Efficient and orthogonal transcription regulation by chemically

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Efficient and orthogonal transcription regulation by chemically inducible artificial transcription factors Wataru Nomura, Daisuke Matsumoto, Taisuke Sugii, Takuya Kobayakawa, and Hirokazu Tamamura Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00741 • Publication Date (Web): 26 Oct 2018 Downloaded from http://pubs.acs.org on October 29, 2018

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Biochemistry

Efficient and orthogonal transcription regulation by chemically inducible artificial transcription factors

Wataru Nomura1,*, Daisuke Matsumoto1, Taisuke Sugii1, Takuya Kobayakawa1, and Hirokazu Tamamura1 1

Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan

* To whom correspondence should be addressed. Tel: +81-3-5280-8038; Fax: +81-3-5280-8039; Email: [email protected] ORCID Wataru Nomura: 0000-0001-8348-7544 Hirokazu Tamamura: 0000-0003-2788-2579

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ABSTRACT The DNA-binding specificity of genome editing tools can be applied towards gene regulation. Recently, multiple artificial transcription factors (ATFs) were shown to synergistically and efficiently regulate gene expression. Chemically triggered protein associations are useful for functional regulation at specific timings. A combination of several inducible protein association systems could enable the regulation of multiple genes at different loci with independent timing. We applied the FKBP-rapamycin-FRB and the GAI-Gibberellin-GID systems for gene regulation using multiple TALEs and dCas9. By the combined use of currently available systems, reporter gene assays were performed; the results indicated that gene expression was regulated by rapamycin or gibberellin in the presence of the FRB-, or GAI-effector domains, respectively. Furthermore, the activation of endogenous genes was differentially regulated by the system. This success suggests the usability of the chemically inducible multiple ATFs for the time-dependent regulation of multiple genes, such as the case for cellular phenomena that are dependent on the programmable timing of expression and on the differential expression of multiple genes.

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Biochemistry

INTRODUCTION The selective and precise control of the gene expression of an organism’s complex genome is an increasingly realistic goal in the fields of biotechnology and synthetic biology, and genome editing systems are important tools for this purpose. Three major platforms of genome editing, ZFN (1), TALEN (2, 3), and the CRISPR-Cas9 system (4, 5), have been developed (6). These approaches enable the manipulation of genomes on demand and have been applied to many organisms, including animal models of disease. With the guidance of sequence-specific nucleases, targeted genes are cleaved in a highly specific manner, introducing insertion and deletion mutations, often resulting in the knockout of a specific gene’s ability to function (6). Another application of the components of these platforms is gene regulation, by creating transacting artificial transcription factors (ATFs) coupled with sequence-specific DNA-binding domains (7). Such gene regulation techniques could be the best option for targeted gene therapy or studying gene functions especially when the gene product is critical for cell survival, such as in metabolic pathways in which a gene knockout could lead to the death of the cell or organism. In addition, precise manipulation of gene expression could be utilized in research on cell lineage control and other complex processes (8). Gene regulation studies using designed ATFs have been conducted for the past two decades (9), and the recent expansion of options for sequence-specific DNA-binding domains has created more avenues for the robust control of endogenous gene expression in cells (10, 11). It has been suggested that the use of multiple ATFs on the promoter or enhancer region could result in a synergistic effect on gene expression control (12). Additionally, the regulation of protein functions by chemicals or light has enabled spatiotemporal gene regulation and editing (13-19). Chemically controlled systems could be applied for in vivo studies, while light-triggered systems have advantages in optogenetics research. Several chemically controlled systems have been developed, such as the FKBPrapamycin-FRB system (20). In this system, the tight affinities of the domains for each other and for rapamycin allow the efficient assembly of a functional protein complex after its components are expressed in cells. Gene regulation or genome editing regulated by rapamycin has been reported (21, 22); in addition, a recent study that used fluorescent reporter genes showed that a combination of chemicals can orthogonally regulate two genes, instead of an endogenous gene (23). By combining this technique with chemically controlled protein associations, such as the GAI-Gibberellin-GID system (24,25), the multiplexed control of genes in a single cell is

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theoretically possible. As the first step towards the on-demand orthogonal endogenous gene regulation by chemicals, multiple uses of rapamycin-inducible ATFs targeting the same endogenous proximal promoter were tested. C-terminally FKBP-fused TALE and dCas9, the DNA-binding parts of ATFs, were designed and synthesized, and FRB-fused effector domains were commonly utilized in experiments. When targeting and regulating the different promoter regions, dCas9 might be difficult to use in two target loci at the same time, as the Cas9 recognition sequence is commonly shared in the sgRNA. To overcome this problem, it is possible to use other Cas9 orthologs with different PAM target sequences (26). However, these orthologs mostly have longer PAM sequences than S. pyogenes Cas9 (SpCas9), thus limiting the possible selection of target sites. Hence, in this study, currently available and reliable systems, with a combination of the dCas9, derived from SpCas9, and TALE domains were tested for controlling different genes in an orthogonal manner, using distinct chemicals. For the GAIGibberellin-GID system, the GID domain was C-terminally fused to TALE and dCas9, and the GAI domain was fused to the effector domains. Co-transfection of two distinct chemical regulation systems showed that orthogonal gene regulation is possible in a temporally specific manner (Figure 1). Our successful results indicate the potential of chemical control systems for the manipulation of endogenous genes in a temporally specific manner, in combination with robust gene regulation by multiplexed ATFs.

Figure 1. Schema of distinctly chemically inducible ATF systems regulated by rapamycin and gibberellin. The orange and green boxes show DNA-binding domains such as TALE or dCas9

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Biochemistry

with a domain for chemically induced dimerization. The red spheres show the effector domains for gene regulation with another domain for association. The upper and lower chemical structures are those of rapamycin and gibberellin, respectively.

MATERIAL AND METHODS Construction of TALE-effector, dCas9-effector, and guide RNA expression vectors TALE domains targeting IL-1RN were designed following a previously published protocol (10). The genes for TALE domains were constructed by the Golden-gate method, following the methods provided in a previous report (27) with minor optimization. The constructed TALE domains were ligated to the pcDNA vector containing DNA coding for 3×NLS, 3×FLAG tags, and the FKBP domain. The expression of the TALE-FKBP domains was confirmed by western blotting (Figure S1). VP64 and SID×4 were utilized as the effector domains for the upregulation and downregulation of genes, respectively. The synthetic genes for the domains were incorporated into pcDNA3.1, which contained the FRB gene. The expression of FRB-VP64 and FRB-SID×4 was also confirmed by western blotting (Figure S1). The dCas9 gene was constructed by introducing D10A and H840A mutations via site-directed mutagenesis. Similarly, the gene was introduced into pcDNA with TALEs. Expression of dCas9-FKBP was confirmed by western blotting (Figure S1). The target sequences for sgRNAs were designed using the CRISPRdirect software tool (28). The gene for sgRNA expression was constructed as previously reported (29). The functioning of sgRNAs in mammalian cells was confirmed by co-expression with hCas9 (29), and the cleavage of the target sequences was detected by a surveyor nuclease assay (Figure S2). Transcription regulation by TALE- and dCas9-based ATFs was assessed using the luciferase reporter assay. Direct fusions with the effector domains were prepared as positive controls, and their expression was confirmed by western blotting (Figure S3). Western blotting analysis Transfection of 293A cells was performed in 6-well plates using Lipofectamine LTX (Invitrogen), following the manufacturers’ protocol. 293A cells were transfected with expression plasmids (1 μg) and collected after being incubated for 72 hours. The collected cells were treated with RIPA buffer and subjected to standard blotting procedure. The target protein was detected with HRP-conjugated antiFLAG M2 antibodies (Sigma-Aldrich, St. Louis, Missouri, U.S.A) or anti-HA monoclonal antibodies (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). HRP-conjugated Goat Anti-Mouse IgG (H + L) (EMD Millipore, Burlington, Massachusetts, U.S.A) was used as the secondary antibody for the antiHA primary antibody.

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Luciferase reporter gene assay Transfection of 293A cells was performed in 24-well plates using Lipofectamine LTX (Invitrogen, Waltham, Massachusetts, U.S.A), following the manufacturers’ protocol. Reporter plasmid (400 ng) were transfected in 293A cells with various combinations of expression plasmids (450 ng) and pRL-CMV (100 ng) as an internal control. The cells were extracted, and gene expression levels were analyzed by a dualluciferase reporter assay system (Promega, Fitchburg, Wisconsin, USA) utilizing the Genios Pro (TECAN, Männedorf, Switzerland) plate reader. Endogenous gene expression analysis Transfection of 293A cells was performed in 6-well plates using Lipofectamine LTX (Invitrogen, Waltham, Massachusetts, U.S.A), following the manufacturers’ protocol. Amounts of various combination of expression plasmids were 2 μg. Endogenous gene expressions of IL-1RN and CEACAM5 were analyzed using the StepOnePlus real-time qPCR system (Applied Biosystems, Waltham, Massachusetts, U.S.A). Messenger RNA was extracted with IsogenII (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and cDNA was obtained using the Superscript III DNA first strand synthesis system (Invitrogen, Waltham, Massachusetts, USA), following the manufacturer’s instructions. Taqman gene expression assay kits (Assay IDs Hs00893626_m1 for IL-1RN, Hs00944023_m1 for CEACAM5, and Hs01011487_g1 for ribosomal protein S13 as standard) were purchased from Applied Biosystems (Waltham, Massachusetts, U.S.A). Synthesis of gibberellin derivatives: general information All reactions utilizing air- or moisture-sensitive reagents were performed in dried glassware under an atmosphere of nitrogen, using commercially supplied solvents and reagents unless otherwise stated. CH2Cl2 was distilled from CaH2 and stored over molecular sieves 4A. TLC was performed on Merck 60F254-precoated silica gel plates; the samples were visualized by fluorescence quenching under UV light and by staining with phosphomolybdic acid, p-anisaldehyde, or ninhydrin. Flash column chromatography was carried out with “Biotage Isolera One®” (Biotage, Uppsala, Sweden) equipped with “SNAP Ultra Silica Cartridge” or “SNAP Ultra C18 Cartridge”. 1H NMR (500 MHz), and 13C NMR (125 MHz) spectra were recorded using a Bruker Avance II spectrometer. Chemical shifts are reported in δ (ppm) relative to Me4Si (in CDCl3) as the internal standard. Infrared (IR) spectra were recorded on a JASCO FT/IR 4100 (JASCO, Tokyo, Japan), and are reported as wavenumber (cm–1). Low- and high-resolution mass spectra were recorded on Bruker Daltonics micrOTOF focus (ESI-MS) spectrometers in the positive and negative detection mode. Optical rotations were measured on a JASCO P-2200 polarimeter with a 100-mm path

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Biochemistry

length cell, operating at the sodium D line, and were reported as follows: [α]D (concentration (g/100 mL), solvent). Detailed experimental procedures for the synthesis and characterization of gibberellin derivatives have been described in the supplementary information section.

RESULTS AND DISCUSSION Reporter gene activation and suppression by rapamycin-inducible system using TALE-and dCas9-based ATFs The positions of target sequences for TALEs A to C and sgRNAs, named CR-1 to -4, in the IL1RN promoter region are depicted in Figure 2A and Figure S4. Transcription regulation in 293A cells was evaluated by a luciferase reporter system using transient expression. When TALE-VP64 or dCas9 fusions were used in combination, transcription activation was more efficient (Figure 2B, gray bars), in line with the results from a previous report (10). In rapamycin-induced gene activation, the combination of TALE B+C showed better activation than the combination of TALE A+B+C, which indicates that the number of binding domains does not determine the activity of ATFs. This phenomenon can be understood considering the results obtained using TALE-FKBP and FRB-VP64 without rapamycin. TALE-A-FKBP showed decreased gene expression compared to the control. It is not clear whether the binding of TALEA inhibits the functioning of the transcription machinery or the binding of native transcription factors, but it seems that when a reduction in the basal level of gene expression upon the binding of an ATF in the absence of inducer was observed, positive activation by the ATF in the presence of an inducer was lower than that by an ATF that does not reduce basal expression. This fact could be a clue to efficiently explore the DNA-binding domains of ATFs to achieve better gene activation. The four newly designed sgRNAs (CR1–4) for dCas9 were tested for genome editing with Cas9, resulting in the successful introduction of mutations, as revealed by the surveyor assay (Figure S2). Fifteen sets of sgRNA combinations, including single sgRNAs, were tested using dCas9-based direct-fusion ATFs. In experiments with dCas9-VP64, the combinations CR2+4 and CR1+2+4 showed the best enhancement of gene expression level: up

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to 50-fold. Luciferase expression by these combinations was higher than that by the combination of all four sgRNAs. In the absence of rapamycin, dCas9-FKBP and FRB-VP64 did not cause a decrease in gene expression, as was seen in case of TALE-A-FKBP, although an approximately a 5-fold increase in activation was observed with the combinations of sgRNAs CR2+4 and CR1+2+4 in the absence of rapamycin. This effect was similar to that seen in case of TALE domains, which might have stemmed from weak interactions existing between FKBP and FRB. After the addition of rapamycin, these combinations of sgRNAs showed approximately a 50-fold activation, which was the most efficient among all the sgRNA combinations. In comparison with the direct fusion dCas9-based ATFs, the activation of gene expression by these sgRNA combinations was almost the same, indicating that dCas9-based FKBP-FRB functions as efficiently as the direct fusions.

Figure 2. (A) Relative positions of target sequences for TALEs and sgRNA for dCas9 on the IL1RN promoter sequence on the luciferase reporter gene. (B) Results for reporter gene activation by TALE A–C and dCas9 using sgRNA CR1–4. Activation by direct fusion with VP64 is shown by gray bars. Results of rapamycin-induced activation are shown by yellow and orange bars,

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Biochemistry

without or with rapamycin (1 μM), respectively. Error bars show standard deviation (SD) of triplicated data. Combinations of TALE domains or sgRNA are indicated below. (C) Results for reporter gene repression by TALE A–C and dCas9 using sgRNA CR1–4. Gene repression by direct fusion with SID×4 is shown by gray bars. Results of rapamycin-induced repression are shown by light blue and blue bars, without or with rapamycin (1 μM), respectively. Error bars show SD of triplicated data. Combinations of TALE domains or sgRNA are indicated below.

Four repeats of the mSin3 interaction domain (SID) were utilized as a potent effector domain for gene suppression (30). For TALE-SID×4, enhanced suppression was observed when multiple TALEs were transfected (Figure 2C). The basal gene expression in the absence of the inducer rapamycin showed different profiles for TALE-FKBP and FRB-VP64. When TALE-C-FKBP and FRB-SID×4 were transfected into cells, the basal expression level of the reporter gene was suppressed to half of the control level, in contrast with FRB-VP64, which did not affect basal expression or slightly enhanced it. The suppression of basal expression could be explained by a weak interaction between FKBP and FRB in the absence of rapamycin, similar to the effect observed in the dCas9-FKBP experiments presented in Figure 2C. The addition of rapamycin induced repression, but the efficiency was lower than that of the sets of direct fusions of TALESID×4 in most cases, with the exception of TALE A or C alone. The combination of TALESID×4 A to C showed the strongest suppression (RLA = 0.05) compared to that of each TALE alone (A, B, and C yielded RLA values of 0.3, 0.5, and 0.56, respectively.) As indicated above, TALE-A showed suppression of the target by itself in the form of FKBP fusion. The combination containing the TALE-A-binding domain showed enhanced repression compared to that containing TALEs B and C. The dCas9-SID×4 fusion showed only a small suppression even with combinations of multiple sgRNAs. For dCas9-FKBP and FRB-SID×4, suppression of basal expression without rapamycin was observed, as seen in Figure 2B, suggesting a weak interaction between FKBP and FRB. Upon the addition of rapamycin, the suppression level was enhanced and reached the similar low levels as seen in case of the dCas9-SID×4 direct fusion with the same combinations of sgRNA. Overall, the suppression levels did not reach those attained by the TALE-based ATFs. The rapamycin-inducible TALE- and dCas9-based ATFs showed distinctive properties in the activation and suppression of gene expression. The common principle for ATFs is the effect of DNA binding on basal gene expression, which could govern the efficiency of gene regulation, as

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shown by the FKBP fusion without rapamycin. When the DNA binding of a TALE-based ATF inhibits gene expression significantly, the ATF did not efficiently activate gene expression. On the contrary, in such cases, gene suppression was efficiently induced. One of the dCas9-based ATFs, dCas9-FKBP, did not interfere with gene expression in the presence of FRB-VP64. Another factor that affected basal gene expression could stem from a weak interaction between FKBP and FRB in the absence of rapamycin. In addition, it was remarkable that gene activation by dCas9-based ATFs worked as efficiently as the direct fusion of dCas9 with VP64.

Expansion of the scope of chemical regulation for the endogenous IL-1RN gene As the next step, gene regulation of an endogenous gene, IL-1RN, was assessed utilizing combinations of TALE- and dCas9-based ATFs that showed the strongest gene activation in the reporter gene assay. Rapamycin was added to the cells 24 h after plasmid transfection. For the analysis of mRNA levels of IL-1RN, cells were collected at day 3 or day 7, and the total RNA was extracted (Figure 3A). Rapamycin-induced cells were cultured for a further seven days, with or without rapamycin. The gene activation levels were evaluated utilizing real-time qPCR and a specific Taqman probe. The activation of an endogenous gene by rapamycin was successfully observed in both systems (Figure 3B). When rapamycin was no longer provided after three days of culture, gene expression decreased to almost the basal level (7.4-fold for TALE (B+C)) at day 7, but the decrease was more significant when the combination of TALE (A+B+C) was used (1.9-fold) (Figure S5). When the cells were cultured with rapamycin continuously for seven days, the expression level of IL-1RN was maintained at approximately 10% of the level at day 3. Plasmid degradation is a plausible explanation for the observed decrease of gene expression. However, the difference in gene expression levels caused by the presence of rapamycin is encouraging. It was significant that the endogenous mRNA level was induced by as much as more than 800-fold and 300-fold at day 3 by TALE- and dCas9-based ATFs, respectively. Furthermore, a change in gene expression level was successfully induced by the addition or removal of rapamycin, with effects measurable even after seven days. A more elaborate analysis varying the duration of culture or the amount of rapamycin is required, but this system has the potential to be added to the toolbox of stimuli-inducible gene regulation systems.

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Biochemistry

Figure 3. Rapamycin-induced endogenous IL-1RN gene activation. (A) Scheme for the activation and evaluation of the endogenous IL-1RN gene. (B) Results of gene activation by rapamycin addition using TALE- or dCas9-based inducible ATFs. R(-) and R(+) indicate the absence or presence of rapamycin (1 μM). TALE domains and sgRNA for dCas9 are indicated below. The Y axis is shown using a logarithmic display. Error bars show SD of triplicated data. Statistical analysis by Student’s t-test was applied to normalized Ct (ΔCt) values. P