Real-Time Sensing of TET2-Mediated DNA Demethylation in Vitro by

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Real-Time Sensing of TET2-Mediated DNA Demethylation in Vitro by Metal-Organic Framework-Based Oxygen Sensor for Mechanism Analysis and Stem-Cell Behavior Prediction Yuzhi Xu, Si-Yang Liu, Jie Li, Li Zhang, Danping Chen, JiePeng Zhang, Yanhui Xu, Zong Dai, and Xiaoyong Zou Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01941 • Publication Date (Web): 06 Jul 2018 Downloaded from http://pubs.acs.org on July 6, 2018

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Analytical Chemistry

Real-Time Sensing of TET2-Mediated DNA Demethylation in Vitro by Metal-Organic Framework-Based Oxygen Sensor for Mechanism Analysis and Stem-Cell Behavior Prediction Yuzhi Xu,†,# Si-Yang Liu,†,# Jie Li,‡ Li Zhang,† Danping Chen,† Jie-Peng Zhang,*,† Yanhui Xu,‡,§ Zong Dai,*,† and Xiaoyong Zou† †

School of Chemistry, Sun Yat-sen University, Guangzhou 510275, PR China.

‡

Fudan University Shanghai Cancer Center, Institute of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai, 200032, PR China.

§

State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, 200433, PR China.

Supporting Information Placeholder ABSTRACT: Active DNA demethylation, mediated by O2-dependent ten-eleven translocation (TET) enzymes, has essential roles in regulating gene expression. TET kinetics assay is vital for revealing mechanisms of demethylation process. Here, by a metal-organic framework (MOF)-based optical O2 sensor, we present the first demonstration on real-time TET2 kinetics assay in vitro. A series of luminescent Cu(I) dialkyl-1,2,4-triazolate MOFs were synthesized, which were noble-metal-free and able to intuitively response to dissolved O2 in a wide range from cellular hypoxia (≤ 15 μM) to ambient condition (~257 μM). By further immobilization of the MOFs onto transparent silicon rubber (MOF@SR) to construct O2 film sensors, and real-time monitoring of O2 consumption on MOF@SR over the reaction time, the complete TET2-mediated 5-methylcytosine (5mC) oxidation process were achieved. The method overcomes the limitations of the current off-line methods by considerably shortening the analytical time from 0.5 – 18 h to 10 min, and remarkably reducing the relative standard deviation from 10% – 68% to 0.68% – 4.2%. As a result, the Michaelis-Menten constant (Km) values of TET2 for 5mC and O2 in ascorbic acid-free (AA–) condition were precisely evaluated to be 24 ± 1 and 43.8 ± 0.3 μM, respectively. By comparative study on AA-containing (AA+) conditions, and further establishing kinetics models, the stem-cell behavior of TETs was successfully predicted, and the effects of key factors (AA, O2, Fe2+) on TETs were revealed, which were fully verified in mouse embryonic stem (mES) cells. The method is promising in wide application in kinetics analysis and cell behavior prediction of other important O2-related enzymes.

DNA methylation is an epigenetic modification that regulates gene expression. Ten-eleven translocation dioxygenase 1-3 (TET13) influence DNA methylation status and mediate active DNA demethylation by an O2-dependent 5mC oxidation reaction. Active DNA demethylation is critical for cellular process. Facilitated by TETs, 5-methylcytosine (5mC) is stepwise oxidized to 5hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5carboxylcytosine (5caC), concomitant with the decarboxylation of α-ketoglutarate (α-KG) to CO2 and the consumption of O2.1-4 However, the mechanisms followed by TETs are poorly discovered. To reveal the mechanism of active DNA demethylation, a well accepted way is to evaluate TET kinetics in vitro. By comparative study on the kcat/Km (kcat is catalytic rate, Km is Michaelis-Menten constant) of TET2 for 5mC/5hmC/5fC-containing DNA (5mC/5hmC/5fC-DNA) in vitro, TET2 was revealed to have higher activity for 5mC-DNA than for 5hmC-DNA or 5fC-DNA.5 However, inconsistent conclusions were also raised when different Km values were achieved. Based on the Km of TETs for O2 (denoted as Km(O2)) in vitro, Laukka et al. concluded that TETs retained good activity in hypoxic condition,6 but Thienpont et al. soon

declared that TETs were inactive under tumor hypoxic condition.7 The conflict is still unsolved, which desperately needs precise study of TET dynamics. Despite its importance and practical needs, precise analysis of TET dynamics is difficult and arduous. The basic protocol for determining Km values of TETs relies on the Michealis-Menten equation by running a series of enzymatic assays in vitro at varying concentrations of substrate while keeping other components saturated, and measuring the initial reaction rate (V0). The current methods are mostly based on off-line assays, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS),5,8 liquid scintillation counting (LSC),6,9 selective chemical labeling strategy10,11 and enzyme-linked immunosorbent assay (ELISA),7,12 which are discontinuous and quantify cytosine derivatives or CO2 only after quenching the demethylation reaction at desirable times (end-point method). Beyond laboriousness, time and reagent consumption, multiple tests carried out in different batches may increase signal variation and reduce reproducibility of assay. Most importantly, precise kinetics study relies on accurate evaluation of the maximal V0, which can be only achieved by fast kinetics assay

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with high sampling frequency. Off-line assays can only provide average V0. A method capable of real-time tracking TET-mediated 5mC oxidation process is essential for more accurate, rapid, complete and reproducible results. However, no efficient approach has been proposed until now. Besides the formation of oxidation derivatives and the release of CO2, the entire DNA demethylation process also accompanies the consumption of O2. It implies that TET kinetics could be analyzed facilely by real-time monitoring of the consumption of O2 over 5mC oxidation time, if a suitable O2 sensor can be constructed with high sensitivity, good biocompatibility, and intuitive response to O2 without consuming O2 during enzymatic reaction. Herein, we present the first demonstration on real-time monitoring of TET2mediated 5mC oxidation process in vitro. A series of luminescent Cu(I) dialkyl-1,2,4-triazolate metal–organic frameworks (MOFs) were synthesized. The MOFs were noble-metal-free and able to response to dissolved O2 intuitively in wide range from cellular hypoxia to ambient condition. By further immobilizing the MOFs onto a transparent, O2-permeable, and nonluminescent silicon rubber (MOF@SR), optical O2 film sensors were fabricated. Complete reaction curves were achieved more rapidly by real-time monitoring of the consumption of O2 on MOF@SR over 5mC oxidation time, and thus reproducible and precise Km values were obtained. Based on TET dynamics assay and mouse embryonic stem (mES) cell experiments, the cellular behavior of TETs was successfully predicted and the effects of key factors (ascorbic acid (AA), O2, Fe2+) on TETs were revealed.

EXPERIMENTAL SECTION Reagents and Materials. Glucose oxidase (GOx) from Aspergillus niger, catalase from bovine liver, glucose were purchased from Sigma-Aldrich. Oligonucleotides were purchased from Takara Biotech. Co., Ltd. (Dalian, China), of which the sequences are 5′TTT CAG CTC CGG TCA CAC-3′ for unmethylated DNA (CDNA) and 5′-TTT CAG CTC mCG GTC ACAC-3′ for 5mCDNA. The ligands Hdetz, Hdptz and Hdbtz (Hdetz = 3,5-diethyl1,2,4-triazole, Hdptz = 3,5-dipropyl-1,2,4-triazole, Hdbtz = 3,5dibutyl-1,2,4-triazole) were synthesized according to the reference.13 Room temperature vulcanization SR RTV-118 was obtained from Momentive Performance Materials Co., Ltd. Recombinant TET2 dioxygenases were provided by professor Yanhui Xu (Fudan University Shanghai Cancer Center, Institute of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai, China), which were produced essentially as previously reported.14 All of the other reagents were purchased from Sigma-Aldrich and used without further purification. All solutions were prepared with doubly distilled water. Preparation of MOFs and MOFs@SR O2 Sensors. Three MOF powders were synthesized according to the protocol.15 In brief, 0.5 mmol Cu(NO3)23H2O, 1 mmol AA and 1 g NaOH were mixed in 30 mL water at 25 °C, resulting the formation of Cu2O nanoparticles. After centrifugation and washing, the Cu2O nanoparticles were added into a 20 mL of 1 mM mixed-ligands composed of pre-synthesized Hdetz, Hdptz and Hdbtz (Hdetz:Hdptz = 58 : 42 for P46:MAF-2; pure Hdetz for MAF-2; Hdptz:Hdbtz = 75 : 25 for B31:MAF-2P) in deoxygenized ethanol in a sealed glass tube., and incubated at 90 °C for 6 h. The resultant solution was filtrated and the product was vacuumed to dry. In

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addition, the exact ratios of the ligands were different from the feed ratio, which were verified by 1H-NMR. MOF@SR film sensor was fabricated by our previously developed counter-diffusion crystal-growth method.16 The room temperature vulcanization SR RTV-118 was put into a plexiglas clamping apparatus and cured at ambient atmosphere overnight to form transparent membrane (thickness ≈ 1 mm). The membrane gradually swelled and absorbed the ligand solution when immersed in a CH2Cl2 solution of 0.5 M mixed-ligands for 10 min (the same ratio of ligands as that in powder synthesis). The swelled membrane was immediately transferred into a 100 mL of lowly stirring (60 rpm) solution containing 0.1 M [Cu(NH3)2]OH in aqueous ammonia/methanol (1:1). The ligands leaked out of the membranes and reacted with [Cu(NH3)2]OH to form the desired microcrystals on the solid/liquid interface. After 5 min, the shrunk and frosted membranes were taken out, washed by methanol to give the desired MOF@SR membranes (Figure S1). Characterization Methods. Powder X-ray diffraction (PXRD) patterns were collected by a Bruker D8 ADVANCE X-ray powder diffractometer (Cu-Kα). Scanning electron microscope (SEM) images were obtained on a Hitachi FE-SEM SU8010. 1H-NMR was tested on a Mercury Plus 300. Determination of Dissolved O2 with MOFs. MOF particles were dispersed in water (1 mg mL−1) and pipetted into a 100 μL custom-made quartz fluorescence cuvette. The MOF aqueous was equilibrated by bubbling with O2/N2 gas mixtures (0 – 100 mL mass flow controller, Alicat) for 10 min at room temperature, and the cuvette was then quickly sealed with Parafilm. 5% Na2SO3 aqueous solution was applied for complete deoxygenization. Luminescence spectra at each amount of dissolved O2 were measured on a F-7000 Spectrofluorophotometer (Hitachi, Japan). The amount of dissolved O2 was quantified by a dissolved O2 meter (JPB-607A, Shanghai INESA Scientific Instrument Co., Ltd., China). In Vitro Enzymatic Assays with MOF@SR Sensors. The assays were performed at 25 °C in a 100 μL cuvette with a piece of MOF@SR positioned inside at an angle of 45o. The GOx assays were carried out in 50 mM sodium acetate solution (pH 5.5, 100 μL) consisted of 126 nM catalase and 0.5 – 100 mM glucose. The Km(glucose) was determined by varying the concentration of glucose at saturating concentrations of other components, except the O2 concentration was that of air. The Km(O2) was determined by varying O2 concentrations with the appropriate gas mixture (N2/O2) for 10 min at room temperature. The reaction was initiated once 47 nM GOx was injected into the cuvette through the Parafilm. The changes of luminescent intensity over time at different concentration of glucose were monitored real-timely in 1 s intervals and stopped at 2600 s. The TET2 dioxygenases assays were carried out essentially as described above at 37 °C in 50 mM HEPES (pH 7.9, 50 μL) with P46:MAF-2@SR unless otherwise specified. The reaction was initiated by the addition of TET2 solution [30 mg mL−1 in 20 mM Tris buffer (pH 8.5) containing 120 mM NaCl and 3 mM DTT] into a solution of 50 μM Fe(NH4)2(SO4)2, 1 mM α-KG and 25 – 200 μM methylated DNA (5mC), and last for 2600 s. Cell Culture and Treatment. R1 mES cells were cultured feeder-free at 37 °C in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 15% fetal bovine serum (FBS) (ESC quality; Hyclone), 1,000 U mL−1 leukemia inhibitory factor

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(LIF) (ESGRO; Chemicon), 0.1 mM non-essential amino acids (NEAA), 0.1 mM β-mercaptoethanol, 1% nucleoside mix, 2 mM Lglutamine, and penicillin (100 U mL−1) and streptomycin (100 μg mL−1). For hypoxia and AA (L-ascorbic acid 2-phosphate, Sigma) exposed cells, both control and treated cells were seeded 18 – 24 h prior to the exposure at 35% – 45% confluency for 24 h. For normoxic cultured cells, cells were grown at atmospheric O2 concentrations (21%) with 5% CO2. For hypoxic cultured cells, cells were incubated in an atmosphere of 0.5% O2, 5% CO2 and 94.5% N2 in a custom-made seal chamber which was filled with the gaseous mixture every 8 h and then properly sealed. For AA treatment, 100 μg mL−1 of AA was added to fresh culture medium. Immunofluorescent Staining. Cultured cells were plated on glass-bottom dishes and stained after treatments of 24 h. First, cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min followed by another PBS wash. Then, cells were permeabilized with 0.1% Triton X-100 for 20 min and further incubated with 2 N HCl for 15 min at room temperature to denature double-stranded DNA (dsDNA) into single-stranded DNA (ssDNA), and placed in 0.1 mM Tris-HCl (pH 8.5) for 10 min. After washed with PBS and blocked with 10% goat serum in PBS for 1 h at 37 °C, cells were incubated with anti-5hmC rabbit polyclonal IgG (Active Motif, Cat. No. 39770, 1:250) overnight at 4 °C. The dishes were washed with PBS and incubated with Alexa Fluor 555-conjugated anti-rabbit IgG (1:300, #4413, CST) for 2 h at 37 °C. Flowed by PBS wash, cells were then counterstained with 4′,6-Diamidino-2-phenylindole (DAPI) for 3 min. Fluorescence images were acquired with a Zeiss LSM710 confocal microscope. Intensity of 5hmC immunofluorescence staining was analyzed by ImageJ software. Genomic DNA Isolation and Dot Blot. Genomic DNA extraction was performed with a Biospin Cell Genomic DNA Extraction Kit (Hangzhou Bioer Co. Ltd.) according to the manufacturer’s instructions. The extracted DNA was quantified with a Quawell Q6000UV and denatured with heat at 95 °C for 10 min, immediately followed by transferring the denatured DNA samples to ice. 1 μL of each DNA sample in 2-fold serial dilutions was manually spotted onto a positively charged nylon membrane (Hybond N+ membrane, Beyotime Institute of Biotechnology) and dried in air for 10 min. The membrane was blocked with 5% BSA and incubated with rabbit antibody to 5hmC (Active Motif, Cat. No. 39792, 1:2000) for 1.5 h at room temperature. Horseradish peroxidase-conjugated antibody to rabbit (Active Motif, #100612, 1:4000) was used as a secondary antibody and incubated for 1 h at room temperature. The membrane was visualized by ScanWizard Bio Software. The density of each dot signal was quantified by Image-Pro Plus 6.0. Quantitative Reverse-Transcription Polymerase Chain Reaction (qRT-PCR) Analysis for Expression of TETs and Marker Genes. Total RNA was extracted from cells with RNAiso Plus (Takara Biotechnology Co., Ltd.). 3 μg RNA was reversely transcribed to cDNA with Bestar qPCR RT Kit (DBI Bioscience). Real-time PCR was performed using a Bestar SybrGreen qPCR Master Mix (DBI Bioscience) following the manufacturer’s protocol on a Stratagene Mx3000P real-time PCR System (Aglient Technologies). The mRNA levels of TET-related and pluripotent genes were examined and the relative expression of genes was normalized to the gene β-actin. Primer sequences (Shanghai Sangon Biological Engineering Technology and Services Co. Ltd.) were described under Table S1.

Scheme 1. (a) Principle of real-time TET2 kinetics assay by an O2-sensitive MOF@SR sensor; (b) Presumption of kinetics models for 5mC and O2

RESULTS AND DISCUSSION Principle for in Vitro Real-Time TET2 Kinetics Assay and mES Cell Demethylation Prediction. The basic idea for realtime TET kinetics assay is that DNA demethylation process accompanies the consumption of stoichiometric O2, which is promisingly monitored in real time. We synthesized luminescent Cu(I) dialkyl-1,2,4-triazolate MOFs which were constructed by interconnecting the square-planar [Cu2(triazolate)2] units with different dihedral angles. The porosity of the MOFs can be tailored from 0% to > 50% depending on the proportions and types of the alkyl chain of the mixed-ligands. The typical 3MLCT (metal to ligand charge transfer) of these MOFs can be quenched readily and colligatively by molecules with a triplet ground state (such as O2). And because of the tunable porosity and structures of these MOFs, they exhibited detection range from 68 ppb to ambient O2 concentration in gas phase. Similar phenomena can be found in water phase. The luminescence of MOF@SR film sensor lighted rapidly and strongly in O2-free condition without consumption of O2 (Figure S1a). As the TET2-mediated 5mC oxidation reaction took place in a sealed cuvette, the 5hmC-DNA product continuously generated, along with the consecutive consumption of the dissolved O2, causing the increase of the luminescent intensity of MOF@SR sensor with the reaction time. Based on Stern-Volmer equation,17 the complete curve of TET-mediated 5mC oxidation process was profiled, which showed clear lag (1), linear (2) and nonlinear phases (3) (Scheme 1a). As the prominent features of MOF sensors and real-time sensing property, the sampling time was considerably shortened. More importantly, from the complete and continuous enzyme reaction curve, an accurate V0 can be provided from the linear phase and thus Km values can be accurately obtained according to the Michaelis-Menten equation18 and Lineweaver-Burk plot.19 As the Km represents the substrate dependence of enzymes, one can evaluate the TET2 behavior in different methylation pattern (hypo-/hyper-methylation) under the effects of key factors (AA, O2) from comparative study of the Km value of TET2 for 5mC-DNA and O2 in AA-free (AA−) or AAcontaining (AA+) conditions. We evaluated the Km(5mC) and Km(O2) in AA− under air atmosphere and low-O2 condition, respectively, denoted as Km(air, 5mC-DNA) and Km(hypoxia, O2).



By further establishing kinetics models according to these Km values, the relationship of DNA demethylation rate with gene methylation pattern, AA and O2 was simulated (Scheme 1b). That is, the fold change of demethylation rate (also the 5hmC level) relative to AA− decreasing with the increase of 5mC and O2. Based on the simulation, we predicted that: 1) enzymatic activities of TETs are highly stimulated by AA particularly in hypoxia-cultured cells; 2) hypomethylated promoters and hypoxia-cultured cells are influenced much more by AA in demethylation compared to hypermethylated promoters and nornoxia-cultured cells, respectively. The predictions were further confirmed by mES cell experiments. These results provide guidance for further research on regulatory mechanisms of varied factors on TET-mediated demethylation in stem cells which follow a predictable pattern. O2-Sensitive Properties of MOFs. We synthesized three [Cu(detz)], MOFs, [Cu(detz)54%(dptz)46%], and [Cu(dptz)69%(dbtz)31%], denoted as P46:MAF-2, MAF-2, and B31:MAF-2P, respectively, and immersed them in water. Based on the previous16 and present studies, the PXRD patterns of the three MOFs after being immersed in water were the same with the original ones (Figure S2). As expected, the luminescent intensities of these MOF suspensions were significantly quenched with increasing the concentration of dissolved O2 (Figure S3a-c). The I0/I ratio (in which I0 and I are the luminescent intensities in the absence and presence of O2, respectively) of the MOF suspensions showed good linear correlations with the concentration of dissolved O2 (Figure S3d-f), which were well fitted with the linear Stern-Volmer equation (Eq-S1). The P46:MAF-2 suspension showed the highest sensitivity to dissolved O2 among these MOFs, with a high KSV of 0.19 μM−1 and a low limit of detection (LOD) of 0.053 μM (KSV is the quenching efficiency, and LOD was evaluated at 1% quenching where I0/I = 1.01).17 The MAF-2 suspension had the second highest sensitivity (KSV = 0.096 μM−1, LOD = 0.10 μM), and the B31:MAF-2P suspension was the third (KSV = 0.015 μM−1, LOD = 0.67 μM) (Table S2). The preferable detection range (PDR), defined as the concentrations giving 20% to 80% of the intensity measured in the absence of O2, was also evaluated.15 The PDRs of P46:MAF-2, MAF-2, and B31:MAF-2P suspensions for dissolved O2 were calculated to be 1.3 – 21, 2.6 – 42, and 17 – 267 μM, respectively, which cover dissolved O2 ranging from cellular hypoxia (≤ 15 μM, 0.2% – 5%), tissue normoxia (30 – 60 μM) to ambient condition (~ 257 μM) (Figure 1).20

Figure 1. Outline of tissue O2 concentration and PDRs of P46:MAF-2, MAF-2, and B31:MAF-2P suspensions.

MOF@SR Sensors Characterization. As nano- and macro-particles are easy to aggregate and deposit in solutions, neither nanoscale MOFs nor bulk MOFs are suitable for continuous assay. We further immobilized P46:MAF-2 and B31:MAF-2P on SR film (P46:MAF2@SR and B31:MAF-2P@SR, respectively). As shown in the

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top/cross views of SEM, the crystals with diameters of 2 – 7 μm were densely crystalized on the surface of SR film for P46:MAF-2@SR and B31:MAF-2P@SR (Figure S4). The PXRD patterns indicate that the in situ grown MOFs on films are isostructural to their microcrystalline ones (Figure S2). The phosphorescence of the two films exhibited stable and good reproducible response to the changes of O2 concentration for multiple cycles (Figure 2a, Figure S5). It took about 45 and 160 s (O2 sensing rate 5.69 and 1.60 μM s−1) for P46:MAF2@SR and B31:MAF-2P@SR, respectively, to accomplish 95% of total phosphorescent intensity variation from air-saturated water to O2depleted solution (Figure 2b, Figure S6), implying that both P46:MAF-2@SR and B31:MAF-2P@SR responded fast toward O2. The stable intensities of two film sensors in air-saturated water and after reaching the maximum in O2-depleted solution indicated that the dissolved O2 did not influence the luminescence detection and the gas leakage was negligible. The Stern-Volmer plot of B31:MAF-2P@SR showed the same good linearity and sensitivity as those B31:MAF-2P suspension had (Figure S7, Table S2).

Figure 2. (a) Reversible luminescent response of P46:MAF-2@SR upon alternating exposure to O2-depleted solution and air-saturated water. (b) Time-dependent luminescent intensity of P46:MAF-2@SR in O2-depleted solution and air-saturated water.

Besides, the Michaelis-Menten parameters of GOx, which have been well defined in ambient condition, were tested as a standard model (Figure S8). B31:MAF-2P@SR was employed because it had PDR in ambient condition. The Km(glucose) of 20 ± 2 mM and Km(O2) of 267 ± 37 μM achieved with B31:MAF-2P@SR were well consistent with those obtained with a Clark electrode [Km(glucose) = 26 mM; Km(O2) = 200 μM]21 and a fiber-optic O2sensing system [Km(glucose) = 23 ± 2 mM; Km(O2) = 140 ± 10 μM]22 (Table S3), suggesting that the MOF@SR was able to precisely determine enzymatic kinetics. Real-Time TET2 Kinetics Assays in Vitro. According to the Lineweaver-Burk plot (Eq-S3), the y-axis is 1/V0, which would enlarge small errors in measurement, especially the errors in the low substrate level (the large 1/[Substrate] in x-axis). Therefore, the accurate and sensitive measurement of V0 in the lower O2 level has a crucial effect on the determination of Km(O2). P46:MAF-2 suspension had PDR in the hypoxic range and thus its hybrid film, P46:MAF-2@SR, was applied to TET2 dynamics analysis. The Km values of TETs in AA+ condition have been studied.5−7 In our assay, we performed the comparative study in AA– condition. In order to achieve reliable results, the reduction of the catalytic efficiency of TET2, which was caused by the absence of AA, was compensated by augmenting the amount of TET2 (180 μg in 50 μL) and 5mCDNA (200 μM) to insure a sufficient O2-consumption rate (Figure S9).


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Table 1. Comparison of Analytical Performance and the Kinetics Parameters for TET2 Methods LC-MS/MS 14

Radioisotope assay of CO2

Analytical Km(5mC) time μM[a]

Addition Sampling Implementation of AA interval

V0

Yes

Avg > 18 h

Yes

End-point End-point

2.5 min 20 min

Avg ~ 0.5 h

0.48 ± 0.19

Km(O2)

R.S.D

μM

%

/

0.125 ± 0.085 30 ± 3

[b]

[c]

[Ref]

40

5

10 – 68

6

Global 5hmC quantification kit

Yes

End-point

3 min

Avg > 8 h

/

~3

/

7

MOF-based O2 sensor

No

Real-time

≤1s

Max < 10 min

24 ± 1

43.8 ± 0.3[b]

0.68 – 4.2

This work

[a] tested under air atmosphere. [b] tested under modest hypoxia (2% –5% O2). [c] tested under tumor hypoxia (0.2% – 0.5% O2).

It should be noted that the initial phosphorescence of P46:MAF2@SR was quenched by 50.7% by a large amount of DNA (Figure S10), possibly induced by the obstacle from DNA that prevented O2 from accessing to P46:MAF-2. In this case, the phosphorescence quenching behavior of P46:MAF-2@SR followed nonlinear Stern-Volmer relationship, which can be described by the two-site model (Figure S11, Figure S12, fitted by Eq-S4).17 P46:MAF-2@SR still showed excellent sensitivity to dissolved O2 (KSV1 = 0.18 μM−1, LOD = 0.060 μM), and its PDR (1.5 – 30 μM) just covered the range of cellular hypoxia. Thus, P46:MAF-2@SR is suitable for TET2 kinetics assay especially in hypoxic condition. Before kinetics assay, a control experiment was performed with C-DNA, which is not the substrate of TET2. Upon the addition of TET2, the luminescent intensity of P46:MAF-2@SR only increased by 4.2% (Figure S13a). The response was subtracted as a background, which was resulted from the oxidation of Fe2+ and DTT. In contrast, with the TET2 substrate 5mC-DNA, P46:MAF2@SR in air-saturated solution showed remarkably increased phosphorescence by up to 50.0% upon the addition of TET2. As increasing the concentration of 5mC-DNA, the phosphorescence of P46:MAF-2@SR enhanced more rapidly (Figure S13a). By converting luminescent intensity into O2 concentration from the fitted equation (Figure S12), the real-time O2 consumption during TET-mediated 5mC oxidation was obtained. The oxidation process showed clear lag, linear and nonlinear phases over time (Figure 3a, Figure S14). The O2 consumption rate was less than 0.15 μM s−1 which was much slower than the O2 sensing rate (5.69 μM s−1) of P46:MAF2@SR. It suggested that the fast respond of P46:MAF-2@SR towards O2 enables the real-time monitoring of TET-mediated 5mC oxidation. From the plot of the substrate concentration and V0 values (obtained from the linear phase of O2 consumption at different concentrations of 5mC-DNA), the Km(air, 5mC) was evaluated to be 24 ± 1 μM (Figure 3c). Similarly, the interaction between TET2 and O2 was investigated under various concentrations of O2 (Figure S13b, Figure 3b), and the Km(hypoxia, O2) was achieved to be 43.8 ± 0.3 μM (Figure 3d). The catalytic efficiency (kcat/Km) was calculated as 0.20 mM−1 s−1 for 5mC-DNA and 0.016 mM−1 s−1 for O2, which were less than those in AA+ condition [kcat/Km(5mC) = 4.42 mM−1 s−1],5 confirming that the absence of AA reduces the catalytic efficiency of TET2.

Figure 3. Dynamic plots of O2 consumption over TET2-mediated 5mC oxidation time at different concentrations of (a) 5mC-DNA and (b) O2. Michaelis-Menten curves and Lineweaver-Burk plots (insets) of TET2 for (c) 5mC-DNA and (d) O2. Error bars indicate standard deviation (n = 3).

Notably although several MOFs which are mostly based on noble metal have been reported to real-timely sense O2 even in cells,23,24 they cannot be directly applied for TET kinetics assay in cells due to the complex intracellular environment and interference from reducing substances or other O2-consuming biological processes. In vitro assay is still the primary method and can also provide rich information. The real-time assay overcomes the limitations of the common methods for TET kinetics study in terms of accessibility, detection speed and accuracy (Table 1). The sampling time was considerably shortened from minutes to less than 1 second. As the linear phase was within 10 min (Figure 3a and b), the total analytical time of our assay was less than 10 min, which was noticeably faster than other methods. The relative standard deviation (R.S.D) of the detection was greatly reduced from 10% – 68% to 0.68% – 4.2%, resulted from the simplified measurement procedure. More importantly, the V0 we obtained from the complete and continuous enzyme reaction curve is relatively accurate, while the end-point methods only can provide an average V0 value (Figure S14).

5

ACS Paragon Plus Environment


Figure 4. (a) Michaelis–Menten model for 5mC under conditions of Km1 ≫ Km2 and VAA+ > VAA−. Fold change is the ratio of VAA+/VAA−. (b) Analysis of 5mC and 5hmC DIP-seq data from ref. 25. (c) Box plot analyzed from (b) showing 5mC density for all methylated promoters and dramatically demethylated promoters (fold change of V > 200), *P < 0.05 by analysis of variance (ANOVA). (d) Fold change in gene expression after 72 h of AA treatment decreasing with the increase in 5mC DIP-seq reads (data from ref. 25)

Prediction of Stem-Cell Behavior of TETs. As Km presents the substrate concentration at which half the active sites of enzyme are occupied by substrate, larger Km value means that the enzyme is not easily to be saturated by the substrate in cell, and the reaction rate (also enzyme activity) will be more sensitive to the variation of the intracellular substrate concentration. It is noteworthy that our Km(5mC) value was 50 − 200-fold larger than those obtained in AA+ condition (Table 1). To illustrate this difference, we presumed a Michaelis–Menten model: (1) Km1(5mC) ≫ Km2(5mC) (eg. Km1(5mC) = 100 × Km2(5mC)], where Km1(5mC) and Km2(5mC) are the Km values from AA− and AA+ treatment, respectively; (2) VAA+ > VAA−. As can be seen, the fold change in demethylation rate (VAA+/VAA−) significantly declined with the increase in 5mC concentration (Figure 4a). Neither Km1(5mC) < Km2(5mC) nor Km1(5mC) = Km2(5mC) show this trend (Figure S15). As TET1 shows similar Michaelis-Menten kinetics with TET2,5−7 the model can also be applied for TET1. Therefore, we speculated that AA would stimulate TET activities (TET1 and TET2) more strongly in hypomethylated genes compared with hypermethylated ones, thus influence more the expression of hypomethylated genes. To testify the validity of our hypothesis, we investigated the relationship between demethylation rate and 5mC density in 1045 methylated promoters in AA+/AA− mES cells by analyzing the published data of DIP-seq (DNA immunoprecipitation followed by deep sequencing),25 which is a preferable model as mES cells express both TET1 and TET2 but barely TET3.26 The demethylation rate was considered as the 5hmC DIP-seq reads (reads per kilobase per million, RPKM) at methylated promoters after 12 h culture (V, RPKM 12 h−1, y-axis of Figure S16). The 5mC density was calculated by adding the gain of 5hmC to the DIP-seq reads of 5mC remained in 12 h untreated mES cells. As can be seen, the demethylation rate increased in both AA− and AA+ mES cells

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with the increase in 5mC density (Figure S16), suggesting that TET1 and TET2 are regulated by 5mC concentration in both AA− and AA+ cells. This was in line with the fact that TET1 and TET2 are highly redundant in mES cells and are not saturated by 5mC.25 The fold change of VAA+/VAA− remarkably decreased with the increase in 5mC density (Figure 4b), which well supported our kinetics model. Furthermore, the promoters dramatically demethylated by AA (fold change >200) had lower 5mC density (Figure 4c), suggested that the gene expression changed by AA treatment would be associated with the 5mC density in promoters. From the microarray data of the 1045 methylated promoters, approximately 20 genes particularly 5 germline-associated genes (related to gene ontology term ‘reproduction’) changed obviously (more than 2fold) in expression after 72 h of AA treatment.25 Therefore, we analyzed these 5 germline genes including Asz1, Wfdc15a, Dazl, Fkbp6 and Dpep3, and revealed that AA upregulates more the expression of germline genes with less methylated promoters compared to those with higher methylated promoters (Figure 4d). Hypoxia-Repressed TET Activities can be Rescued by AA. The kinetics model can be employed to not only predict the stemcell behavior of TETs, but also resovle the conflict of TET activities in hypoxic condition. The Km(O2) value we obtained was 1.5 − 15fold larger than those previously achieved (Table 1). We presumed a Michaelis–Menten model following the conditions: (1) Km1(O2) > Km2(O2) (eg. Km1(O2) = 5 × Km2(O2)); (2) VAA+ > VAA−. As can be observed, the fold change (VAA+/VAA−) declined with the increase in O2 concentration (Figure 5a), but the downward trend was not distinct compared to that in model for 5mC (Figure 4a) due to a much smaller difference between Km1 and Km2. Therefore, we speculated that (1) TET activities would be more sensitive to O2 concentration and in turn downregulated by hypoxia in AA– cells; (2) TET activities in hypoxia-treated cells would be raised more by AA treatment than in normoxia-treated cells. For verification, we investigated the simultaneous effect of AA and hypoxia on DNA demethylation in mES cells. The mES cells of R1 were exposed to 21% O2 (normoxia) or 0.5% O2 (hypoxia) for 24 h in AA+/AA− condition. The global 5hmC levels in these cells were visualized by immunofluorescence (IF) staining experiments using 5hmC-specific antibody (Figure 5b). Compared with that in normoxia/AA− cells, the global 5hmC level decreased in hypoxia/AA− cells, which is in line with the previous observation.7 In hypoxia/AA+ and normaxia/AA+ mES cells, the global 5hmC level significantly increased, affirming the promotion of AA towards TET activities. The reasons for the change of global 5hmC level were further investigated. As the possible change of TETs would cause the change of global 5hmC level, the expression of TETs in mES cells was profiled from TET genes by qRT-PCR analysis. As can be found, three TETs did not significantly change in treated cells (Figure 5c). Besides TET expression, the possible cell differentiation would also cause 5hmC change.27,28 However, compared with that in normoxia/AA− (untreated) mES cells, the levels of Oct4, Nanog, and Sox2, representing cell pluripotency, almost kept constant, and the level of the ectoderm differentiationrelated Fgf5 was down-regulated in treated cells (Figure 5d). The results indicated that AA or hypoxia treatment for 24 h did not impair TET expression or stem cell maintenance.


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Figure 5. (a) Michaelis–Menten model for O2 under conditions of Km1 > Km2 and VAA+ > VAA−. (b) IF images of nuclei DAPI staining, 5hmC, and merged images in R1 mES cells with (+) or without (−) AA in hypoxia or normoxia. Scale bar, 50 μm. qRT-PCR analysis for (c) TET transcript levels relative to TET1 in N/AA– (set to 1) and (d) marker genes in mES cells relative to N/AA– (set to 1). Error bars indicate standard deviation for triplicate experiments from three independent assays, ****P < 0.0001 by t-test. (e, f) Dot-blot assay and quantification by Image-Pro Plus 6.0 indicated an overall increase of 5hmC upon treatment of AA in mES cells under hypoxic (H) and normoxic (N) conditions. Error bars indicate standard deviation (n = 3 biological replicates), *P < 0.05, **P < 0.01 by t-test. (g) Analysis of relative 5hmC levels from (f): Fold change in relative 5hmC levels for cells in normoxia (VN) relative to hypoxia (VH). Error bars indicate standard deviation (n = 3 biological replicates). (h) Analysis of the outcome of (f).

The intensities of 5hmC dot blot further confirmed our model (Figure 5e, f). The fold change in 5hmC from nomorxia relative to hypoxia (VN /VH) was close to 1 in AA+ cells, but larger in AA− cells (Figure 5g). The results suggest that with promotion of AA, TETs in hypoxia can retain the same good activities as those in normoxia. Once AA was removed, the TET activities were downregulated by hypoxia, leading to an increased fold change of VAA+/VAA− in hypoxia (Figure 5h). The log2 of the fold change of VAA+/VAA− in hypoxia (~1.4) was much smaller than that in hypomethylated genes (~10) in Figure 4b, fully in conformity with the downward trend varied in models for 5mC and O2 (Figure 4a, Figure 5a). Overall, we conclude that AA directly rescues the oxidative activities of TETs from hypoxia independently of changes in cell pluripotency or TET expression, which has not been discovered yet. While without AA, hypoxia reduces TET activities in mES cells which was in accordance with the previous research.7 Mechanism of O2 Influence on TET2 Kinetics. Based on our study, a new mechanism of O2 indirect influence on TET2 kinetics was derived (Figure 6). In detail, when TET-mediated 5mC oxidation is performed under air atmosphere, the high concentration of O2 makes Fe2+ more likely to be oxidized to Fe3+ spontaneously.29 The TET2 activity relies more on AA to rescue Fe2+. Therefore, the absence of AA greatly increased Km(air, 5mC) (50 – 200-fold). In contrast, under low-O2 or hypoxic condition, Fe2+ is more stable due to the inactivation of its spontaneous oxidation, thus Km(hypoxia, O2) was affected much less (1.5 – 15fold) by AA. Even in the same AA+ solutions, the Km(O2) obtained in modest hypoxia (2% – 5% O2)6 is 10-fold larger than that obtained in tumor hypoxia (0.2% – 0.5% O2),7 indicating that

TET2 has higher affinity to substrate in tumor hypoxia than in modest hypoxia due to a better reservation of Fe2+ by O2 shortage.

Figure 6. Outline of the indirect influence of O2 on TET2 activity in the absence of AA via Fe2+ oxidation.

O2 Dependence of TET2. For a long time, it was unclear whether TETs act as a physiological O2 sensor like other members of the Fe2+- and α-KG- dependent dioxygenases, such as hypoxiainducible factor (HIF)-prolyl-hydroxylase domain proteins (PHDs) and factor inhibiting HIF (FIH). Under the same AA− condition, the mean values of Km(O2) for PHD2, FIH, taurine dioxygenase (TauD) and mature phytanoyl-CoA hydroxylase (mPAHX) were reported in the range from 76 – 240 μM, indicating that the activities of these dioxygenases are closely dependent on O2.22 In comparison, the Km(O2) of TET2 obtained here (43.8 ± 0.3 μM) showed a relatively low value (Table S4), supporting the model that TET2 is less sensitive to the change of O2 content in modest hypoxia and may not act as a physiological O2 sensor.

CONCLUSIONS



In summary, we present the first demonstration on real-time TET2 kinetics assay by a MOF-based O2 film sensor. The O2sensitive MOFs show perfectly tunable sensitivity to dissolved O2, ranging from cellular hypoxia to air atmosphere, which meets the requirements of enzyme analysis in different environments. The assay overcomes the limitations of current off-line methods, providing more precise and reproducible results. From comparative study of Km, the stem-cell behavior of TETs was successfully predicted, and the effect mechanisms of factors (AA, O2, Fe2+) on TETs were revealed. These results would provide guidance for further research on regulatory mechanisms of varied factors on TET-mediated demethylation in stem cells that follow a predictable pattern, and researchers can just use this kind of kinetics model to study other substrate dependences of enzymes while without doing cell experiments. Besides, the significance of our work is not limited to the functional elucidation of these influential factors and the application of kinetics parameters to stem cell research. For instance, our finding that the influential extent of AA on germline gene expression is associated with promoter methylation, could provide guidance for the treatment of certain reproductive diseases. The method is believed to be promising in wide application on kinetics analysis and cell behavior prediction of other important O2-related enzymes.

ASSOCIATED CONTENT Supporting Information This material is available free of charge via the Internet at hppt://pubs.acs.org. Expression and purification of TET2, material characterization, and additional figures and tables (PDF)

AUTHOR INFORMATION Corresponding Author *Z.D.: E-mail: [email protected]. *J.-P.Z.: E-mail: [email protected].

Author Contributions #

Y.X. and S.-Y.L contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We acknowledge Prof. Liang Xu for his insightful discussion. We also acknowledge Prof. Junjun Ding for providing us with R1 mES cells. This work was supported by the National Natural Science Foundations of China (21775169, 91622109, and 21675180), the Scientific Technology Project of Guangzhou City (201604020145), and the Scientific Technology Project of Guangdong Province (2016B010108007, 2015A030401033).

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Real-Time Sensing of TET2-Mediated DNA Demethylation in Vitro by

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