rinsed thoroughly with PBS and permeabilized with 0.5% Triton X-100 for 20 min, followed by a blocking step with 3% (wt/vol) BSA (Sigma). Cells were probed with a ZF-specific antibody, washed with 4% (vol/vol) Tween, and incubated with FITC-conjugated secondary antibody (sc-2777, Santa Cruz). Cells were washed again in 4% (vol/vol) Tween before being placed on slides with mounting media containing DAPI (Vecta Shield). Fluorescence was visualized using a fluorescence microscope (Leica).

Cell Culture and Transfections/Transductions.

MEL and Phoenix A cells were cultured in DMEM (Cellgro) supplemented with 10% (vol/vol) FBS and 1% (vol/vol) penicillin/streptomycin (Cellgro). Cells were grown at 37 °C with 5% CO₂ and maintained at a density of 2.0 × 10⁵ cells/mL. Induction of erythroid differentiation was achieved by the addition of 2% (wt/vol) DMSO to the media following a 72-h incubation period. Retroviral-mediated creation of stable MEL cell lines was achieved by transfecting the packaging cell line Phoenix A with pMSCV-neo-90-β-ZF-DBD or empty vector via Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. After 48 h, the supernatant containing live replication-deficient virus was harvested and centrifuged at 913 × g for 5 min. The supernatant was treated with 2 μg/mL polybrene (Sigma) and added to MEL cells. Cells were incubated for 48 h before the addition of 100 μg/mL geneticin (Cellgro) for selection.

RNA Isolation and Analysis.

RNA was isolated from MEL cells and mouse fetal liver using the RNeasy kit (Qiagen) according to the manufacturer’s instructions. The RNA was reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad). The cDNA analysis was performed as previously described in Liang et al. (39). The following primers were used to amplify cDNA: mouse β-minor, 5′-TACGTTTGCTTCTGATTCT-3′ (upstream [US]) and 5′-GCAGAGGCAGAGGATAGGTC-3′ (downstream [DS]); mouse dematin (band 4.9), 5′-ACCGCATGAGGCTTGAGAGG-3′ (US) and 5′-TCTTCTTAAGTTCGTTCCGCTTCC-3′ (DS); mouse β-spectrin, 5′-GCTTAAGGAACGCCAGACTCCAG-3′ (US) and 5′-ATTTCTCCTGCTCGTCTTTGT-3′; and mouse β-actin, 5′-GTGGGCCGCTCTAGGCACCA-3′ (US) and 5′-TGGCCTTAGGGTGCAGGGGG-3′ (DS). All RT-qPCR data were normalized to mouse β-actin expression levels.

ChIP.

ChIP assays were performed as described previously (38, 39) with the following modifications. A minimum of 2.0 × 10⁷ cells were isolated, cross-linked, and sonicated to produce 200–500 bp chromatin fragments. Lysates were precleared with mouse IgG (Santa Cruz; sc-2025) for 2 h at 4 °C with gentle rotation following a subsequent preclearing step with protein A Sepharose beads (GE Healthcare; CL-4B). Lysates were centrifuged at 1,700 × g for 10 min at 4 °C to pellet beads and the supernatant was incubated with specific antibodies overnight at 4 °C with gentle rotation. Sheared chromatin was incubated with 10 μL of ZF antibody. Alternatively, 2 μg of the following antibodies were used: anti-mouse RNA-Pol II (CTD45H8; Upstate Biotechnology, Inc.) and anti-mouse KLF1 (Abcam; 2483, and a gift from James Bieker, Mount Sinai Hospital, New York, NY). DNA was purified by phenol/chloroform/isoamyl alcohol and subsequent chloroform extractions and precipitated by adding 2.5× volume of 100% (vol/vol) ethanol in the presence 10 μg glycogen (Invitrogen) overnight at −20 °C. DNA precipitates were washed in 70% ethanol, resuspended in 10 mM Tris-Cl pH 8.5, and analyzed by RT-qPCR as previously described (24). The following primer pairs were used: mouse βmin-promoter, 5′-GCCATAGCCACCCTGTGTAG-3′ (US), 5′GAGACAGCAGCCTTCTCAGA-3′ (DS); dematin (band 4.9) promoter, 5′-AATGACGGCAGGGGTCAG (US), 5′-CTTGGT CATGCCCATAGCTT-3′; and mouse HS2, 5′-TGCAGTACCACTGTCCAAGG-3′ (US), 5′-ATCTGGCCACACACCCTAAG-3′ (DS).

Benzidine Staining.

Benzidine (Sigma) stock solution containing 3% benzidine (wt/vol) and 90% (vol/vol) glacial acetic acid was used to prepare a fresh working solution comprising a ratio of 1:1:5 for benzidine stock solution, H₂O₂, and ddH₂O, respectively. Induced MEL cells (1.0 × 10⁶) were centrifuged at 282 × g for 5 min. The cell pellet was resuspended in 500 μL PBS, and 100 μL of the benzidine working solution was added. After a 5-min incubation period, cells were centrifuged at 282 × g for 5 min and resuspended in 500 μL PBS. Cells were counted under a light microscope (Van guard) using a hemocytometer (Bright-Line), and blue cells were calculated as a percentage of total cells. Cells were then imaged using the Scope Image 9.0 software (Life Scientz Bio-tech).

Generation of Transient Transgenic Mice.

The plasmid pMSCV-neo containing the −90 β-ZF-DBD was linearized with Acl I (NEB), and purified from an agarose gel using the Qiagen gel extraction kit, resuspended in injection buffer at a concentration of 2 ng/μL, and injected into FvB oocytes as described previously (40). After transfer to pseudopregnant recipients, embryos were taken at day 13.5 dpc and imaged with a stereomicroscope (Leica MZ16-FA, Houston, TX) and processed with Q-capture software (Q-imaging, BC, Canada). RNA and DNA was extracted and analyzed as described previously (40). The cDNA was analyzed by qPCR using primers specific for the βmin-globin gene as described previously and using primers specific for the −90 β-ZF-DBD cDNA: 5′-CTCGAGCCCGGGGAGAAAC-3′ (US) and 5′-TCACTTGTCATCGTCGTCCT-3′ (DS). The animal work was approved by the University of Florida Institutional Animal Care and Use Committee.

Acknowledgments

We thank our colleagues in the J.B. laboratory, especially Blanca Ostmark, and in the laboratory of Dr. Suming Huang (University of Florida) for technical assistance; Dr. Carlos Barbas (Scripps Research Institute) for ZF-DBD–specific antibodies, Dr. James Bieker (Mount Sinai Hospital) for KLF1-specific antibodies, and Dr. David Reisman (University of Florida) for Brg1-specific antibodies; Dr. Ross Hardison (Pennsylvania State University) for help with sequence annotations; and Dr. Brian Harfe (University of Florida) for microscope availability and Scope Image 9.0 software. This work was supported by National Institutes of Health Grants R01DK052356, R01DK052356-16S1, and R01DK083389 and by a University of Florida alumni graduate student fellowship (to J.J.B.). J.M. was an undergraduate student in the University of Florida Howard Hughes Medical Institute program.

Footnotes

↵¹To whom correspondence should be addressed. E-mail: jbungert{at}ufl.edu.

Author contributions: J.J.B. and J.B. designed research; J.J.B., J.M., and J.B. performed research; J.J.B. and J.B. analyzed data; and J.J.B. and J.B. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1207677109/-/DCSupplemental.

Freely available online through the PNAS open access option.

References

↵
1. Roeder RG
(2005) Transcriptional regulation and the role of diverse coactivators in animal cells. FEBS Lett 579(4):909–915.
OpenUrl CrossRef PubMed
↵
1. Orphanides G,
2. Reinberg D
(2002) A unified theory of gene expression. Cell 108(4):439–451.
OpenUrl CrossRef PubMed
↵
1. Reya T,
2. Grosschedl R
(1998) Transcriptional regulation of B-cell differentiation. Curr Opin Immunol 10(2):158–165.
OpenUrl CrossRef PubMed
↵
1. McLean C,
2. Bejerano G
(2008) Dispensability of mammalian DNA. Genome Res 18(11):1743–1751.
OpenUrl Abstract/FREE Full Text
↵
1. Arnone MI,
2. Dmochowski IJ,
3. Gache C
(2004) Using reporter genes to study cis-regulatory elements. Methods Cell Biol 74:621–652.
OpenUrl PubMed
↵
1. Capecchi MR
(2005) Gene targeting in mice: Functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet 6(6):507–512.
OpenUrl CrossRef PubMed
↵
1. Venter JC,
2. et al.
(2001) The sequence of the human genome. Science 291(5507):1304–1351.
OpenUrl Abstract/FREE Full Text
↵
1. Jamieson AC,
2. Kim SH,
3. Wells JA
(1994) In vitro selection of zinc fingers with altered DNA-binding specificity. Biochemistry 33(19):5689–5695.
OpenUrl CrossRef PubMed
↵
1. Choo Y,
2. Klug A
(1997) Physical basis of a protein-DNA recognition code. Curr Opin Struct Biol 7(1):117–125.
OpenUrl CrossRef PubMed
↵
1. Choo Y,
2. Sánchez-García I,
3. Klug A
(1994) In vivo repression by a site-specific DNA-binding protein designed against an oncogenic sequence. Nature 372(6507):642–645.
OpenUrl CrossRef PubMed
↵
1. Segal DJ,
2. Barbas CF 3rd.
(2001) Custom DNA-binding proteins come of age: Polydactyl zinc-finger proteins. Curr Opin Biotechnol 12(6):632–637.
OpenUrl CrossRef PubMed
↵
1. Corbi N,
2. Libri V,
3. Onori A,
4. Passananti C
(2004) Synthetic zinc finger peptides: Old and novel applications. Biochem Cell Biol 82(4):428–436.
OpenUrl CrossRef PubMed
↵
1. Urnov FD,
2. Rebar EJ,
3. Holmes MC,
4. Zhang HS,
5. Gregory PD
(2010) Genome editing with engineered zinc finger nucleases. Nat Rev Genet 11(9):636–646.
OpenUrl CrossRef PubMed
↵
1. Gupta A,
2. Meng X,
3. Zhu LJ,
4. Lawson ND,
5. Wolfe SA
(2011) Zinc finger protein-dependent and -independent contributions to the in vivo off-target activity of zinc finger nucleases. Nucleic Acids Res 39(1):381–392.
OpenUrl Abstract/FREE Full Text
↵
1. Stamatoyannopoulos G
(2005) Control of globin gene expression during development and erythroid differentiation. Exp Hematol 33(3):259–271.
OpenUrl CrossRef PubMed
↵
1. Siatecka M,
2. Bieker JJ
(2011) The multifunctional role of EKLF/KLF1 during erythropoiesis. Blood 118(8):2044–2054.
OpenUrl Abstract/FREE Full Text
↵
1. Perkins AC,
2. Sharpe AH,
3. Orkin SH
(1995) Lethal beta-thalassaemia in mice lacking the erythroid CACCC-transcription factor EKLF. Nature 375(6529):318–322.
OpenUrl CrossRef PubMed
↵
1. Nuez B,
2. Michalovich D,
3. Bygrave A,
4. Ploemacher R,
5. Grosveld F
(1995) Defective haematopoiesis in fetal liver resulting from inactivation of the EKLF gene. Nature 375(6529):316–318.
OpenUrl CrossRef PubMed
↵
1. Orkin SH,
2. Antonarakis SE,
3. Kazazian HH Jr.
(1984) Base substitution at position -88 in a beta-thalassemic globin gene. Further evidence for the role of distal promoter element ACACCC. J Biol Chem 259(14):8679–8681.
OpenUrl Abstract/FREE Full Text
↵
1. Dreier B,
2. et al.
(2005) Development of zinc finger domains for recognition of the 5′-CNN-3′ family DNA sequences and their use in the construction of artificial transcription factors. J Biol Chem 280(42):35588–35597.
OpenUrl Abstract/FREE Full Text
↵
1. Mandell J,
2. Barbas CF 3rd.
Zinc finger (ZF) tools, version 3.0. Available at www.scripps.edu/mb/barbas/zfdesign/zfdesignhome.php.
↵
1. Cathomen T,
2. Segal DJ,
3. Brondani V,
4. Müller-Lerch F
(2008) Generation and functional analysis of zinc finger nucleases. Methods Mol Biol 434:277–290.
OpenUrl CrossRef PubMed
↵
1. Gusella J,
2. Geller R,
3. Clarke B,
4. Weeks V,
5. Housman D
(1976) Commitment to erythroid differentiation by friend erythroleukemia cells: A stochastic analysis. Cell 9(2):221–229.
OpenUrl CrossRef PubMed
↵
1. Li G,
2. et al.
(2012) Extensive promoter-centered chromatin interactions provide a topological basis for transcription regulation. Cell 148(1-2):84–98.
OpenUrl CrossRef PubMed
↵
1. Hodge D,
2. et al.
(2006) A global role for EKLF in definitive and primitive erythropoiesis. Blood 107(8):3359–3370.
OpenUrl Abstract/FREE Full Text
↵
1. Zhou Z,
2. et al.
(2010) USF and NF-E2 cooperate to regulate the recruitment and activity of RNA polymerase II in the beta-globin gene locus. J Biol Chem 285(21):15894–15905.
OpenUrl Abstract/FREE Full Text
↵
1. Leach KM,
2. et al.
(2001) Reconstitution of human beta-globin locus control region hypersensitive sites in the absence of chromatin assembly. Mol Cell Biol 21(8):2629–2640.
OpenUrl Abstract/FREE Full Text
↵
1. Vieira KF,
2. et al.
(2004) Recruitment of transcription complexes to the beta-globin gene locus in vivo and in vitro. J Biol Chem 279(48):50350–50357.
OpenUrl Abstract/FREE Full Text
↵
1. Johnson KD,
2. et al.
(2003) Highly restricted localization of RNA polymerase II within a locus control region of a tissue-specific chromatin domain. Mol Cell Biol 23(18):6484–6493.
OpenUrl Abstract/FREE Full Text
↵
1. Porcu S,
2. Poddie D,
3. Melis M,
4. Cao A,
5. Ristaldi MS
(2005) β-Minor globin gene expression is preferentially reduced in EKLF Knock-Out mice. Gene 351:11–17.
OpenUrl CrossRef PubMed
↵
1. Zhang F,
2. et al.
(2011) Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat Biotechnol 29(2):149–153.
OpenUrl CrossRef PubMed
↵
1. Sanjana NE,
2. et al.
(2012) A transcription activator-like effector toolbox for genome engineering. Nat Protoc 7(1):171–192.
OpenUrl CrossRef PubMed
↵
1. Räägel H,
2. Säälik P,
3. Pooga M
(2010) Peptide-mediated protein delivery-which pathways are penetrable? Biochim Biophys Acta 1798(12):2240–2248.
OpenUrl PubMed
↵
1. Johnson RM,
2. Harrison SD,
3. Maclean D
(2011) Therapeutic applications of cell-penetrating peptides. Methods Mol Biol 683:535–551.
OpenUrl CrossRef PubMed
↵
1. Manwani D,
2. Galdass M,
3. Bieker JJ
(2007) Altered regulation of beta-like globin genes by a redesigned erythroid transcription factor. Exp Hematol 35(1):39–47.
OpenUrl CrossRef PubMed
↵
1. Lam KN,
2. van Bakel H,
3. Cote AG,
4. van der Ven A,
5. Hughes TR
(2011) Sequence specificity is obtained from the majority of modular C2H2 zinc-finger arrays. Nucleic Acids Res 39(11):4680–4690.
OpenUrl Abstract/FREE Full Text
↵
1. Krizek BE,
2. Amann BT,
3. Kilfoil VJ,
4. Merkle DL,
5. Berg JA
(1991) A consensus zinc finger peptide: Design, high-affinity metal binding, a ph-dependent structure, and a His to Cys sequence variant. J Am Chem Soc 113:4518–4523.
OpenUrl CrossRef
↵
1. Leach KM,
2. et al.
(2003) Characterization of the human beta-globin downstream promoter region. Nucleic Acids Res 31(4):1292–1301.
OpenUrl Abstract/FREE Full Text
↵
1. Liang SY,
2. et al.
(2009) Defective erythropoiesis in transgenic mice expressing dominant-negative upstream stimulatory factor. Mol Cell Biol 29(21):5900–5910.
OpenUrl Abstract/FREE Full Text
↵
1. Bungert J,
2. et al.
(1995) Synergistic regulation of human beta-globin gene switching by locus control region elements HS3 and HS4. Genes Dev 9(24):3083–3096.
OpenUrl Abstract/FREE Full Text

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Proceedings of the National Academy of Sciences: 109 (44)

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Submit

[1] ↵
Roeder RG
(2005) Transcriptional regulation and the role of diverse coactivators in animal cells. FEBS Lett 579(4):909–915.
OpenUrl CrossRef PubMed

[2] Roeder RG

[3] ↵
Orphanides G,
Reinberg D
(2002) A unified theory of gene expression. Cell 108(4):439–451.
OpenUrl CrossRef PubMed

[4] Orphanides G,

[5] Reinberg D

[6] ↵
Reya T,
Grosschedl R
(1998) Transcriptional regulation of B-cell differentiation. Curr Opin Immunol 10(2):158–165.
OpenUrl CrossRef PubMed

[7] Reya T,

[8] Grosschedl R

[9] ↵
McLean C,
Bejerano G
(2008) Dispensability of mammalian DNA. Genome Res 18(11):1743–1751.
OpenUrl Abstract/FREE Full Text

[10] McLean C,

[11] Bejerano G

[12] ↵
Arnone MI,
Dmochowski IJ,
Gache C
(2004) Using reporter genes to study cis-regulatory elements. Methods Cell Biol 74:621–652.
OpenUrl PubMed

[13] Arnone MI,

[14] Dmochowski IJ,

[15] Gache C

[16] ↵
Capecchi MR
(2005) Gene targeting in mice: Functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet 6(6):507–512.
OpenUrl CrossRef PubMed

[17] Capecchi MR

[18] ↵
Venter JC,
et al.
(2001) The sequence of the human genome. Science 291(5507):1304–1351.
OpenUrl Abstract/FREE Full Text

[19] Venter JC,

[20] et al.

[21] ↵
Jamieson AC,
Kim SH,
Wells JA
(1994) In vitro selection of zinc fingers with altered DNA-binding specificity. Biochemistry 33(19):5689–5695.
OpenUrl CrossRef PubMed

[22] Jamieson AC,

[23] Kim SH,

[24] Wells JA

[25] ↵
Choo Y,
Klug A
(1997) Physical basis of a protein-DNA recognition code. Curr Opin Struct Biol 7(1):117–125.
OpenUrl CrossRef PubMed

[26] Choo Y,

[27] Klug A

[28] ↵
Choo Y,
Sánchez-García I,
Klug A
(1994) In vivo repression by a site-specific DNA-binding protein designed against an oncogenic sequence. Nature 372(6507):642–645.
OpenUrl CrossRef PubMed

[29] Choo Y,

[30] Sánchez-García I,

[31] Klug A

[32] ↵
Segal DJ,
Barbas CF 3rd.
(2001) Custom DNA-binding proteins come of age: Polydactyl zinc-finger proteins. Curr Opin Biotechnol 12(6):632–637.
OpenUrl CrossRef PubMed

[33] Segal DJ,

[34] Barbas CF 3rd.

[35] ↵
Corbi N,
Libri V,
Onori A,
Passananti C
(2004) Synthetic zinc finger peptides: Old and novel applications. Biochem Cell Biol 82(4):428–436.
OpenUrl CrossRef PubMed

[36] Corbi N,

[37] Libri V,

[38] Onori A,

[39] Passananti C

[40] ↵
Urnov FD,
Rebar EJ,
Holmes MC,
Zhang HS,
Gregory PD
(2010) Genome editing with engineered zinc finger nucleases. Nat Rev Genet 11(9):636–646.
OpenUrl CrossRef PubMed

[41] Urnov FD,

[42] Rebar EJ,

[43] Holmes MC,

[44] Zhang HS,

[45] Gregory PD

[46] ↵
Gupta A,
Meng X,
Zhu LJ,
Lawson ND,
Wolfe SA
(2011) Zinc finger protein-dependent and -independent contributions to the in vivo off-target activity of zinc finger nucleases. Nucleic Acids Res 39(1):381–392.
OpenUrl Abstract/FREE Full Text

[47] Gupta A,

[48] Meng X,

[49] Zhu LJ,

[50] Lawson ND,

[51] Wolfe SA

[52] ↵
Stamatoyannopoulos G
(2005) Control of globin gene expression during development and erythroid differentiation. Exp Hematol 33(3):259–271.
OpenUrl CrossRef PubMed

[53] Stamatoyannopoulos G

[54] ↵
Siatecka M,
Bieker JJ
(2011) The multifunctional role of EKLF/KLF1 during erythropoiesis. Blood 118(8):2044–2054.
OpenUrl Abstract/FREE Full Text

[55] Siatecka M,

[56] Bieker JJ

[57] ↵
Perkins AC,
Sharpe AH,
Orkin SH
(1995) Lethal beta-thalassaemia in mice lacking the erythroid CACCC-transcription factor EKLF. Nature 375(6529):318–322.
OpenUrl CrossRef PubMed

[58] Perkins AC,

[59] Sharpe AH,

[60] Orkin SH

[61] ↵
Nuez B,
Michalovich D,
Bygrave A,
Ploemacher R,
Grosveld F
(1995) Defective haematopoiesis in fetal liver resulting from inactivation of the EKLF gene. Nature 375(6529):316–318.
OpenUrl CrossRef PubMed

[62] Nuez B,

[63] Michalovich D,

[64] Bygrave A,

[65] Ploemacher R,

[66] Grosveld F

[67] ↵
Orkin SH,
Antonarakis SE,
Kazazian HH Jr.
(1984) Base substitution at position -88 in a beta-thalassemic globin gene. Further evidence for the role of distal promoter element ACACCC. J Biol Chem 259(14):8679–8681.
OpenUrl Abstract/FREE Full Text

[68] Orkin SH,

[69] Antonarakis SE,

[70] Kazazian HH Jr.

[71] ↵
Dreier B,
et al.
(2005) Development of zinc finger domains for recognition of the 5′-CNN-3′ family DNA sequences and their use in the construction of artificial transcription factors. J Biol Chem 280(42):35588–35597.
OpenUrl Abstract/FREE Full Text

[72] Dreier B,

[73] et al.

[74] ↵
Mandell J,
Barbas CF 3rd.
Zinc finger (ZF) tools, version 3.0. Available at www.scripps.edu/mb/barbas/zfdesign/zfdesignhome.php.

[75] Mandell J,

[76] Barbas CF 3rd.

[77] ↵
Cathomen T,
Segal DJ,
Brondani V,
Müller-Lerch F
(2008) Generation and functional analysis of zinc finger nucleases. Methods Mol Biol 434:277–290.
OpenUrl CrossRef PubMed

[78] Cathomen T,

[79] Segal DJ,

[80] Brondani V,

[81] Müller-Lerch F

[82] ↵
Gusella J,
Geller R,
Clarke B,
Weeks V,
Housman D
(1976) Commitment to erythroid differentiation by friend erythroleukemia cells: A stochastic analysis. Cell 9(2):221–229.
OpenUrl CrossRef PubMed

[83] Gusella J,

[84] Geller R,

[85] Clarke B,

[86] Weeks V,

[87] Housman D

[88] ↵
Li G,
et al.
(2012) Extensive promoter-centered chromatin interactions provide a topological basis for transcription regulation. Cell 148(1-2):84–98.
OpenUrl CrossRef PubMed

[89] Li G,

[90] et al.

[91] ↵
Hodge D,
et al.
(2006) A global role for EKLF in definitive and primitive erythropoiesis. Blood 107(8):3359–3370.
OpenUrl Abstract/FREE Full Text

[92] Hodge D,

[93] et al.

[94] ↵
Zhou Z,
et al.
(2010) USF and NF-E2 cooperate to regulate the recruitment and activity of RNA polymerase II in the beta-globin gene locus. J Biol Chem 285(21):15894–15905.
OpenUrl Abstract/FREE Full Text

[95] Zhou Z,

[96] et al.

[97] ↵
Leach KM,
et al.
(2001) Reconstitution of human beta-globin locus control region hypersensitive sites in the absence of chromatin assembly. Mol Cell Biol 21(8):2629–2640.
OpenUrl Abstract/FREE Full Text

[98] Leach KM,

[99] et al.

[100] ↵
Vieira KF,
et al.
(2004) Recruitment of transcription complexes to the beta-globin gene locus in vivo and in vitro. J Biol Chem 279(48):50350–50357.
OpenUrl Abstract/FREE Full Text

[101] Vieira KF,

[102] et al.

[103] ↵
Johnson KD,
et al.
(2003) Highly restricted localization of RNA polymerase II within a locus control region of a tissue-specific chromatin domain. Mol Cell Biol 23(18):6484–6493.
OpenUrl Abstract/FREE Full Text

[104] Johnson KD,

[105] et al.

[106] ↵
Porcu S,
Poddie D,
Melis M,
Cao A,
Ristaldi MS
(2005) β-Minor globin gene expression is preferentially reduced in EKLF Knock-Out mice. Gene 351:11–17.
OpenUrl CrossRef PubMed

[107] Porcu S,

[108] Poddie D,

[109] Melis M,

[110] Cao A,

[111] Ristaldi MS

[112] ↵
Zhang F,
et al.
(2011) Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat Biotechnol 29(2):149–153.
OpenUrl CrossRef PubMed

[113] Zhang F,

[114] et al.

[115] ↵
Sanjana NE,
et al.
(2012) A transcription activator-like effector toolbox for genome engineering. Nat Protoc 7(1):171–192.
OpenUrl CrossRef PubMed

[116] Sanjana NE,

[117] et al.

[118] ↵
Räägel H,
Säälik P,
Pooga M
(2010) Peptide-mediated protein delivery-which pathways are penetrable? Biochim Biophys Acta 1798(12):2240–2248.
OpenUrl PubMed

[119] Räägel H,

[120] Säälik P,

[121] Pooga M

[122] ↵
Johnson RM,
Harrison SD,
Maclean D
(2011) Therapeutic applications of cell-penetrating peptides. Methods Mol Biol 683:535–551.
OpenUrl CrossRef PubMed

[123] Johnson RM,

[124] Harrison SD,

[125] Maclean D

[126] ↵
Manwani D,
Galdass M,
Bieker JJ
(2007) Altered regulation of beta-like globin genes by a redesigned erythroid transcription factor. Exp Hematol 35(1):39–47.
OpenUrl CrossRef PubMed

[127] Manwani D,

[128] Galdass M,

[129] Bieker JJ

[130] ↵
Lam KN,
van Bakel H,
Cote AG,
van der Ven A,
Hughes TR
(2011) Sequence specificity is obtained from the majority of modular C2H2 zinc-finger arrays. Nucleic Acids Res 39(11):4680–4690.
OpenUrl Abstract/FREE Full Text

[131] Lam KN,

[132] van Bakel H,

[133] Cote AG,

[134] van der Ven A,

[135] Hughes TR

[136] ↵
Krizek BE,
Amann BT,
Kilfoil VJ,
Merkle DL,
Berg JA
(1991) A consensus zinc finger peptide: Design, high-affinity metal binding, a ph-dependent structure, and a His to Cys sequence variant. J Am Chem Soc 113:4518–4523.
OpenUrl CrossRef

[137] Krizek BE,

[138] Amann BT,

[139] Kilfoil VJ,

[140] Merkle DL,

[141] Berg JA

[142] ↵
Leach KM,
et al.
(2003) Characterization of the human beta-globin downstream promoter region. Nucleic Acids Res 31(4):1292–1301.
OpenUrl Abstract/FREE Full Text

[143] Leach KM,

[144] et al.

[145] ↵
Liang SY,
et al.
(2009) Defective erythropoiesis in transgenic mice expressing dominant-negative upstream stimulatory factor. Mol Cell Biol 29(21):5900–5910.
OpenUrl Abstract/FREE Full Text

[146] Liang SY,

[147] et al.

[148] ↵
Bungert J,
et al.
(1995) Synergistic regulation of human beta-globin gene switching by locus control region elements HS3 and HS4. Genes Dev 9(24):3083–3096.
OpenUrl Abstract/FREE Full Text

[149] Bungert J,

[150] et al.

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Neutralizing the function of a β-globin–associated cis-regulatory DNA element using an artificial zinc finger DNA-binding domain

Abstract

Results

Discussion

Methods

ZF Design and Plasmid Construction.

Expression and Purification of the ZF-DBD.

EMSA.

Immunoblot Analysis.

Immunofluorescence Microscopy.

Cell Culture and Transfections/Transductions.

RNA Isolation and Analysis.

ChIP.

Benzidine Staining.

Generation of Transient Transgenic Mice.

Acknowledgments

Footnotes

References

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Main menu

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Abstract

Results

Discussion

Methods

ZF Design and Plasmid Construction.

Expression and Purification of the ZF-DBD.

EMSA.

Immunoblot Analysis.

Immunofluorescence Microscopy.

Cell Culture and Transfections/Transductions.

RNA Isolation and Analysis.

ChIP.

Benzidine Staining.

Generation of Transient Transgenic Mice.

Acknowledgments

Footnotes

References

Citation Manager Formats

Article Classifications

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