NTP Concentration Switches Transcriptional Activity by Changing the

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Communications NTP Concentration Switches Transcriptional Activity by Changing the Large-Scale Structure of DNA Tatsuo Akitaya,† Kanta Tsumoto,‡,§ Ayako Yamada,‡ Naoko Makita,† Koji Kubo,† and Kenichi Yoshikawa*,†,‡, Department of Physics, Graduate School of Science, Kyoto University, and CREST, Kyoto 606-8502, Japan Received January 15, 2003; Revised Manuscript Received May 19, 2003

It is becoming clearer that genetic activity is closely associated with the intracellular energy state. However, the mechanisms of this association are still unclear. In this study, we focused on large-scale changes in the structure of DNA to examine the effect of the NTP concentration on the transcription reaction with T7 RNA polymerase and compared the results with long duplex DNA to those with a short persistent-length1 fragment. The transcriptional activity dramatically changed only for long duplex DNA within a narrow range of NTP concentrations associated with changes in the large-scale structure of DNA. This result suggests that the energy state may play an essential role in regulating ON/OFF switching on transcriptional activity. Introduction There has recently been growing interest in the fact that gene activation, i.e., replication, recombination, transcription and gene expression, is heavily dependent on the energy state of the cell. Cells require ATP as an energy source in ordinary biological processes, including replication and transcription. Other nucleotides also coordinate the supply of energy in the cell. The rate of protein synthesis strongly depends on the ATP and GTP concentrations, and the initial stage of transcription is affected by the NTP concentration.2-4 It has also been shown that a change in the ATP concentration can induce the packing of diffuse DNA into compact chromosomes in the process from interphase to mitosis during cell division.5,6 The dynamics of chromatin in interphase nuclei in yeast are exquisitely sensitive to ATP depletion and to changes in the metabolic status in the cell.7,8 It has also been reported that the nutritional status, i.e., energy state, of the cell entrains the expression of abundant genes concomitantly with a circadian clock.9,10 These studies not only suggest that NTP acts as an energy source for reactions linked to gene activation but also suggest that the intracellular energy state directly regulates gene activation by some process that involves changes in the large-scale structure of DNA. Previous studies11,12 showed that transcriptional activity with condensed plasmid DNA varied relating to concentration * To whom correspondence should be addressed. Fax: (81) (75) 7533779. E-mail: [email protected]. † CREST. ‡ Kyoto University. § Present address: Department of Chemistry for Materials, Faculty of Engineering, Mie University, Tsu 514-8507, Japan.

or type of condensing agent. However, relationships between higher-order structures of individual DNA molecules and transcriptional activity still remain complicated. It has recently been established that individual giant DNA chains exhibit a large discrete transition, or switching, between elongated unfolded and compact folded states,13,14 depending on the concentrations of various kinds of condensation agents, such as polyamines,15 multivalent metal cations,16,17 and other polycationic or hydrophilic polymers.13,14 It has also been shown that this folding transition of large DNA inhibits the action of a restriction endonuclease.18 We recently found that the ATP/ADP ratio induces switching of the higher-order structure of DNA.19 This finding raises the possibility that this transition in long DNA gives rise to all-or-none, i.e., ON/OFF, switching of largescale gene activity which is dependent on the intracellular energy state. In this study, we observed the effect of the NTP concentration on the transcription reaction with T7 RNA polymerase using a long duplex DNA as the first step, to understand the correlation between the conformational change of DNA and metabolic state in intracellular environment. We found that ON/OFF switching occurs for transcription on long duplex DNA associated with changes in the large-scale structure of DNA. Experimental Section Materials. Lambda ZAP II DNA (40.82 k base pairs (bp)) provided with the Lambda ZAP II Undigested Vector Kit was purchased from Stratagene Cloning Systems (La Jolla,

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Figure 1. Top: Map of the Lambda ZAP II insertion vector DNA showing the location of phagemid pBluescript SK(-). Numbers above the map indicate the base pair number of the (+) strand. Middle: Map of pBluescript SK(-) in which the position and direction of replication (arrow) of the T7 promoter and the forward and reverse primers for PCR are indicated. Numbers below the map represent the base number of the (-) strand of pBluescript SK. Bottom: Sequences of the T7 promoter, and the forward and reverse primers indicated in the map.

CA). T7 RNA polymerase (T7 RNAP) was from Invitrogen (Gaithersburg, MD). Four kinds of ribonucleoside triphosphate (NTP: ATP, CTP, GTP, and UTP, lithium salts) were purchased from Roche Diagnostics (Basel, Switzerland). 5 X T7 buffer (0.2 M Tris-HCl (pH8.0), 40 mM MgCl2, 10 mM spermidine-(HCl)3 and 125 mM NaCl) and 0.1 M dithiothreitol (DTT) stock solutions provided together with T7 RNAP from Invitrogen were used for the transcription reaction. Spermidine trichloride to be added to the transcription reaction solution was purchased from Nakalai Tesque (Kyoto, Japan). Japanese Pharmacopoeia distilled water for injection (Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan) was exclusively used as nuclease-free water throughout the experiment: we previously ensured that RNA was stable in this medium. TE-saturated phenol solution (DNasefree, RNase-free; Nippon Gene Co., Ltd., Toyama, Japan) was used for extraction after transcription. To visualize DNA and RNA, 4′,6-diamidino-2 -phenylindole dihydrochloride (DAPI) was purchased from Wako Pure Chemicals Industries, Ltd. (Osaka, Japan) and ChromaTide BODIPY TMR14-UTP (BODYPI-UTP) was obtained from Molecular Probes, Inc. (Eugene, OR). Deoxyribonuclease (RT grade; Nippon Gene Co., Ltd., Toyama, Japan) and a RiboGreen RNA Quantitation Kit (Molecular Probes, Inc. Eugene, OR) were purchased to estimate transcriptional activities. In this study, a 140-bp fragment containing the T7 promoter sequence was used as a persistent-length template for comparison with a long double-stranded DNA, Lambda ZAP II. The 140-bp fragment DNA was prepared by PCR from Lambda ZAP II DNA as a template. Figure 1 shows a map of the Lambda ZAP II insertion vector and inserted phagemid pBluescript SK(-) and indicates the locations of the T7 promoter and the forward and reverse primers in the 140-bp fragment. Based on the sequence around the T7 promoter on Lambda ZAP II DNA provided on-line (Stratagene), forward and reverse primers were designed to obtain a 140bp region that included the T7 promoter. Both the forward and reverse primers (5′-TAAAA CGACG GCCAG TGAGC3′ and 5′-CCGCT CTAGA ACTAG TGGAT-3′, respec-

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tively) were synthesized commercially and provided by Amersham Biosciences K. K. (Tokyo, Japan). TaKaRa Taq Hot Start Version (Takara Shuzo Co., Ltd., Otsu, Japan) was used for PCR with a thermoregulatory apparatus (iCycler; Bio-Rad Laboratories, Hercules, CA). The PCR solution obtained from the first-round PCR was electrophoresed in agarose LE (Nakalai Tesque) gel and separated from the gel by conventional methods; the PCR product was then eluted with Ultra-free DE filter (Millipore). The PCR product was purified by phenol extraction and ethanol precipitation according to conventional methods. The PCR product was dissolved and used in the second-round PCR, with purification using the methods described above. The concentration of 140-bp fragments was finally estimated with a UV-visible spectrophotometer (BioSpec-mini; Shimadzu Corporation, Kyoto, Japan). Measurement of Transcriptional Activity. Lambda ZAP II DNA and the 140-bp fragment were used as templates for transcription by T7 RNAP. Transcription of Lambda ZAP II DNA or 140-bp fragment was carried out in 50 µL of reaction solution containing 40 mM Tris-HCl, 25 mM NaCl, 8 mM MgCl2, 5 mM spermidine-(HCl)3, 5 mM DTT, 50600 µM of each (0.2 to 2.4 mM total) NTP and 2 µg/mL template DNA with/without 50 U of T7 RNAP, adjusted with 0.1 M NTP stock solution and 20 mM spermidine stock solution. The transcription reaction solutions with/without T7 RNAP were incubated at 37 °C for 1 h. After incubation, 100 µL of TE-saturated phenol solution was added to terminate the transcription reaction, and the mixture of transcribed RNA and template DNA was then recovered by ethanol precipitation according to conventional methods, dried up, and dissolved with 50 µL of distilled water. To remove remaining template DNAs, the solutions were treated with 1 U of Deoxyribonuclease at 37 °C for about 1.5 h. Fifty microliters of the solution and 200-fold diluted RiboGreen working solution in TE were mixed to give 100 µL of sample, which was allowed to stand at room temperature for about 15 min. The fluorescence intensity (AU) of the sample was measured by a spectrofluorometer (FP-750; JASCO Corporation, Hachioji, Japan) with excitation at 500 nm and emission at 525 nm. The fluorescence intensity obtained for the negative control was subtracted from that for transcribed RNA. Each value was normalized so that the value at 2.4 mM NTP corresponded to 1.0. Fluorescence Microscopy. The large-scale structure of Lambda ZAP II DNA was directly observed by fluorescence microscopy as follows. Four hundred microliters of transcription reaction mixture without T7 RNA under various concentrations of NTP were incubated at room temperature for 30 min and then mixed with the fluorescence dye DAPI and observed with an inverted fluorescence microscope (the final concentration of DAPI was 0.1 µM). DAPI molecules in the sample solutions were excited by UV light (365 nm), and fluorescence images of DNAs were observed at room temperature using a Carl Zeiss Axiovert 135 TV microscope. Images were recorded on videotape through a high-sensitivity Hamamatsu SIT TV camera and an Argus 10 image processor (Hamamatsu Photonics). For observation, an aliquot of each sample solution was loaded on a chamber

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Figure 2. Relative transcriptional activity of T7 RNA polymerase in terms of the amount of RNA transcribed from (a) Lambda ZAP II DNA and (b) a 140-mer short DNA fragment, as a function of the NTP concentration in transcription reaction solution. Data were normalized so that the amount of RNA transcribed at 2.4 mM NTP is shown as 1.0. An abrupt change within a narrow range of NTP concentrations and complete suppression under 1.0 mM NTP were observed for transcriptional activity on Lambda ZAP II DNA (a) in marked contrast to the gradual change for the 140-mer short DNA fragment (b).

composed of glass slips, which were cleaned very carefully (burned at 500 °C for an hour). Because of the blurring effect of fluorescence light, the apparent size in DNA images is about 0.6 µm larger than that of the actual DNA.20 Results and Discussion A folding transition typically takes place on duplex DNA that is sufficiently longer13,14 than the persistent length of about 50 nm, i.e., 150 bp. DNA molecules longer than several tens of kbp are large enough for microscopic observation of their individual structures. In this study, we examined Lambda ZAP II DNA (40.82 kbp) and a 140-bp DNA fragment amplified from the region in Lambda ZAP that included the T7 promoter. Figure 2 shows the relative transcriptional activity of T7 RNA polymerase in terms of the amount of RNA transcribed from Lambda ZAP II DNA and a 140-bp short DNA fragment, as a function of the total NTP concentration in the transcription reaction solution. Transcriptional activity was defined in terms of the relative fluorescence intensity of RiboGreen bound to RNA. The data shown are from a representative experiment. With Lambda ZAP II DNA (Figure 2a), an abrupt change in transcriptional activity was observed at an NTP concentration of between 1.0 and 1.6, and transcriptional activity was almost completely suppressed below an NTP concentration of 0.8 mM, indicating an all-

Figure 3. Upper panels: Fluorescence microscopic images of Lambda ZAP II DNA molecules adsorbed on the surface of a cover glass obtained using a 100× objective lens. Scale bar ) 5µm. The ON/OFF state of transcriptional activity corresponding to each image is indicated above the images. Upper left: Condensed structures of Lambda ZAP II DNA molecules in a transcription reaction with 0.2 mM NTP, which corresponds to completely suppressed transcriptional activity (see Figure 2a). Upper right: Unfolded coil structures of Lambda ZAP II DNA molecules in a transcription reaction with 2.4 mM NTP, which corresponds to high transcriptional activity (see Figure 2a). Most of the large spherical structures (upper left, top) were observed on the glass surface, and they gradually increased in number while small particles (upper left, bottom) were predominantly found in the liquid phase. Lower panels: Schematic representations of the actual conformations of condensed collapsed (left) and single unfolded (right) double-stranded DNA molecules in the upper panels. The apparent sizes in the fluorescence microscopic images represented by a broken line are larger than the actual sizes due to a blurring effect.20

or-none biphasic phenomenon. In a repeated experiment under the same experimental conditions, we noted a slight shift in the transition profile of around 0.5 mM. However, we confirmed that the biphasic profile remained essentially constant. In marked contrast to Lambda ZAP II DNA, the 140-bp fragment showed a gradual, rather than an abrupt, change in transcriptional activity at an NTP concentration above 0.6 mM, with a steeper slope below 0.6 mM (Figure 2, b), which coincides with the region of reduced RNA synthesis due to a shortage of NTPs.4 These data clearly suggest that an all-or-none, i.e., ON/OFF, switching mechanism exists for transcription with large-scale DNA, and this depends on the NTP concentration, which closely relates to the metabolic energy state in living cells. Figure 3 shows fluorescence microscopic images (top) and schematic representations of the actual conformation (bottom) of Lambda ZAP II DNA molecules adsorbed on the surface of a cover glass, observed with a transcription reaction solution without T7 RNAP, but with 0.2 (left) and 2.4 mM (right) of NTP, which correspond to the OFF and ON states of transcriptional activity, respectively, as shown in Figure 2. There were marked differences in the structure of DNA molecules between the ON and OFF states.

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Spherical bright structures (top, left) predominantly appeared both on the glass surface and in the liquid phase (data not shown) at an NTP concentration of 0.2 mM, i.e., OFF state, although a small number of diffused DNA molecules were observed in some experiments. On the other hand, diffused DNA molecules (top, right) were predominantly observed both on the glass surface and in the liquid phase under the active (ON) state of transcription with 2.4 mM NTP. The bright spherical structures observed in the OFF state ranged in size from several-hundred nm to ca. 3 µm in diameter. There was also a large difference in the nature of the Brownian motion of DNA molecules in the liquid phase between the ON and OFF states. For spherical DNA in the OFF state, significant translational motion for small particles, with slower motion for larger particles, was observed without apparent intrachain fluctuation. In contrast, the diffused DNA molecules in the ON state exhibited relatively slow translational fluctuation along with marked interchain thermal motion. The results of previous studies15,21 suggested that the small spherical and diffused structures correspond to the collapsed globule and elongated unfolded structures of a single DNA molecule, respectively. Many small particles remained in the liquid phase, whereas most of the large spheres existed on the glass surface. The number of large spheres on the glass surface was shown to increase gradually under the same experimental conditions. Therefore, the large spherical structures may be interchain aggregates of DNA molecules, as illustrated in the schematic representations of the actual conformation (bottom, left), which develop under a high ionic concentration.13 It is almost clear that the ON/ OFF switching of transcriptional activity occurs based on a discrete biphasic change in the large-scale structure of DNA. Previous studies on the energy-dependent regulation of transcription have reported that transcriptional activity is controlled by nucleosomal disruption and the enhanced binding of activator to nucleosomes, mediated by protein complexes (e.g., SWI/SNF, NURF) that hydrolyze ATP.5,6,22-26 These reports have postulated that ATP does not directly interact with DNA but rather acts as a reactor or energy source for modulating proteins that interact with DNA, supposing that there is only local interaction between an activator protein and DNA. On the other hand, gene activation essentially occurs in an all-or-none, i.e., ON/OFF, manner in large-scale DNA.27 The expression of many genes is regulated spatiotemporally.10 Moreover, the injection of exogenous genes into nuclei of cells led to efficient transcription in the early S phase but apparently repressed transcription in the late S phase.28 This supports a chromatinbased transcription-factor-independent manner of transcription. What mechanism can explain the large-scale biphasic nature of gene expression? This bimodal nature may be due to cooperation in multisite binding between proteins and template DNA.29,30 However, this cooperative model cannot describe the steep change within a narrow concentration range and the discrete biphasic, i.e., ON/OFF, manner of switching, as shown in our data. Our results strongly suggest that NTP induced an intramolecular structural transition that significantly affected the ability of T7 RNA polymerase to access DNA.

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It has been previously indicated that the folding transition of giant DNA induced by a polyvalent cation is mostly driven by ion exchange between monovalent and polyvalent cations, as the counterions for negatively charged phosphate groups along double-stranded DNA.15 Therefore, the relative stabilities of the unfolded and folded states of the DNA structure substantially depend on the chemical potential of the free polyvalent cation (spermidine in the present study). Because NTPs are multivalent anions and spermidine is a multivalent cation, binding equilibria can be expected in a transcription solution. Thus, we can postulate that NTP behaves as an antagonist toward spermidine with regard to its effect on the switching of the higher-order structure of DNA molecules. Based on our previous study,15 we can speculate that polyamines displace the transition and act with NTP in relation to their electrical charge. It is highly expected that environmental chemical species such as potassium and chloride ions have a significant influence on the higher order structure and the transcriptional activity of giant DNA molecules through the similar scenario as in the effect of nucleotide triphosphates. Studies to examine such effect of the environmental species are now under progress.

Conclusions Both NTP and polyamines such as spermidine are ubiquitous in the cytoplasmic environment in which the energy is generated and from which the energy in the nucleus is supplied. Therefore, the present results suggest that gene expression may be regulated by the energy state in the cytoplasmic fluid, in addition to the well-established mechanism of regulation through key-lock interaction. This proposes a hierarchical gene activation mechanism that governs large-scale ON/OFF activation dependent on the cytoplasmic energy state, incorporated with the previously known mechanism that controls local activation by interaction between a specific activating protein and DNA. References and Notes (1) Persistent length l is defined as 〈cos ϑ(s)〉 ) exp(-s/l) where the magnitude 〈cos ϑ(s)〉 is the mean cosine of the angle ϑ(s) between the chain segments separated by the length s on the polymer chain.32 Memory of chain direction is retained on length scales shorter than l, but lost once l is exceeded. Persistent length is a constant for each given polymer chain, e.g., ca. 50 nm for duplex DNA, and the basic characteristic of polymer flexibility. (2) Roberts, J. Science 1997, 278, 2073. (3) Gaal, T.; Bartlett, M. S.; Ross, W.; Turnbough, C. L., Jr.; Gourse, R. L. Science 1997, 278, 2092. (4) Guajardo, R.; Lopez, P.; Dreyfus, M.; Sousa, R. J. Mol. Biol. 1998, 281, 777. (5) Murray, A. W. Science 1998, 282, 425. (6) Kimura, K.; Hirano, M.; Kobayashi, R.; Hirano, T. Science 1998, 282, 487. (7) Heun, P.; Laroche, T.; Shimada, K.; Furrer, P.; Gasser, S. M. Science 2001, 294, 2181. (8) Gasser, S. M. Science 2002, 296, 1412. (9) Rutter, J.; Reick, M.; Wu, L. C.; McKnight, S. L. Science 2001, 293, 510. (10) Ueda, H. R.; Chen, W.; Adachi, A.; Wakamatsu, H.; Hayashi, S.; Takasugi, T.; Nagano, M.; Nakahama, K.; Suzuki, Y.; Sugano, S.; Iino, M.; Shigeyoshi, Y.; Hashimoto, S. Nature 2002, 418, 534. (11) Baeza, I.; Gariglio, P.; Rangel, L. M.; Chavez, P.; Cervantes, L.; Arguello, C.; Wong, C.; Montanez, C. Biochemistry 1987, 26, 6387.

Communications (12) Baeza, I.; Ibanez, M.; Wong, C.; Chavez, P.; Gariglio, P.; Oro, J. Orig. Life EVol. Biosph. 1991-92, 21, 225. (13) Yoshikawa, K. AdV. Drug DeliVery ReV. 2001, 52, 235. (14) Yoshikawa, K.; Yoshikawa, Y. In Pharmaceutical PerspectiVes of Nucleic Acid-Based Therapeutics; Mahato, R. I., Kim, S. W., Eds.; Taylor & Francis: New York, 2002; pp 136-163. (15) Takahashi, M.; Yoshikawa, K.; Vasilevskaya, V. V.; Khokhlov, A. R. J. Phys. Chem. B 1997, 101, 9396. (16) Yoshikawa, K.; Kidoaki, S.; Takahashi, M.; Vasilevskaya, V. V.; Khokhlov, A. R. Ber. Bunsen-Ges. Phys. Chem. 1996, 100, 876. (17) Yamasaki Y.; Yoshikawa, K. J. Am. Chem. Soc. 1997, 119, 10573. (18) Oana, H.; Tsumoto, K.; Yoshikawa, Y.; Yoshikawa, K. FEBS Lett. 2002, 530, 143. (19) Makita, N.; Yoshikawa, K. FEBS Lett. 1999, 460, 333. (20) Yoshikawa, K.; Matsuzawa, Y. Physica D 1995, 84, 220. (21) Minagawa, K.; Matsuzawa, Y.; Yoshikawa, K.; Masubuchi, Y.; Matsumoto, M.; Doi, M.; Nishimura, C.; Maeda, M. Nucleic Acids Res. 1993, 21, 37. (22) Tsukiyama, T.; Becker, P. B.; Wu C. Nature 1994, 367, 525.

Biomacromolecules, Vol. 4, No. 5, 2003 1125 (23) Cote, J.; Quinn, J.; Workman, J. L.; Peterson, C. L. Science 1994, 265, 53. (24) Kwon, H.; Imbalzano, A. N.; Khavari, P. A.; Kingston, R. E.; Green, M. R. Nature 1994, 370, 477. (25) Varga-Weisz, P. D.; Blank, T. A.; Becker, P. B. EMBO J. 1995, 14, 2209. (26) Imbalzano, A. N.; Schnitzler, G. R.; Kingston, R. E. J. Biol. Chem. 1996, 271, 20726. (27) Felsenfeld, G. Cell 1996, 86, 13. (28) Walters, M. C.; Fiering, S.; Eidemiller, J.; Magis, W.; Groudine, M.; Martin, D. I. K. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 7125. (29) Zhang, J.; Xu, F.; Hashimshony, T.; Keshet, I.; Cedar, H. Nature 2002, 420, 198. (30) Adams, C. C.; Workman, J. L. Mol. Cell. Biol. 1995, 15, 1405. (31) Polach, K. J.; Widom, J. J. Mol. Biol. 1996, 258, 800. (32) Grosberg, A. Y.; Khokhlov, A. R. In Statistical Physics of Macromolecules; AIP Press: New York, 1994.

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