Expression of Rat DNA (cytosine-5) Methyltransferase (DNA MTase) in Rodent Trophoblast Giant Cells: Molecular Cloning and Characterization of Rat DNA MTase

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.

253, 495–501 (1998)

RC989802

Expression of Rat DNA (cytosine-5) Methyltransferase (DNA MTase) in Rodent Trophoblast Giant Cells: Molecular Cloning and Characterization of Rat DNA MTase Hiromichi Kimura, Toyokazu Takeda, Satoshi Tanaka, Tomoya Ogawa, and Kunio Shiota Laboratory of Cellular Biochemistry, Animal Resource Science/Veterinary Medical Science, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received October 29, 1998

Methylation of genomic DNA is involved in the basic methanism of gene inactivation, chromatin organization, X chromosome inactivation and genomic imprinting. A pattern of DNA methylation is maintained in mitotic cells by DNA (cytosine-5) methytransferase (DNA MTase). The DNA MTase has been shown to be also expressed in postmitotic cells such as neurons. In the present report, as an approch to analyzing mechanisms underlying regulation of DNA MTase expression, we first isolated rat DNA MTase cDNA. The isolated cDNA encoded a protein of 1,622 amino acid residues showing 88.3% and 64.2% of homology with mouse and human DNA MTase, respectively. Northern blot analysis showed that DNA MTase mRNA was highly expressed in placenta during mid- to late- pregnancy. We then analyzed the expression of DNA MTase in Rcho-1 cells, a rat choriocarcinoma-derived cell line, which cease cell division but keep replicating genomic DNA when differentiated in vitro. We found that the expression of DNA MTase protein was decreased in terminally differentiated Rcho-1 cells whereas DNA MTase mRNA was consistently expressed. This result suggested posttranscriptional regulation of DNA MTase activity in Rcho-1 cells. The Rcho-1 cells would be a valuable model for studying the regulation of gene expression and function of DNA MTase in postmitotic, differentiated cells. © 1998 Academic Press

DNA (cytosine-5) methytransferase (DNA MTase) is the methylating enzyme in CpG dinucleotides and its cDNA has been cloned in the mouse and human (1)(2). Methylation of genomic DNA is involved in the basic mechanism of gene inactivation, chromatin organization, X-chromosome inactivation and genomic imprinting, which are related to the various biological events

including embryogenesis (3), DNA repair (4) and tumour progression (5)(6)(7)(8) in mammals. DNA MTase was also expressed in postmitotic cells such as neurons (9), suggesting that DNA MTase plays a role in not only mitotic cells but also postmitotic cells and there seem to be several mechanisms in the regulation of DNA MTase activity depending on the cell type. DNA MTase gene expression is regulated in a cell cycle-dependent manner with a peak at S phase (10) and is greatly decreased in differentiated cells in vitro (11)(12). The pattern of DNA methylation is inherited directly by the daughter DNA strands through DNA replication in mitotic cells. In contrast to the usual diploid cells, rodent placenta contains trophoblast giant cells (TGC), whose genome has been shown to be amplified up to 100 times that of the haploid genome (13)(14). We previously showed that this genome is constructed of polyploidal DNA, and the differentiation process of rat TGC involves a change in the methylation pattern of CpG islands (15). TGCs are therefore unusual and distinct from general diploid cells because DNA synthesis does not cease at the start of differentiation. The Rcho-1 cell is a choriocarcinoma cell line derived from a spontaneous tumor in WKA-strain rat, and it was originally isolated by Teshima et. al. (16). Rcho-1 cells have been shown to possess the similar characteristics to TGC and continue to replicate genomic DNA while mitotically arrested during the differentiation, so that Rcho-1 cells have been used to study the differentiation of TGC by analyzing the gene expression of placental prolactin (PRL) like hormones as a differentiation molecular marker (17). Various differentiation states can be induced in Rcho-1 cells depending on culture conditions: (i) proliferative in the growth medium, (ii) not proliferative and differentiated with continuous DNA synthesis by short term culture in the differentiation medium (day 4 of differentiation in the

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text) and (iii) not proliferative, terminally differentiated without DNA synthesis by long term culture in the medium (day 8 of differentiation). In the present study, we cloned cDNA for rat DNA MTase, prepared the specific antibody by using recombinant rat DNA MTase and investigated the expression of DNA MTase in rat placenta and Rcho-1 cells in each differentiation state. MATERIALS AND METHODS Probes for screening. To use as probes, three DNA fragments of rat DNA MTase cDNA were amplified by RT-PCR in which 1mg of total RNA from rat adult brain or spleen was first reverse transcribed with 20 units of RNaseH- reverse transcriptase (Superscript; GIBCO BRL) and 0.5mg of oligo (dT) 12–18 primer, then amplified with 0.5units of rTaq polymerase (TOYOBO) employing the oligonucleotide primers; sense, 59-TGGAGAGCAGAAATGGCAG-39, antisense, 59-GTCGTTTTTCGTCTCTTCTC-39 for probe A, sense, 59-GATGAACCCCAGATGTTGAC-39, antisense, 59-GCTGTGACCCTGGCTAGATA-39 for probe B, sense, 59-CCACCAAACCATGCCCGCAG-39, antisense, 59-AGGGGTGGTGGCACAGCATT-39 for probe C. The PCR products were subcloned into pGEM-T or pGEM-T Easy vector (Promega) and sequenced by the standard protocol (18). Library screening and sequencing. The oligo (dT) and random primed 8-12 week-old rat brain cDNA library (lgt11 59-stretch) was purchased from Clontech. An oligo (dT)-primed 14 day-old rat placenta cDNA library in lZAP was constructed from the mRNA obtained from the placentae of day 14 of pregnancy. The lgt11 library of 5 3 105 plaque-forming units (pfu) and the lZAP library of 3 3 105 pfu were screened with three RT-PCR fragments as described in the text (Fig. 1A). Positive clones for each probe were subcloned into the splI site of pUC119 or pBluescript and sequenced by the standard protocol (18). Northern blot analysis. Total RNA was isolated from various adult rat tissues, and proliferating or differentiating Rcho-1 cells with TRIzol (GIBCO BRL), fractionated in 1.0% denaturing agarose gels containing 2.2M formaldehyde and blotted onto nylon membranes. The blots were probed at high stringency with antisense RNAs for the rat DNA MTase catalytic domain, PL-I (19), PLP-D (17) and rat GAPDH cDNA (20) labeled with digoxigenin-UTP (DIG) by in vitro transcription with T7 or SP6 RNA polymerases. Specific signals were detected with a DIG Luminescent Detection Kit (BOEHRINGER MANNHEIM). Cell cultures. Rcho-1 trophoblast cells were maintained in Rcho-1 medium: NCTC 135 (Sigma) supplemented with 20% FBS, 10mM HEPES, 50mM b-mercaptoethanol, 1mM sodium pyruvate. To induce differentiation, subconfluent Rcho-1 cells were spilit in half and cultured overnight in the Rcho-1 medium, after which the cells were rinsed twice with phosphate-buffered saline (PBS) and further cultured in NCTC135 supplemented with 10% Horse Serum (JRH), 10mM HEPES, 50mM b-mercaptoethanol and 1mM sodium pyruvate (Differentiation medium). NRK49F (NRK) cells were maintained in Dulbecco’s modified essential medium (DMEM) (GIBCO BRL) supplemented with 5% FBS (DMEM-5FBS). To arrest mitosis, NRK cells were cultured for 6hr in DMEM-5FBS followed by incubation in a serum-free DMEM for 36hr. All culture media were also supplemented with 100U/ml of penicillin and 100mg/ml of streptomycin (Sigma). All cells were cultured at 37°C in 5% CO2/95% air. Antibody production. Recombinant partial rat DNA MTase proteins (residues 109-318) fused to thioredoxin protein containing 6xHistidine (TRX/6His protein) was expressed by using the pET TRX Fusion System 32 (Novagen) and were roughly purified with Ni21 agarose culumns (QIAGEN). The recombinant proteins were purified further by preparative SDS-polyacrylamide gel electro-

phoresis (SDS-PAGE) and used to immunize rabbits. Antibodies were depleted of anti-TRX/6His protein antibodies with TRX/6His protein linked to Ni21 agarose. Resulted antisera were affinitypurified with rProtein A-sepharose FF (Pharmacia Biotech) for immunoprecipitation. The concentration of antibody was determined by the UV method (21). Western blot and immunoprecipitation. Cell extracts were prepared in NP-40 buffer [50mM Tris, pH 8, 400mM NaCl, 1% Nonidet P- 40 (NP-40) ] with the addition of 1mM PMSF, 5mg/ml leupeptin, 1% aprotinin, 5mg/ml pepstatin, 1mM Na3VO4, 0.5mM EDTA and 0.5mM NaF. The concentration of whole cell extract was quantitated by the BCA method (PIERCE). Extracted proteins (20mg) were fractionated on 7.5% SDS-PAGE, then transfered to PVDF membrane by a semidry transfer method in 100mM Tris, 192mM glycine (for 1hr at 1.0 mA/cm2). After blocking with 5% skim milk in TBST (0.1M Tris, pH 7.6, 150mM NaCl, 0.1% Tween 20), membranes were probed with the rabbit anti-DNA MTase antiserum diluted at 1:10,000 with 0.5% skim milk in TBST for 1hr at room temperature (RT). The secondary antibody was peroxidase-conjugated goat anti-rabbit IgG antibody (Jackcon Immunoreserch) at 1:5,000-20,000 in 5% skim milk in TBST. Signals were detected by ECL (Amarsham). Whole cell extract was immunoprecipitated with 5mg of the appropriate antibodies bound to 10ml of protein A-sepharose CL-4B (Pharmacia Biotech) in IP buffer [50mM Tris, pH 8, 150mM NaCl, 1% Nonidet P-40 (NP-40)] with the addition of 1mM PMSF, 5mg/ml leupeptin, 1% aprotinin, 5mg/ml pepstatin, 1mM Na3VO4, 0.5mM EDTA, 0.5mM NaF. Immunecomplexes washed three times with washing buffer (50mM Tris pH 7.2, 150mM NaCl, 1% Triton X-100, 1% Sodium deoxcolate, 0.1% SDS) were separated by SDS-PAGE prior to immunoblot with the appropriate antibodies. BrdU incorporation. The 5-bromo-29-deoxuridine (BrdU, final 10mM) was added to the culturing well and incubated at 37°C in 5% CO2/95% air for 2hr. The amount of BrdU incorporated was measured with a Cell Proliferation ELISA, BrdU (colorimetric) kit (BOEHRINGER MANNHEIM) according to instructions in the manufacturer’s manual. Nuclear staining. Rcho-1 cells were fixed in 4% paraformaldehyde (PFA) for 20min. The fixed cells were incubated in 0.5%Triton X-100 in PBS for 5min followed by an incubation in 0.05% Tween 20 in PBS for 5min, after which the cells were washed with PBS and incubated with 10ng/ml DAPI in PBS for 30min. The samples were washed twice with PBS before observation. All operations were parformed at RT.

RESULTS Molecular cloning of rat DNA MTase cDNA. To isolate rat DNA MTase cDNA, three fragments of rat DNA MTase cDNA were cloned by RT-PCR with primers designed for conserved regions between mouse and human, and were used as probes (Fig. 1A). By screening the rat cDNA libraries, three, three and six positive clones for probe A, B and C, respectively, were obtained from the cDNA libraries. The clones containing the longest insert for each probe were subcloned and sequenced. These clones overlapped (Fig. 1B), and the overall length of the assembled cDNA sequence for rat DNA MTase from the above clones is 5,253 bases, including 22 nucleotides of poly(A) tail. A single long open reading frame (ORF) encodes a polypeptide of 1,622 amino acid residues having a predicted moleculer weight of approximately 186 kDa (Fig. 1C). The amino acid sequence of the ORF shows 88.3% and

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FIG. 1. Cloning of rat DNA MTase (A) Position of three probes; probes A, B and C partially contain a sequence correspond to the nuclear localization signal (NLS), DNA replication foci-targeting sequence and catalytic domain of reported animal DNA MTase, respectively. (B) Three isolated cDNA fragments analyzed further. (C) The amino acid sequence of rat DNA MTase (D) Schematic comparison of the amino acid sequences among rat, mouse, human DNA MTase and bacterial cytosine methyltransferase (28). NLS, nuclear localization signal; PBHD, Polybromo-1 protein homologous domain. 497

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FIG. 2. Expression of DNA MTase mRNA in tissues. (A) Tissue distribution of rat DNA MTase mRNA. Total RNA was harvested from brain (B), placenta (Pl), liver (Li), kidney (Ki), lung (Lu), heart (Ht) and spleen (Sp). Each sample contains 5mg of total RNA was separated on 1.0% denaturing agarose gel, transferred to the membrane and hybridized to DIG-labeled probe C (see FIG. 1A). The size of the band is 5.2kb. (B) Expression of DNA MTase mRNA in the placenta during pregnancy. Total RNA was harvested from the placentae on days 12, 16 and 20 of pregnancy. Each sample contains 10mg of total RNA was separated on 1.0% denaturing agarose gel, transferred to the membrane and hybridized to DIG-labeled probe C. The expression of GAPDH mRNA is shown as an internal control.

64.2% of homology with that of mouse and human DNA MTase, respectively. A putative catalytic domain of the amino acid sequence showed higher homology with that of mouse (93.8%) and human (89.5%) DNA MTase, respectively. Characteristic motifs were also found in the amino terminal domain of rat DNA MTase, such as nuclear localization signal (NLS), DNA replication focitargeting sequence, Zn-binding motif, Polybromo-1 protein homologous domain (PBHD) and GK-repeats (Fig. 1D). Expression of DNA MTase mRNA in tissues. Northern blot analysis with the probe A used at high stringency showed the presence of hybridizing RNA of 5.2kb in all tissues analysed (Fig. 2A). The 5.2kb mRNA was present at relatively high levels in the brain, placenta, lung, and spleen and at low levels in the liver. DNA MTase mRNA was therefore expressed in all tissues tested, although there was variation in the level of expression. The expression of DNA MTase mRNA was the highest in the placenta. The expression of DNA MTase mRNA was investigated in the placenta obtained from mid- to latepregnant rat (Fig. 2B). Northern blot analysis showed that DNA MTase mRNA is highly expressed and the level is constant during mid- to late-pregnancy. These findings indicate that the mRNA of DNA MTase is strongly expressed in the placenta throughout pregnancy.

Nuclear size and DNA synthesis in differentiating Rcho-1 cells. To investigate whether expression of DNA MTase is associated with DNA replication in placenta, we used Rcho-1 cells that is only one rodent placental cell line. The cells were induced to differentiate by changing culture conditions (see Materials and Methods). The differentiated Rcho-1 cells showed TGC-like properties (Fig. 3A). The nuclear size of differentiating Rcho-1 cells showed an increase on day 4 of the culture in the differentiation medium and further increase was attained by day 8 of the culture. In the culture conditions, Placental Lactogen-I (PL-I), a marker for TGC in early/mid-pregnancy (22)(19)(23), is expressed on day 4 of differentiation, whereas Prolactin-Like Protein-D (PLP-D), a marker for TGC in late-pregnancy (17), is expressed on day 8 of differentiation (Fig. 3B). To evaluate the DNA synthesis activity in differentiating Rcho-1 cells, we measured BrdU incorporation in the differentiating Rcho-1 cells and in the serumstarved NRK cells and compared them. The BrdU incorporation index of Rcho1 cells on day 4 of differentiation reached 45–50% of that of undifferentiated, proliferating Rcho-1 cells (column 2 in Fig. 3C). On days 8 and 12 of differentiation, the BrdU incorporation index was decreased by 83–90% (columns 3 and 4 in Fig. 3C). In NRK cells, high activity of BrdU incorporation was observed in the proliferative condition and the activity was dramaticaly suppressed in the serum-starved condition. DNA synthesis was still higher in differentiating Rcho-1 cells (day 12 of differentiation) than that of mitotically arrested NRK cells (p,0.01) (column 5 in Fig. 3C). Therefore, DNA synthesis persists in differentiated Rcho-1 cells at least till day 12 of differentiation. Expression of DNA MTase in differentiating Rcho-1 cells and serum-arrested NRK cells. Western blot analysis of whole cell extract harvested from Rcho-1 cells revealed only a 186kD band in size (“WCE” lane, Fig. 4A). The antibody was also applied for immunoprecipitation from whole cell extract harvested from Rcho-1 cells. The specific band of DNA MTase was not observed in the experiment with preimmune serum as a negative control (“PI” lane, Fig. 4A). In contrast, a specific 186kD band identical to the Western blot analysis of immunoprecipitates from whole cell extract harvested from Rcho-1 cells was detected (“anti-MTase” lane, Fig. 4A). The antibody against rat DNA MTase is useful for both Western blot and immunoprecipitation. Western blot analysis showed that DNA MTase protein was expressed in growing NRK cells, but DNA MTase protein was not detectable in serum-arrested NRK cells (Fig. 4B). In serum-arrested fibroblast, Balb/c 3T3 cells, expression of DNA MTase mRNA was not detectable (10). These results suggest that the level of expression of DNA MTase protein is correlated with

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FIG. 3. (A) Nuclear size in differentiating Rcho-1 cells. a and 1, proliferating Rcho-1 cells; b and 2, day 4 of differentiation; c and 3, day 8 of differentiation. All photos are of the same magnification. The intensity was measured by means of NIH Image software. Values are standardized to that of proliferating Rcho-1 cells. Bars indicate the mean value for each treatment, vertical lines indicate the SEM (n54). (B) Total RNA was harvested from proliferating Rcho-1 cells or differentiating Rcho-1 cells. Each sample contains 20mg of total RNA was separated on 1.0% denaturing agarose gel, transferred to the membrane and hybridized to DIG-labeled probe PL-I (19) and PLP-D (17) as mid- and late-differentiation molecular marker. The expression of GAPDH mRNA is shown as an internal control. P, proliferating Rcho-1 cells, D 4, day 4 of differentiation; D 8, day 8 of differentiation. (C) BrdU incorporation in differentiating Rcho-1 cells and NRK cells. 1, proliferating Rcho-1 cells; 2, day 4 of differentiation; 3, day 8 of differentiation; 4, day 12 of differentiation; 5, serumarrested NRK cells; 6, the growing NRK cells. Values are standardized to that of proliferating Rcho-1 cells. Bars indicate the mean value for each treatment, and vertical lines indicate the SEM (n53). *, Significantly different from serum-arrested NRK cells (p,0.01).

FIG. 4. Expression of DNA MTase protein and mRNA. (A) Detection of rat DNA MTase protein by anti-rat DNA MTase antiserum. WCE, twenty micrograms of whole cell extract of undifferentiated, proliferating Rcho-1 cells; PI, protein immunoprecipitated with preimmune antiserum from 200mg of whole cell extract of the Rcho-1 cells; anti-MTase, protein immunoprecipitated with antiDNA MTase antiserum from 200mg of whole cell extract of the Rcho-1 cells. (B) Expression of DNA MTase protein in NRK cells. NRK-A, protein immunoprecipitated with anti-DNA MTase antiserum from whole cell extract of 1x106 of serum-arrested NRK cells; NRK-G, protein immunoprecipitated with anti-DNA MTase antiserum from whole cell extract of 1x106 of growing NRK cells. The arrow indicates the approximately 186kD DNA MTase protein. (C) Expression of DNA MTase protein in Rcho-1 cells. DNA MTase protein was detected with anti- rat DNA MTase antiserum in the protein immunoprecipitated by anti-DNA MTase antiserum from 200mg of whole cell extract of undifferentiated, proliferating Rcho-1 cells (P), Rcho-1 cells at day 4 of differentiation (D 4) and at day 8 of differentiation (D 8). (D) Northern blot analysis of the DNA MTase in Rcho-1 cells. Total RNA (20mg) was separated on 1.0% denaturing agarose gel, transferred to the membrane and hybridized to DIGlabeled probe C (see FIG. 1A). P, proliferating cells; D 4, day 4 of differentiation; D 8, day 8 of differentiation. Photograph of the gel stained with ethidium bromide after electrophoresis is shown at the bottom.

that of the expression of DNA MTase mRNA in fibroblasts such as NRK cells. In differentiating Rcho-1 cells, DNA MTase protein was not decreased on day 4 of differentiation as in the proliferating Rcho-1 cells (“D 4” lane, Fig. 4C), but DNA MTase protein was decreased on day 8 (“D 8” lane, Fig. 4C). We tested whether this decrease in DNA MTase protein in differentiating Rcho-1 cells on day 8 of dif-

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ferentiation is due to a decrease in DNA MTase mRNA. DNA MTase mRNA was constantly expressed on both day 4 and day 8 of differentiation at a comparable intensity to that of proliferating cells (Fig. 4D), so that the level of DNA MTase protein was not correlated with that of DNA MTase mRNA in the differentiating Rcho-1 cells. Together, these findings indicate that DNA MTase is regulated at the posttranscriptional level in terminally differentiating Rcho-1 cells. DISCUSSION The amino acid sequence deduced from the sequence of cDNA for putative rat DNA MTase (Accession No. AB012214) isolated in this study is highly homologous to those of mouse (88.4%) and human (64.6%) DNA MTase. Functionally important motifs of DNA MTase such as NLS, DNA-replication foci targeting sequence, Zn-binding motif, PBHD and GK-repeats are highly conserved in the cDNA clone. The antibody raised against the recombinant N-termimal portion (109 –318 a.a.) in the isolated cDNA reacted with a protein which has a predicted size of DNA MTase, 186 kD. The cDNA is, therefore, concluded to encode the rat DNA MTase. A decrease in the enzyme activity has been reported in differentiating erythroleukemia cells (24), myoblasts (25) and teratocarcinoma cells (12). In this study, the disappearance of DNA MTase protein was observed in serum-arrested NRK cells, confirming the results of previous studies with Balb/c 3T3 cells (10), so that in general, DNA synthesis ceases when cells are forced to stop proliferation or induced to differentiate (26). In contrast to the general diploid cells, the DNA MTase protein was abundant and not decreased in the Rcho-1 cells even after the onset of differentiation (day 4 of differentiation). Even though DNA synthesis activity was decreased to the basal level on and after day 8 of differentiation, DNA MTase was still expressed in both mRNA and protein levels in the differentiating Rcho-1 cells. Since DNA synthesis occurs continuously without cell division in differentiating Rcho-1 cells, expression of DNA MTase may continue because of having DNA synthesis activity as endoreduplication. The increase in DNA MTase activity in the late G1/ early S phase is correlated with an increase in the expression of DNA MTase mRNA in Balb/c3T3 cells (10). During differentiation of cells including mouse myoblasts (11) and F9 teratocarcinoma cells (12), there is a decrease in DNA MTase mRNA. In addition, there is an increase in the turnover rates of both DNA MTase protein and its mRNA associated with the differentiation of myoblasts (11), suggesting that down-regulation of DNA MTase in differentiating cells occurs essentially at both transcriptional and posttranscriptional steps, depending on the cell type. Interestingly, the

DNA MTase mRNA level was constant regardless of the status of DNA synthesis in Rcho-1 cells although the protein level was decreased on day 8 of differentiation, so that posttranscriptional regulation seems to be involved in the down-regulation of DNA MTase in the Rcho-1 cells. It is of interest to note that the DNA MTase mRNA is highly expressed in the placenta. The mice lacking DNA MTase do not survive past mid-pregnancy, which is when placentation occurs, although the embryonic stem cells do not show signs of any defects in proliferation or survival (27). In addition, the mutant ES cells can grow normally but be eliminated when cells are induced to differentiate. We previously reported that the methylation of genomic DNA may be involved in the differentiation in the trophoblast lineage (15). These reports support the concept that the placenta is one of the main tissues utilizing DNA MTase to express or maintain cellular function. In conclusion, we isolated the coding regions of rat DNA MTase gene, and showed that the expression of DNA MTase was abundant in placenta and in Rcho-1 cells, a trophoblast derived cell line. The Rcho-1 cells will be a valuable model for studying the regulation of gene expression and function of DNA MTase in postmitotic, differentiated cells. ACKNOWLEDGMENTS This work was supported by Research for the Future Program, the Japan Society for the Promotion of Science (JSPS-RFTF97L00904), and Grants-in-Aid for Scientific Research (08556047).

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