Appl Biochem Biotechnol DOI 10.1007/s12010-016-2187-4

Transient Tcf3 Gene Repression by TALE-Transcription Factor Targeting Junko Masuda 1,2 & Hiroshi Kawamoto 3,4 & Warren Strober 2 & Eiji Takayama 5 & Akifumi Mizutani 1 & Hiroshi Murakami 1 & Tomokatsu Ikawa 3,6 & Atsushi Kitani 2 & Narumi Maeno 1 & Tsukasa Shigehiro 1 & Ayano Satoh 1 & Akimasa Seno 1 & Vaidyanath Arun 1 & Tomonari Kasai 1 & Ivan J. Fuss 2 & Yoshimoto Katsura 3,7 & Masaharu Seno 1

Received: 11 February 2016 / Accepted: 4 July 2016 # Springer Science+Business Media New York 2016

Abstract Transplantation of hematopoietic stem and progenitor cells (HSCs) i.e., selfrenewing cells that retain multipotentiality, is now a widely performed therapy for many hematopoietic diseases. However, these cells are present in low number and are subject to replicative senescence after extraction; thus, the acquisition of sufficient numbers of cells for transplantation requires donors able to provide repetitive blood samples and/or methods of expanding cell numbers without disturbing cell multipotentiality. Previous studies have shown that HSCs maintain their multipotentiality and self-renewal activity if TCF3 transcription

* Junko Masuda [email protected]

1

Division of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan

2

Mucosal Immunity Section, Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA

3

Laboratory for Lymphocyte Development, RIKEN Research Center for Allergy and Immunology, Yokohama 230-0045, Japan

4

Department of Immunology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan

5

Department of Oral Biochemistry, Asahi University School of Dentistry, Hozumi 1851, Gifu 501-0296, Japan

6

Laboratory for Immune Regeneration, RIKEN Center for Integrative Medical Sciences, Yokohama 230-0045, Japan

7

Division of Cell Regeneration and Transplantation, Advanced Medical Research Center, School of Medicine, Nihon University, Tokyo 173-8610, Japan

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function is blocked under B cell differentiating conditions. Taking advantage of this finding to devise a new approach to HSC expansion in vitro, we constructed an episomal expression vector that specifically targets and transiently represses the TCF3 gene. This consisted of a vector encoding a transcription activator-like effector (TALE) fused to a Krüppel-associated box (KRAB) repressor. We showed that this TALE-KRAB vector repressed expression of an exogenous reporter gene in HEK293 and COS-7 cell lines and, more importantly, efficiently repressed endogenous TCF3 in a human B lymphoma cell line. These findings suggest that this vector can be used to maintain multipotentiality in HSC being subjected to a long-term expansion regimen prior to transplantation. Keywords TCF3 (E2A) . Artificial transcription factor . TALE technology

Introduction Hematopoietic stem and progenitor cells (HSCs) are self-renewing cells that retain the multipotent capacity to differentiate into distinct hematopoietic blood cells such as lymphoid T and B cells, natural killer (NK) cells, and dendritic cells [1, 2]. Consequently, clinical transplantation of HSCs has been widely performed as therapy for leukemias, malignant lymphomas, and even some types of solid tumor [3–5]. In addition, mature immune cells derived from HSCs have been transplanted into cancer patients as adoptive T cell and NK cell immunotherapy [6]. It should be noted, however, that HSCs constitute only a small fraction of bone marrow cells (between 0.05 and 0.1 % of total murine BM cells) [7, 8], and whereas primary HSCs can be cultured and expanded ex vivo, replicative senescence prevents longterm expansion [9–11]. This problem is compounded by the fact that most mature immune cells derived from HSC are short-lived and thus patients undergoing autologous immune cell therapy require repetitive blood drawing to obtain sufficient numbers of cells for expansion in vitro prior to transplantation [11]. In view of these difficulties, umbilical cord blood (UCB) has been considered as a possible alternative source of HSCs, particularly in the light of the fact that these cells have a greater survival than BM HSCs in HLA-mismatched recipients who lack a source of matched donor cells [12–14]. However, the use of this possible HSC source is limited by the fact that the number of HSCs obtainable from UCB is quite reduced compared to BM and may be insufficient for sequential transplantation of adult patients. Overall, then, there are considerable barriers to the provision of a steady supply of both HSCs and immune cells derived from HSCs for treatment of potential patients [12]. TCF3, also known as E2A, a member of the E-protein family of basic helix–loop–helix (bHLH) transcription factors, is involved in B and T cell lineage differentiation and, as such, in the development of multipotent stem cells [15, 16]. Splice variants of E2A, E12, and E47 form homo- or heterodimeric complexes with other E-proteins and then bind to a consensus CANNTG motif in promoter elements, (referred to as an E-box sites), to bring about gene transcription [17]. Such transcriptional activity, however, is blocked by members of the Id family of bHLH proteins, such as Id3, that function as inhibitors of E-protein transcription because they have the capacity to form heterodimeric complexes with E-proteins but lack a DNA binding domain [18]. Recently, it has been shown that HSCs maintain their multipotentiality and self-renewal capacity if TCF3 transcription function is blocked by overexpression of Id3 under B cell differentiating conditions [19, 20]. The Id3-induced hematopoietic progenitor (IdHP) cells thus

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obtained are capable of expanding exponentially for several months in vivo while retaining the capacity to differentiate into all types of immune cells when cultured under appropriate inductive conditions. Thus, IdHP cells generated from the peripheral blood of individual patients can conceivably be used in HLA-matched adoptive T cell and NK cell-based immunotherapy. One potential difficulty with this approach, however, is that attenuation of TCF3 function has so far been accomplished by transduction of a randomly integrating retrovirus that could cause disruption of endogenous genomic organization [21]. Therefore, virus-free generation of cells with blocked TCF3 is needed for clinical application of IdHP cells. One approach to achieving this goal is the use of transcription activator-like effectors (TALEs), i.e., DNA binding proteins that have transcriptional suppressor properties. TALEs are naturally produced by phytopathogenic bacteria of the genus Xanthomonas [22] and consist of proteins with DNA binding domains containing a combination of the four most common tandem repeats (i.e., repeat-variable di-residues (RVDs) capable of binding to one of the four nucleotide bases [23]. These properties allow the use of TALE technology to (1) customize binding repeats so as to obtain TALEs that rapidly bind to desired target sequences; (2) fuse with various effector proteins that enable chromatin modifications or gene regulation relating to specific target genes; and (3) allow exogenously expressed TALE-binding proteins from an episomal vector that does not have transgene integration [24–26]. On this basis, TALE technology can conceivably be applied to the generation of cells with IdHP properties. In this paper, we describe the construction of a novel TALE expression vector that attenuates TCF3 expression in human cells. This vector expresses TALE protein fused with transcriptional repressor that binds to a proximal promoter region upstream of TCF3 gene and suppresses the expression of this gene. As such, it can be used to create large numbers of hematopoietic progenitor cells for potential use in transplantation.

Materials and Methods Cell Culture African Green Monkey SV40-transfected kidney fibroblast cell line (COS-7) and human embryonic kidney (HEK) 293 cell lines were obtained from Dr. Shigekazu Nagata (IFReC, Osaka University) cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich, St. Louis, MO) supplemented with 10 % fetal bovine serum (FBS; CCB, Nichirei Bioscience, Tokyo, Japan) and 1 % antibiotic/antimycotic solution (Gibco, BRL, Life Technologies, Gaithersburg, MD). Human Burkitt’s lymphoma Daudi cell line [27] was purchased from the Japanese Cancer Research Resources Bank (JCRB; 9071) and cultured in RPMI1640 medium (Sigma-Aldrich) supplemented with 20 % FBS and 1 % antibiotic/antimycotic solution.

Design and Construction of TALE Transcription Repressor, Control and Reporter Plasmid for Human (h) TCF3 TALE DNA binding sites (TDBS) was designed against 5′-TCCCAGGCTCTGGACCTCAC3′ located from the −474 to the −455 region of the sense strand of TCF3 (transcript variant 1) using the RVD array NG-HD-HD-HD-NI-NN-NN-HD-NG-HD-NG-NN-NN-NI-HD-HD-

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NG-HD-NI-HD. The TALE transcription repressor plasmid-Krüppel-associated box (pTALEKRAB) was constructed using an EZ-TAL™ Assembly Kit (SBI, Mountain View, California) according to the user manual [24, 28]. This vector was composed of TAL protein N-terminal coding sequence (TAL N), TALE DNA-binding modules, TAL protein C-terminal coding sequence (TAL C), and nuclear localization signal (NLS), KRAB inhibitory domain without FokI effector, Thoseaasigna virus 2A (T2A), followed by red fluorescence protein (RFP) (Figs. 1a and 2a). The KRAB deletion form (ΔKRAB) was constructed using KOD mutagenesis kit (Toyobo, Osaka, Japan) with primers 5′-CATGGGCAAGCTTACCATGG-3′ (forward) and 5′-TTGATCGATCTTCGAAAGGA-3′ (reverse) (Fig. 2a). The deletion form of TAL N, binding repeats, TAL C, and KRAB (ΔTALE, RFP alone) was prepared with primers 5′-TGGATAGCGGTTTGACTCACG-3′ (forward) and 5′-TGGT AAGCTTGCCCATGGTGGCCGTACGCCCCTAT-3′ (reverse) (underlined sequence for

Fig. 1 Schematic illustration of the vector of TALE transcription repressor, translated protein, genomic DNA and reporter plasmid for TCF3. a-d Ori the origin of replication, Ampr the ampicillin resistance gene, pA poly(A) signal, T2A Thoseaasigna virus 2A, Pcmv cytomegalovirus promoter, TAL N TAL protein N-terminal coding sequence, TAL C TAL protein C-terminal coding sequence, NLS nuclear localization signal, KRAB Krüppelassociated box inhibitory domain, TDBS TALE DNA binding sites, Pmcmv cytomegalovirus minimal promoter, FLuc firefly luciferase. e Schematic of the TDBS and the transcription factor binding sites in the 5′-flanking region of TCF3 gene on chromosome 19. The translation initiation site of TCF3 transcript variant 1 is designated as +1. The black, gray, and white boxes represent the transcription initiation sites, TNBS, and putative transcription factor binding sites, respectively

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Fig. 2 TALEs express with RFP. a Schematic illustration of the full-length TALE-repressor protein (TALEKRAB) and deletion forms. Pcmv; cytomegalovirus promoter, TAL N TAL protein N-terminal coding sequence, TAL C TAL protein C-terminal coding sequence, NLS nuclear localization signal, KRAB Krüppel-associated box inhibitory domain, T2A Thoseaasigna virus 2A. TALE-KRAB and RFP are cleaved by T2A; ΔKRAB lacks KRAB and fuses with RFP; ΔTALE remains only RFP; Mock lacks last 11 bp of promoter region and initial 6 bp of RFP. b mRNA expression of RFP. HEK293 cells (5 × 105) was transfected with the indicated plasmids (1 μg) for 48 h. +RT with reverse transcriptase; −RT without reverse transcriptase

HindIII, Fig. 2a). The PCR product was digested with SacI and HindIII and replaced by the corresponding sequence of pTALE-KRAB. Mock was prepared by digesting pTALE-KRAB with SacI and HindIII, followed by Klenow fill-in reaction and self-ligation (Fig. 2a). The insertion of an internal ribosome entry site (IRES)-mouse (m) CD8α (Lyt-2) into pTALEKRAB (pTALE-KRAB-mCD8α) and pΔTALE (pΔTALE-mCD8α) was done with forward primer 5′-ATCCATAATAAGATCTTAATTAAGGATCCC-3′ (the underlined sequence for BglII; the underlined wavy line sequence for multiple cloning site upstream of IRES) and the reverse primer 5′-ATATTATTATAGATCGTCGACTTACACAAT-3′ (the underlined sequence for SalI; the underlined wavy line sequence for mCD8α) inserted into the BglII site of RFP using In-Fusion® HD Cloning Kit (Clontech, Mountain View, CA). Replacement of the cytomegalovirus (CMV) promoter with the elongation factor 1α (EF1α) promoter in the pTALE-KRAB-mCD8α and pΔTALE-mCD8α constructs was performed by digestion with EcoRV and BsiWI. Dual-reporter plasmid for TDBS (pRep) was constructed by gene fusion technology of SBI [29]. pRep was composed of a transcription stopper sequence, three copies of TDBS, green fluorescent protein (GFP), Thoseaasigna virus 2A (T2A) peptide, and firefly luciferase, followed by a poly A signal (core sequence), driven by minimal CMV (mCMV) promoter (Fig. 1). The pRL-SV40 plasmid which expresses Renilla luciferase was purchased from Promega (Madison, WI). Supercoiled plasmids were isolated by ethidium bromide-cesium chloride (EtBr-CsCl) density gradient centrifugation at 90,000 rpm at 20 °C for 24 h using a Beckman centrifuge (Beckman Instruments, Palo Alto, CA) as previously described [30].

Transfection and Fluorescence Microscopy Lipid-based transfection into COS-7 cells and HEK293 cells was performed using the Effectene™ Transfection Reagent (Qiagen, GmbH, Hilden, Germany) according to the manufacturer’s instructions. Electroporation into Daudi cells was performed with serum-free RPMI1640 medium containing 10 mM Hepes in chilled cuvettes (0.4 cm gap Gene Pulser®/ MicroPulser™; Bio-Rad, Hercules, CA) at 280 V and 950 μF using a Gene Pulser II (Bio-Rad) as previously described [31]. Cell images were obtained using an Olympus (New York, NY) LX81 at 20× and processed with a MetaMorph software (Universal Imaging Corp., West Chester, PA).

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Reverse Transcription PCR (RT-PCR) Total RNA was extracted using TRIzol® LS according to the manufacturer’s instruction manual (Trizol, Life Technologies, Inc.). Three hundred nanograms of total RNA was treated with deoxyribonuclease (DNase I, amplification grade, Gibco, BRL, Life Technologies) and then cDNA was synthesized with reverse transcriptase (+RT) or without the enzyme (−RT) using Superscript® III First-Strand Synthesis SuperMix according to the manufacturer’s instruction manual (Gibco, BRL, Life Technologies). RT-PCR was performed with 250 nM primers, 6 ng of cDNA, and Quick TaqTM™ HS DyeMix (Toyobo) in a Master Cycler Gradient (Eppendorf Scientific, Inc., Hamburg, Germany). Samples were subjected to initial denaturation at 94 °C for 2 min, followed by 35 thermal cycles of at 94 °C for 30 s, at 53 °C for 30 s, and at 68 °C for 30 s. PCR amplification was electrophoresed on 2 % agarose gel (Agarose-LE, Classic Type, Nacalai Tesque, Kyoto, Japan). Images were obtained using Atto Light-Capture II (Atto, Tokyo, Japan). Primers were 5′-AGGCTTCAAGTGGGAGAGATTC-3′ (forward) and 5′-TTTTGCATGACAGGGCCATC-3′ (reverse) for RFP; 5′-CAAC G A C C A C T T T G T C A A G C T C - 3 ′ ( f o r w a r d ) a n d 5 ′ - G G T C TA C AT G G C A A CTGTGAGG-3′ (reverse) for GAPDH.

Detections of Episomal Plasmid and Genomic DNAs Total DNAs were isolated from Daudi cells by the method as previously described [32]. Briefly, cells were homogenized with Tris-HCl, pH 8.0, buffer saline containing 10 mM Na2EDTA, 0.1 % SDS, and 100 μg/ml proteinase K, extracted by neutralized phenol and chloroform, and then total DNAs were isolated. Episomal plasmid and genomic DNAs were detected from 100 pg of total DNAs by PCR using 250 nM primers, 10 ng of DNA, and EmeraldAmp MAX PCR Master Mix (TaKaRa Bio, Shiga, Japan) in Mastercycler gradient. Samples were subjected to initial denaturation at 94 °C for 2 min, followed by 35 thermal cycles of at 94 °C for 30 s, at 53 °C for 30 s and at 68 °C for 30 s for RFP; initial denaturation at 94 °C for 2 min, followed by 35 thermal cycles of at 94 °C for 30 s, at 55 °C for 30 s and at 72 °C for 30 s for genomic GAPDH. Primers were 5′G A C C T C A A C TA C AT G G T G A G T G C T- 3 ′ ( f o r w a r d ) a n d 5 ′ - A C T C C T G GAAGATGGTGATGGGAT-3′ (reverse) for genomic GAPDH (the underlined sequence for intron).

Luciferase Assays Luciferase activities were measured using Promega dual-luciferase reporter assay system according to the manufacturer’s instructions. In brief, 2 × 105 of HEK293 cells in 24 wells were washed with phosphate buffered saline (PBS) and lysed in 100 μl passive lysis buffer. Firefly and Renilla luciferase activities from 20 μl of extract were added into 96-well flat-bottomed microplate (SPL Life Sciences, Seoul, Korea) and measured with 100 μl Luciferase Assay Reagent II (556 nm) and Stop & Glo® Reagent (480 nm) respectively using a microplate reader (SH-9000, Corona Electric, Ibaraki, Japan). Firefly luciferase activities were divided by Renilla luciferase units to obtain relative luciferase units (RLU).

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Flow Cytometric Analyses Cells (5 × 106) were washed once with ice-cold staining buffer (PBS containing 2 % FCS, 1 mM Na2EDTA, and 0.1 % sodium azide), incubated with anti-mCD8α-APC (clone 53-6.7, eBioscience) or isotype control (Rat IgG2a κ; clone R35-95; BD Biosciences) at 4 °C for 30 min. For hTCF3 staining, CD8α-expressing cells were collected by immuno-magnetic cell sorting according to the manufacturer’s instructions (CD8α (Ly-2) MicroBeads; Miltenyi Biotec, Bergisch Gladbach, Germany) and then fixed with BD FACS lysing solution (BD Biosciences) for 10 min at room temperature; the cells were then permeabilized with BD FACS permeabilizing solution 2 (BD Biosciences) for 10 min at room temperature then incubated with anti-hTCF mAb (anti-human E47; clone G127-32; BD Biosciences) or isotype control (Mouse IgG1, κ; clone MOPC-21; BD Biosciences) for 30 min on ice. Cells were acquired using a BD Accuri® C6 (BD Biosciences), and analyses were performed using FlowJo® software (Treestar, Inc., San Carlos, CA).

Statistical Analyses Statistical analyses were performed using the GraphPad Prism version 6 software package (GraphPad Software, San Diego, CA). Differences were considered to be significant when P was <0.05.

Results Construction of a Plasmid Expressing a TALE Transcriptional Repressor and a Reporter Construct for Detection of the Suppressor-Expressing Plasmid A pTALE-KRAB plasmid under the control of a CMV promoter and expressing TALE protein fused with a KRAB transcriptional repressor was constructed as indicated in BMaterials and Methods^ section and is depicted in Fig. 1a. This plasmid contains a T2A site encoding a selfcleaving peptide immediately upstream of the RFP encoding sequence that enables independent expression of RFP at the protein level. The expressed suppressor protein contains binding repeats that bind to TDBS in genomic target DNA upstream of the coding sequence of TCF3 (Fig. 1b, c). TCF3 has two transcription start sites giving rise to two variant transcripts, but the translation initiation site at +2081 from transcription initiation site of variant 1 is the same (Fig. 1e). The TDBS was upstream of the start site of both variants and thus positioned to repress both variants while to avoid off-target effects. While STAT3 is highly expressed in HSCs [33, 34], a co-repressor of KRAB negatively regulates STAT3 activation [35]. Thus, TCF transcription could not be initiated by STAT3 in the presence of TALE-KRAB (Fig. 1e). A reporter plasmid (pRep) under the control of a minimal CMV promoter and containing three upstream TDBS sites identical to that in the genomic target DNA was also constructed as indicated in BMaterials and Methods^ section is depicted in Fig. 1d; here again the plasmid contained a T2A site allowing independent expression of GFP and firefly luciferase (FLuc) at the protein level.

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Protein Expression by Cells Transfected with Plasmids Expressing Full-Length Constructs or Deletion Mutant Constructs of the Repressor To gain insight into the repressor function of the TALE-KRAB plasmid described above, we determined such function in HEK293 and COS-7 cells transfected with plasmids expressing the full-length TAL-KRAB construct or with plasmids expressing deletion mutants of this construct. The mutants consisted of constructs with a deletion of the KRAB domain (ΔKRAB), a deletion of the TALE domain (ΔTALE), or a deletion of both the KRAB and TALE domains (Mock). It should be noted, however, that the ΔKRAB construct also contained a deletion of T2A, thus eliminating independent expression of RFP at the protein level; in addition, the ΔTALE construct contained a deletion of the last 11 bp of the promoter region and the initial 6 bp of the RFP coding region. Expression of these plasmids in the transfected cells was determined by RT-PCR of total extracted mRNA using primers in the RFP sequence. The RT-PCR revealed that in HEK293 cells transfected with plasmids expression, each of the constructs expressed a unique and identical RFP amplification band (Fig. 2b). Using the transfected cells, we evaluated RFP expression by cells expressing the full-length TALE-KREB construct or the various deletion mutants of this construct. RFP protein expression, as measured by fluorescence microscopy, was equally robust in cells expressing the fulllength TALE-KREB construct and in cells expressing either the ΔKRAB or ΔTALE constructs, reflecting the RT-PCR results in Fig. 2b. However, RFP protein expression was reduced in cells expressing only RFP (Mock cells), despite the equal expression of RFP mRNA in these cells, probably reflecting the fact that the latter contained promoter and coding region deletions described above that may have impaired mRNA translation (Fig. 3a, b).

Fig. 3 RFP fluorescence and phase contrast images. COS-7 cells (a and c) and HEK293 cells (b) were transfected with the indicated plasmids (0.4 μg) for 48 h. (a and b) Scale bar; 50 μm. c RFP and DAPI fluorescence images of COS-7 cells. Scale bar; 20 μm

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Interestingly, RFP expression in cells transfected with plasmids expressing TALE-KRAB and ΔTALE constructs was present in both cytosol and nucleus; this is likely due to the fact that both of these constructs led to synthesis of free (diffusible) RFP, in the case of the TALEKRAB construct because of the presence of the T2A segment and efficient cleavage of synthesized protein to form free RFP. In contrast, RFP expression in cells transfected with the plasmid expressing the ΔKRAB construct was present only in the nucleus; this probably reflects the fact that the T2A segment had been deleted from this construct with the result that the synthesized RFP remains bound to the TALE protein as well as the fact that the synthesized TALE-RFP protein also contains a nuclear localization signal (NLS) that ensured efficient nuclear localization of this protein (Fig. 3b, c).

Repressor Function of the TALE-KRAB Plasmid Measured by GFP Expression and Luciferase Activity of a Reporter Gene To evaluate the capacity of the TALE-KRAB-expressing plasmid to generate protein that binds to TDBS sequence and represses transcription, we next co-transfected pTALE and pRep plasmids into HEK293 cells and monitored pRep GFP expression. As in a previous study [29], we found that baseline expression of GFP driven by the mCMV promoter in the pRep plasmid was minimal and in the present context was adequate in transfected HEK293, but not in COS-7 cells. We found that HEK293 cells co-transfected with increasing molar ratios of TALE-KRAB to pRep expressing plasmids exhibited decreasing GFP+ cells and decreasing GFP expression measured by both fluorescence microscopy or flow cytometry. In contrast, a decreasing number of GFP+ cells was not observed in cells co-transfected with increasing ratios of ΔTALE to pRep expressing plasmids by fluorescence microscopy or flow cytometry (Fig. 4). Moreover, co-localization of GFP and RFP in TALE-KRAB-transfected cells was dramatically lower than in ΔTALE-transfected cells (Fig. 4; co-localization in merged images is indicated by arrows). This result is expected, since the ΔTALE construct does not express protein that can repress reporter GFP expressed by pRep and therefore GFP expression is not repressed by changing the ratio of ΔTALE/mock.

Fig. 4 Full-length TALE represses reporter GFP expression. HEK293 cells (5 × 105) were co-transfected with the indicated molar ratio of TALE plasmids and Mock (1 pmol in total) and with reporter plasmid (pRep; 0.1 pmol) for 48 h. a and c TALE-KRAB or ΔTALE (RFP), reporter (GFP), and merged images. Arrows indicate yellow in merged image. b and d GFP positive cells were measured by flow cytometry

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In parallel studies, we assessed the repressor activities of the TALE-KRAB and the ΔTALE constructs on luciferase activity generated by the reporter plasmid. We found that even at a low molar ratio of TALE-KRAB to pRep plasmids, very substantial inhibition of luciferase RFU was observed whereas ΔTALE caused no inhibition (Fig. 5). Taken together, these results suggest that the TALE-KRAB protein is capable of binding to TDBS and repressing a reporter gene via its KRAB inhibitory domain; in contrast, ΔKRAB not expressing the TDBS binding domain has no repressor ability.

hTCF3 Repression by the TALE-KRAB Protein To test the repressor function of the TALE-KRAB protein on TCF3 gene expression, we turned to Daudi cells, i.e., a B lymphoma cell line that in contrast to HEK293 cells expresses high levels of TCF3 [36]. Efficient detection of live Daudi cells transfected with the TALE-KRAB-expressing plasmid by flow cytometry was obtained by insertion of an IRES-mCD8α cassette into the pTALE-KRAB gene downstream of RFP so that transfected cells could be identified by expression of CD8α. In addition, the CMV promoter in the original construct was replaced by an EF1α promoter to obtain a higher level of protein expression. A control construct driven by EF1α-expressing ΔTALE and mCD8α was also prepared (Fig. 6a). Transfection of these plasmids into Daudi cells by electroporation followed by 20 h incubation led to mCD8α expression in a fraction of transfected cells that could then be examined for concomitant TCF3 expression by flow cytometry (Fig. 6b). Cells transfected with the plasmid expressing the TALE-KRAB construct modified as indicated above contained a substantial sub-population of mCD8α+ cells in which TCF3 was decreased or absent whereas in cells transfected with the a plasmid expressing the TALE construct alone, this sub-population was not seen (Fig. 6c). In addition, 7 days after transfection of the TALE-KRAB construct, cells exhibited normal TCF3 expression and no genomic integration of the TALE-KRAB construct (Fig. 6d).

Fig. 5 Full-length TALE represses reporter expression of luciferase. HEK293 cells (1.25 × 105) were cotransfected with the indicated molar ratio of TALE plasmids and Mock (1 pmol in total) in the presence of reporter plasmid (pRep; 0.1 pmol) and pRL-SV40 (0.025 pmol) and incubated for 48 h. Firefly luciferase activity was normalized by Renilla luciferase activity. Data are represented as mean ± SD

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Fig. 6 Full-length TALE represses TCF3 expression in Daudi cells. a Schematic illustration of the TALEKRAB-mCD8α and ΔTALE-mCD8α. PEF1α elongation factor 1α promoter, TAL N TAL protein N-terminal coding sequence, TAL C TAL protein C-terminal coding sequence, NLS nuclear localization signal, KRAB Krüppel-associated box inhibitory domain, T2A Thoseaasigna virus 2A, IRES internal ribosome entry site. b TALE-KRAB-mCD8α and ΔTALE-mCD8α (1.2 pmol) under control of the CMV and EF1α promoter were electroporated into Daudi cells (5 × 106) for 20 h stained by anti-mCD8α antibody. c After electroporated with pTALE-KRAB-mCD8α and ΔTALE-mCD8α (EF1α promoter; 6 pmol into 25 × 106), mCD8α-expressed Daudi cells were enriched by magnetic sorting (1 × 106) and stained by anti-TCF3 antibody. Expression of TCF3 was evaluated in mCD8α+ cells of the TALE-KRAB-mCD8α and ΔTALE-mCD8α transfected and in untransfected cells. d pTALE-KRAB-mCD8α in Daudi cells disappeared within 7 days after electroporation. The episomal plasmids from electropolated cells harvested every day were detected by PCR for RFP gene on pTALE-KRAB-mCD8α. The lower panel showed genomic GAPDH detection as a control

Discussion In this study, we describe a vector constructed on the basis of TALE technology that can be used to target and repress the TCF3 gene in human stem (progenitor) cells (HSC) and thus create cells previously called IdHP cells that are capable of self-renewal despite preservation of multipotentiality [19]. To accomplish this goal, we first identified a specific DNA sequence upstream of the TCF3 gene start site that could serve as the binding site for the targeting protein and then constructed a TALE-KRAB-expressing plasmid that could express protein that both binds to this site and mediates KRAB-induced repression of the gene. In addition, we showed that the expressing plasmid produces TALE-KREB protein that is capable of repressing both exogenous reporter genes and endogenous TCF3. As in the case of HSCs, IdHP cells ultimately require expression of TCF3 to differentiate into B and T cells; it is therefore not possible to produce clinically useful IdHP by a genome editing process that leads to TCF3 deletion. In contrast, the strategy for producing IdHP by transient suppression of TCF3, as described here, may overcome this problem since such

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transient repression is said to be sufficient for the development of IdHP cells [19]. The transcriptional repressor used to repress the TCF3 gene in these studies, Krüppel-associated box or KRAB, was originally identified in mammalian organisms as an heptad leucine repeat and was shown to mediate transcriptional repression via recruitment of various transcriptional co-repressors [37–41]. The expression plasmid used results in KRAB fused with a TCF3specific TALE and is thus targeted to the TCF3 gene to cause transient TCF3 repression with a few days of expression plasmid transduction; however, after a few more days, the expression plasmid disappears. The TALE-KRAB expression plasmid used in our initial studies was under control of a CMV promoter; however, the latter promoter has been shown to be weaker in HSCs than the EF1α promoter [42, 43]. In our studies of TCF3 gene repression in Daudi cells, we therefore transduced the cells with a construct in which the CMV promoter was replaced with an EF1α promoter and did in fact obtain increased TALE-KRAB expression. In addition, in these studies, we transduced cells with a plasmid that co-expressed both TALEKRAB and surface mCD8α, the latter to provide a means of identifying or isolating transduced cells; it is possible, however, that other markers of transduced cells will ultimately prove more useful, particularly markers that do not put the stem cells at risk for elimination by residual cytotoxic cells (NK cells) in the stem cell population. This would avoid the need to submit the starting stem cell population (presumably derived from umbilical cord blood) to initial CD34 purification. The delivery of a targeted repressor protein in these studies was accomplished with an episomal plasmid that enables transgene-free transfection with little or no ability to integrate into the host genome [26] and, indeed, is currently being developed to generate induced pluripotent stem cells (iPSCs) for clinical application [44–47]. In the latter respect, the gene repression dependent on the TALE targeting vector constructed in this study requires comparison with methods of gene repression utilizing the RNA-guided CRISPR/Cas9 nuclease system recently modified to serve as a gene editing tool [48–50]. In contrast to TALEN, nuclease Cas9 protein is dependent on an engineered single guide RNA (sgRNA) that binds to a target genomic DNA sequence. In addition, it is dependent on a mutant Cas9 (dCas9) that lacks nuclease activity while retaining its capacity to bind to DNA. Thus, when fused with activator or repressor domains such as VP64 or KRAB, the sgRNA-dCas9 construct can either activate or repress an endogenous target gene [51–54]. To test this system and compare it with the TALE system, Zhang et al. constructed two sgRNAs fused to KRAB that target the c-Kit promoter and showed that the resultant construct inhibits cellular c-Kit activity and c-Kit promoter activity in a luciferase reporter assay. However, the inhibition obtained was not as great as that obtained with a c-Kit-targeted TALE-KRAB construct [55]. In addition, it has been shown that the CRISPR-Cas9 gene editing is associated with a high level of off-target binding in human cells that indeed may be exaggerated with the use of monomeric nucleasedeficient gRNA-dCas9 necessary in gene editing that does not result in gene deletion [56]. Thus, the TALE-based targeting construct may exhibit more specific targeting than a gRNAbased targeting construct. Overall, then, the gene repression obtained with TALE-KRAB appears to have advantages over that obtained with a gRNA-Cas9 both in terms of efficiency and specificity, although this might vary with respect to the nature of the target gene. As discussed above, the ability to expand undifferentiated HSCs ex vivo would greatly facilitate human bone marrow transplantation. This has led to previous studies focused on methods for accomplishing such expansion. Yamaguchi et al. for instance, provided evidence that ex vivo 3-week-expansion system of hematopoietic progenitor cells from UCB could be

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achieved by culturing UCB with cytokines and human AB serum in the presence of human primary bone marrow stromal cells [57]. More recently, Takubo et al. found that stem cells exhibit anaerobic glycolysis driven by a pyruvate dehydrogenase (PDK)-dependent mechanism and the latter serves as a metabolic checkpoint maintaining HSC quiescence and transplantability; thus, mimetics of PDK promote these HSC qualities [58]. Finally, Reya et al. reported that overexpression of activated β-catenin leads to long-term self-renewal and expansion of HSCs [59]. This finding led to considerable exploration of the role of the Wnt pathway in stem cell development and the understanding that this pathway differentially supports either stem cell proliferation/multipotentiality or stem cell differentiation depending on the presence of different β-catenin co-activators [60]. Thus, a major barrier to the ex vivo expansion of HSCs is the lack of sure methods of inducing robust cell proliferation while preventing cell differentiation and loss of transplantibility. The method described here for the creation of HSCs with impaired ability to generate TCF3 that are therefore locked in a multipotential state during a period of possible expansion offers a possible way of overcoming this barrier. Acknowledgments This work was supported in part by Japan Society for the Promotion of Science (JSPS) KAKENHI, grant numbers 25860797, and Okayama Foundation for Science and Technology. Compliance with Ethical Standards Conflict of Interest The authors declare that they have no financial or commercial conflicts of interest.

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