Genome-Wide Screening for Target Regions of Histone Deacetylases in Cardiomyocytes
The acetylation status of core histones in cardiomyocytes has been linked to the development of cardiac hypertrophy and heart failure. Little is known, however, of the genes affected by abnormal histone acetylation in such pathological conditions. We recently developed a genome-wide screening method, differential chromatin scanning (DCS), to isolate genomic fragments associated with histones subject to differential acetylation. We have now applied DCS to H9C2 rat embryonic cardiomyocytes incubated with or without trichostatin A (TSA), a specific inhibitor of histone deacetylase (HDAC) activity. About 200 genomic fragments were readily isolated by DCS on the basis of the preferential acetylation of associated histones in TSA-treated cells. Quantitation of the amount of DNA in chromatin immunoprecipitates prepared with antibodies to acetylated histone H3 revealed that 37 of 38 randomly chosen DCS clones were preferentially precipitated from the TSA-treated cells, thus verifying the high fidelity of DCS. Epigenetic regulation of DCS clones was further confirmed in cells treated with sodium butyrate, another HDAC inhibitor, as well as in cardiac myocytes isolated from neonatal rats. The mRNA level of 9 (39%) of 23 genes corresponding to DCS clones changed in parallel with the level of histone acetylation in H9C2 cells. Furthermore, a physiological hypertrophic stimulus, cardiotrophin-1, affected the acetylation level of histones associated with genomic regions corresponding to certain DCS clones. Our data thus establish a genome-wide profile of HDAC targets in cardiomyocytes, which should provide a basis for further investigations into the role of epigenetic modification in cardiac disorders.
Epigenetic modification of chromatin includes methylation of genomic DNA as well as acetylation, methylation, and phosphorylation of histone proteins. Such epigenetic changes play important roles in the regulation of gene transcriptional activity associated with cell growth and differentiation as well as with organ development.1–3 Acetylation of core histones is mediated by histone acetyltransferases (HATs) and, in many instances, results in relaxation of chromatin structure and transcriptional activation of associated genes.4 Histone deacetylases (HDACs) counteract HAT activity by catalyzing the removal of acetyl moieties from lysine residues in histone tails, thereby inducing chromatin condensation and transcriptional repression.5
Regulation of histone acetylation has been linked to cardiac hypertrophy. The HAT activity of CREB-binding protein (CBP) and p300 is thus required for the induction of hypertrophic changes in cardiac muscle cells by phenylephrine.6 Consistent with this observation, inhibition of HDAC activity results in an increase in the size of muscle cells.7 Furthermore, HDACs of class II (HDAC-4, -5, -7, and -9) suppress cardiac hypertrophy in part by binding to and inhibiting the activity of myocyte enhancer factor 2 (MEF2).8 In contrast, however, HDAC2 together with Hop was found to promote cardiac hypertrophy in vivo in a manner sensitive to systemic administration of the HDAC inhibitor trichostatin A (TSA).9 Moreover, HDAC inhibitors prevent hypertrophy and sarcomere organization in cultured cardiac myocytes,10 suggestive of a positive role for HDACs in cardiac hypertrophy.
These seemingly discrepant findings may be attributable either to differential actions of different classes of HDACs (and, possibly, of HATs) with regard to myocyte hypertrophy or to a dissociation between the deacetylase activity of HDACs and a prohypertrophic function.8 Clarification of the role of HATs and HDACs in hypertrophy would be facilitated by identification of the genes targeted by these enzymes during the induction of hypertrophic changes. Little is known, however, of the genes regulated by HATs or HDACs in myocytes. Induction of the atrial natriuretic peptide (ANP) gene is associated with acetylation of histones (H3 and H4) located in the 3′ untranslated region of the gene.11 Histones bound to the β-myosin heavy chain gene have also been shown to be targeted by HATs in myocytes.8
We have recently established a new technique, differential chromatin scanning (DCS),12 for genome-wide screening of DNA regions associated with histones that are differentially acetylated between a given pair of cell or tissue samples. To isolate target genes of HDACs in cardiac myocytes, we have now applied DCS to a rat embryonic heart–derived myogenic cell line, H9C2, treated or not with TSA. More than 200 genomic fragments were readily isolated by DCS, and genomic regions corresponding to 37 clones of 38 examined were confirmed to be associated with differentially acetylated histones. Furthermore, the expression of genes located in or close to such regions paralleled the associated level of histone acetylation.
Materials and Methods
H9C2 cells were obtained from American Type Culture Collection (Rockville, Md) and were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and 2 mmol/L L-glutamine. For preparation of the tester sample, cells were incubated for 24 hours with 300 nM TSA (Wako). For other treatments, cells were incubated with 2 or 4 mmol/L sodium butyrate (Sigma) for 24 hours or with 1 nM cardiotrophin-1 (Calbiochemy) for the indicated times.
Neonatal cardiac myocytes were prepared as described previously.13 In brief, ventricular tissue was dissected from newborn rats and subjected to digestion overnight at 4°C with trypsin (1 mg/mL; Invitrogen) in Hanks’ balanced salt solution (Invitrogen). Myocytes were harvested by subsequent digestion of the tissue with collagenase (Worthington,) and were centrifuged twice at 50g to remove less dense cells such as fibroblasts. Myocytes were then cultured in DMEM-F12 (Invitrogen) supplemented with 10% fetal bovine serum and 2 mmol/L L-glutamine.
Differential Chromatin Scanning
HDAC targets were screened in H9C2 cells by DCS as described previously.12 In brief, both tester and driver cells were fixed and subjected to immunoprecipitation with antibodies to acetylated histone H3 with the use of a chromatin immunoprecipitation (ChIP) assay kit (Upstate Biotechnology). DNA fragments recovered from the immunoprecipitates were digested with RsaI (New England Biolabs), and the digestion products were ligated to the TAG adapter (5′-CCACCGCCATCCGAGCCTTTCTGCCCGGG-3′/3′-GAAAGACGGGCCC-5′). After polymerase chain reaction (PCR)–mediated amplification with the TAG primer (5′-CCACCGCCATCCGAGCCTTTCTGC-3′), the tester and driver DNA samples were digested with XmaI and SmaI, respectively. The tester DNA (0.5 μg) was ligated to the first subtraction adapter (5′-GTGAGGGTCGGATCTGGCTGGCTC-3′/3′-CGACCGAGGGCC-5′), annealed with 40 μg of the driver DNA at 67°C for 20 to 24 hours, and then subjected to PCR with the first subtraction primer (5′-GTGAGGGTCGGATCTGGCTGGCTC-3′). After digestion of single-stranded DNA with mung-bean nuclease (New England Biolabs), the amplified products were subjected to digestion with XmaI followed by a second round of subtraction PCR with the second subtraction adapter (5′-GTTAGCGGACACAGGGCGGGTCAC-3′/3′-GCCCAGTGGGCC-5′) and second subtraction primer (5′-GTTAGCGGACACAGGGCGGGTCAC-3′). The final products were digested with XmaI and ligated into pBlueScript (Stratagene). Escherichia coli DH5α cell clones transformed with the resulting recombinant plasmids were grown in 96-well plates and subjected to direct plasmid purification in the plates with the use of a Montage Plasmid Miniprep96 Kit (Millipore). The nucleotide sequences of the purified plasmids were then determined by Dragon Genomics Center (Mie) and were used to screen, with the BLAT search program,14 the nucleotide sequence database (http://genome.ucsc.edu/) assembled in June 2003 by the Genome Bioinformatics Group of the University of California at Santa Cruz (UCSC).
Quantitation of DNA
Genomic fragments immunoprecipitated by antibodies to acetylated histone H3 (Upstate Biotechnology) were subjected to PCR with a QuantiTect SYBR Green PCR Kit (Qiagen). The amplification protocol comprised incubations at 94°C for 15 s, 60°C for 30 s, and 72°C for 1 minute. Incorporation of the SYBR green dye into PCR products was monitored in real time with an ABI PRISM 7700 sequence detection system (PE Applied Biosystems), thereby allowing determination of the threshold cycle (CT) at which exponential amplification of PCR products begins. The CT values for DNA molecules in the immunoprecipitates and for those in the original sample before immunoprecipitation were used to calculate the abundance of the former relative to that of the latter. The oligonucleotide primers for PCR were 5′-CCGGAAGAGGTGGTTATGTAAA-3′ and 5′-GCTAAGAAGGGACAGGGCTAAC-3′ for the H9C2T-2_D09 clone, 5′-GTTTGTCTGGAGCCTGTACTCTC-3′ and 5′-AAGTTCTCCGTTTCAGGATTCAC-3′ for the H9C2T-2_C06 clone, 5′-CACATCCTTGGTGCTTCTGA-3′ and 5′-GAGGAGGGTGAGGAGCTGAG-3′ for the H9C2T-1_E03-1 clone, and 5′-CCCGGTGTTCTGTACGTAGG-3′ and 5′-ACTGATGGAGCATCCACATTCT-3′ for the H9C2T-S-1-8 clone.
Quantitation of mRNA
Total RNA was prepared from the tester and driver cells with an RNeasy Mini column (Qiagen) and was subjected to reverse transcription (RT) with PowerScript reverse transcriptase (BD Biosciences Clontech) and an oligo(dT) primer. Portions of the resulting cDNA were subjected to PCR with a QuantiTect SYBR Green PCR Kit. The amplification protocol comprised incubations at 94°C for 15 s, 60°C for 30 s, and 72°C for 1 minute. The CT values for cDNAs corresponding to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA and the mRNAs of interest were used to calculate the abundance of the latter relative to that of the former. The CT values for GAPDH mRNA determined with 10 μg of total RNA from TSA-treated or nontreated cells were 18.5±0.8 and 18.2±0.2 (mean±SD), respectively, validating the use of GAPDH mRNA as an internal control.
The oligonucleotide primers for PCR were 5′-AATGTATCCGTTGTGGATCTGAC-3′ and 5′-ATTGTCATACCAGGAAATGAGCTT-3′ for GAPDH, 5′-GCCTGTGATACTCTGCTTATGTGT-3′ and 5′-CTTGAGGATTTCCTCTTTCTTCTG-3′ for the inositol 1,4,5-trisphosphate receptor type 3 gene (Itpr3), 5′-CAGTACCCTGTTGAGTCATCTCTG-3′ and 5′-GAAAGCAAGGTCTTCTTATTCTGG-3′ for the NAD(P)H dehydrogenase, quinone 1 gene (Nqo1), 5′-GCCTTCTACCTGCATACTACCAAG-3′ and 5′-AGTCTCAAGATACCGGAGCACA-3′ for the metastasis-associated 1 gene (Mta1), and 5′-CTGTTGGTACCTGTGCTGTGTAG-3′ and 5′-ACTGGTAGAGTACGTCCTTGTGG-3′ for the Jagged2 precursor gene (Jag2).
Quantitation of DNA or mRNA was performed in triplicate in at least 2 independent experiments, and data are presented as mean±SD. The statistical significance of differences was analyzed by Student t test, with a probability value of <0.05 being considered significant.
DCS in H9C2 Cells
Given that the ANP gene is a known target of HDAC in myocytes,11 we first examined the effect of TSA on the acetylation level of histones bound to this gene in H9C2 cells. Real-time PCR analysis revealed that the amount of the 3′ untranslated region of the ANP gene that was precipitated by antibodies to acetylated histone H3 from TSA-treated cells was 7.85 times that precipitated from nontreated cells (data not shown), indicating that the ANP gene is indeed a target of HDAC activity in H9C2 cells.
With the use of TSA-treated cells as the tester and nontreated cells as the driver, we then performed DCS, which in effect couples ChIP with subtraction PCR. After the second round of subtraction PCR, we sequenced the isolated genomic clones in a 96-well plate format. Analysis of 3 such plates thus yielded the nucleotide sequences of 288 DCS products. Among these randomly selected products, 222 clones contained >50 bp and were used to screen the nucleotide sequence database of UCSC with the use of the BLAT program.
A total of 195 clones showed >95% sequence identity to the rat genome sequence, and 178 of these clones were located either within protein-coding genes (demonstrated or predicted) or in the vicinity (within 10 kbp) of such genes (119 independent genes) (Table 1; Table SI available in the online supplement at http://circres.ahajournals.org). Forty-two (23.6%) of the 178 clones were assigned to a region spanning the promoter (0 to −2000 bp relative to the transcriptional start site), the first exon, and the first intron of the corresponding genes. Given that protein-coding genes account for only a few percent of the rat genome,15 our data suggest that histone acetylation occurs preferentially at regions of the genome involved directly in transcriptional regulation.
Eleven DCS clones were assigned to overlapping sequences upstream of the Oct11 gene, and 7 clones were mapped to overlapping sequences at chromosomal position 8q24, a region with no annotation information (data not shown). The isolation of such multiple clones for individual genomic regions suggests that the DCS products isolated may represent most HDAC targets in H9C2 cells.
HDAC Targets in a Cardiomyocyte Cell Line
To verify the fidelity of DCS, we randomly selected 38 DCS clones and quantified the corresponding genomic fragments in immunoprecipitates prepared from both tester and driver cells with antibodies to acetylated histone H3. The amount of each DNA fragment in the immunoprecipitate relative to that in the original sample before ChIP was determined by real-time PCR. Selective amplification by DCS proved to be highly reliable (Table 1), with 37 of the 38 clones exhibiting tester-selective precipitation (tester/driver ratio of ≥1.5). It is therefore likely that DCS indeed identified targets of HDAC in myocytes.
To visualize directly the genome-wide distribution of HDAC targets, we mapped to rat chromosome figure our genomic clones whose chromosomal positions were known (Figure 1). The HDAC targets were distributed widely throughout the rat genome, although some “hot spots” for deacetylation were apparent. For example, 7 of the DCS clones mapped to chromosomal position 5q36, and detailed mapping revealed that all of these clones were located within a region spanning 27 Mbp. It is thus possible that regional alterations of chromatin structure result in coordinated transcriptional regulation of genes within the affected region.
Some of the clones listed in Table 1 correspond to loci within or close to rat genes whose products function in intracellular calcium mobilization or antioxidant processes. One such clone (H9C2T-2_D09), for instance, mapped to a region encompassing intron 21 and exon 22 of Itpr3 (Figure 2A), which encodes a receptor for inositol 1,4,5-trisphosphate that plays an important role in Ca2+-mediated signal transduction. The cytosolic concentration of Ca2+ directly regulates muscle contraction and cardiac rhythm and is a determinant of myocyte hypertrophy and heart failure.16 The amount of the genomic fragment corresponding to the H9C2T-2_D09 clone was 6.6-fold greater in the ChIP product of TSA-treated cells than in that of untreated cells (Figure 2B), indicating that the extent of histone acetylation in this region of the genome of the tester cells was 6.6 times that in the driver cells. Furthermore, inhibition of HDAC activity was accompanied by an increase in the amount of Itpr3 mRNA (Figure 2C). These data suggest that HDAC actively deacetylates a chromosomal region corresponding to Itpr3 and thereby suppresses the transcriptional activity of the gene.
Another clone (H9C2T-2_C06) was mapped to the first intron of Nqo1 (Figure 2D), which encodes a reductase that contributes to detoxification of quinones and to regulation of apoptosis.17 We examined whether the acetylation of associated histones and the expression of Nqo1 are regulated by HDAC activity in cardiomyocytes. As with Itpr3, the acetylation level of histones bound to Nqo1 was increased by TSA treatment in H9C2 cells (Figure 2E), and this epigenetic change was accompanied by an increase in the amount of Nqo1 mRNA (Figure 2F).
Nineteen (10.7%) of the 178 clones whose chromosomal location was known were assigned to loci corresponding to at least 2 genes in the rat genome. One such clone, H9C2T-1_E03-1, was mapped to a region corresponding to the first intron of Mta1 and to the last exon of Jag2 (Figure 3A). Histone acetylation in this region might thus affect the transcription of both genes simultaneously. The level of histone H3 acetylation in this region was confirmed to be greater in the tester cells than in the driver cells (Figure 3B). However, although inhibition of HDAC activity by TSA resulted in upregulation of the amount of Jag2 mRNA (Figure 3C), it had no effect on the abundance of Mta1 mRNA (Figure 3D). Histone acetylation in the genomic region that encompasses both Jag2 and Mta1 thus appears to regulate the transcriptional activity of the former gene but not that of the latter. Jag2 is a ligand for the receptor Notch1 and is abundant in the heart.18 Coculture of fibroblasts expressing human JAG2 with murine C2C12 myoblasts resulted in inhibition of myogenic differentiation of the latter cells, implicating JAG2 in regulation of this process.
We selected an additional 19 DCS clones for quantitation of the corresponding mRNAs. Among the genes examined, 6 were preferentially expressed (tester/driver ratio of ≥1.5) in the TSA-treated cells compared with the nontreated cells (Table 1).
Regulation of Histone Acetylation in Neonatal Rat Cardiac Myocytes
Our DCS analysis identified HDAC targets in a cardiomyocyte cell line. To investigate whether the level of histone acetylation at these targets is also affected by HDAC activity in differentiated cardiomyocytes, we isolated cardiac myocytes from neonatal rats and incubated these cells in the absence or presence of TSA. Consistent with the results obtained with H9C2 cells (Figures 2 and 3⇑), TSA treatment resulted in marked increases both in the acetylation level of histones associated with the genomic locus corresponding to clone H9C2T-2_D09 and in the amount of Itpr3 mRNA in neonatal rat cardiac myocytes (Figure 4A). Similarly, TSA increased both the histone acetylation level for genomic DNA corresponding to clone H9C2T-2_C06 and the abundance of Nqo1 mRNA in the rat cardiac myocytes (Figure 4B). Furthermore, TSA increased the level of histone acetylation associated with the genomic locus corresponding to clone H9C2T-1_E03-1 in rat cardiac myocytes and, as in H9C2 cells, it increased the amount of Jag2 mRNA without affecting that of Mta1 mRNA (Figure 4C). Quantitation of genomic DNA corresponding to an additional 19 DCS clones in ChIP products prepared from neonatal rat cardiac myocytes revealed that the acetylation level of bound histones was increased by a factor of >5 by TSA treatment (data not shown). These data thus indicate that the genomic clones identified by DCS in H9C2 cells represent genomic regions whose associated histones are targeted by HDAC activity in differentiated cardiac myocytes. Twelve of the 23 genes that mapped to (or in the vicinity of) the DCS clones examined were also transcriptionally activated by a factor of >1.5 in the TSA-treated neonatal rat cardiac myocytes compared with nontreated cells (data not shown).
Effects of Sodium Butyrate on Histone Acetylation
Various compounds other than TSA inhibit HDAC activity with different target preferences.19 Sodium butyrate, for example, inhibits the catalytic activity of HDACs belonging to class I or IIA, whereas TSA inhibits that of HDACs belonging to class I, IIA, or IIB. We therefore examined whether sodium butyrate also affects the acetylation level of histones associated with our DCS clones.
H9C2 cells or neonatal rat cardiac myocytes were incubated for 24 hours with 0, 2, or 4 mmol/L sodium butyrate and then subjected to ChIP, and the resulting products were subjected to real-time PCR to quantitate the amount of genomic DNA corresponding to the clones H9C2T-2_D09, H9C2T-2_C06, or H9C2T-1_E03-1. Treatment with sodium butyrate at either 2 or 4 mmol/L resulted in marked increases in the amount of DNA corresponding to each clone in the ChIP products from both H9C2 cells (Figure 5A) and neonatal rat cardiac myocytes (Figure 5B). Although the magnitudes of the effects of 2 and 4 mmol/L sodium butyrate were similar in H9C2 cells, the effects of the higher concentration were greater than those of the lower concentration in the differentiated cardiac myocytes. The genomic regions corresponding to these 3 DCS clones were thus epigenetically regulated by both TSA and sodium butyrate.
Regulation of HDAC Targets by a Hypertrophic Stimulus
Given that H9C2 cells retain the ability to undergo hypertrophic changes in response to various stimuli,20,21 we next examined whether stimulation with a physiological hypertrophic agent, cardiotrophin-1 (CT-1), affects the extent of histone acetylation at genomic regions corresponding to DCS clones in these cells. H9C2 cells were incubated with 1 nM CT-1 for various times up to 24 hours and were then subjected to ChIP and real-time PCR analysis. Quantitation of genomic DNA revealed that the level of histone acetylation at the region corresponding to clone H9C2T-1_E03-1 was increased significantly at 6 hours and was still increasing at 24 hours (Figure 6A). Similar to the effects of TSA at this locus (Figure 3), the increase in histone acetylation induced by CT-1 was not accompanied by a change in the amount of Mta1 mRNA (Figure 6B), but was associated with a transient activation of Jag2 transcription apparent after stimulation for 6 hours (Figure 6C).
Finally, we analyzed a different DCS clone for epigenetic regulation by CT-1. The H9C2T-S-1-8 clone corresponds to the promoter region for the Myocilin (Myoc) gene (Figure 6D). This gene encodes a protein involved in cytoskeletal function, and mutations in Myoc have been shown to cause a hereditary form of juvenile-onset open-angle glaucoma.22 We found that CT-1 induced a time-dependent increase in the extent of histone acetylation at the genomic region corresponding to this DCS clone (Figure 6E). Together, these data indicate that the histone acetylation level of genomic regions corresponding to our DCS clones is regulated not only by TSA and sodium butyrate but also by physiological stimuli.
With the use of our recently developed screening method, DCS, we have now identified almost 200 HDAC targets in cardiomyocytes. The acetylation of histones associated with genomic regions corresponding to 37 of 38 randomly chosen DCS clones was confirmed to be induced by treatment of H9C2 cells with TSA. Genomic regions corresponding to DCS clones were also epigenetically regulated by another HDAC inhibitor, sodium butyrate. Furthermore, the histone acetylation level of genomic regions corresponding to all 22 DCS clones examined was shown to be HDAC targets also in freshly isolated cardiac myocytes. Finally, the acetylation level of histones associated with DCS clones was shown to be regulated by a physiological stimulus, CT-1. The genomic loci identified by DCS thus include physiologically relevant targets for epigenetic modification.
The HDAC targets identified in cardiomyocytes include several genes related to cell growth or differentiation that might play an important role in heart function. In addition to Itpr3 and Jag2, such genes include those for G protein–coupled receptors (Senr, Gbl), regulators of cytosolic Ca2+ concentration (phosphodiesterase, Pik4cb, Pacsin1), and mediators of growth signaling (Ccnd1, Runx1, Fosl1, Il11, Oct11, Sox6) (online Table SI). Our data suggest that these various genes are under epigenetic control in cardiomyocytes, a conclusion that was verified for some of them by the demonstration that the level of acetylation of associated histones was regulated by the physiological stimulus CT-1. Although CT-1 has not previously been shown to elicit intracellular signaling that affects histone acetylation, our data now indicate that this is indeed the case.
Many (42 of 178) of our DCS clones mapped to the 5′-regions of genes in the rat genome. Bernstein et al recently attempted to identify loci in the human genome whose associated histones are acetylated.23 These researchers used the “ChIP on chip”24 strategy with high-density genome-tilling microarrays for human chromosomes 21 and 22. Interestingly, consistent with our results, they found that histone H3 acetylated on lysine-9 and lysine-14 was preferentially localized at the 5′ ends of genes, with 58% of such sites of histone acetylation residing in regions encompassing −1.0 to +1.0 kbp of known genes.
Acetylation of core histones is not always associated with the regulation of gene transcription, however. Histone acetylation may thus be related to chromosome replication or to chromatin remodeling unrelated to transcriptional regulation.25 In addition, various types of covalent modification, including acetylation, phosphorylation, and methylation, may exert their effects on histones in a coordinated manner, resulting in the generation of a so-called histone code.26 Consistent with these considerations, only 39% and 52% of the genes corresponding to DCS clones examined showed preferential expression in TSA-treated H9C2 cells and TSA-treated neonatal rat cardiac myocytes, respectively, compared with the corresponding untreated cells.
Although DCS does not readily allow a quantitative comparison of the level of histone acetylation between samples, our method is technically straightforward and does not require any specialized apparatus such as a DNA microarray system. In conclusion, DCS enabled us to efficiently identify HDAC targets in cardiomyocytes. Our present data support the feasibility of determining genome-wide histone acetylation profiles of the cardiovascular system. They should also provide a basis for further characterization of the importance of epigenetic alterations in the development of cardiac hypertrophy or heart failure. Application of DCS to human heart specimens has the potential to highlight such information of clinical relevance.
This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (C) “Medical Genome Science” from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by a grant from Salt Science Research Foundation (#04C7).
Original received February 21, 2005; revision received June 1, 2005; accepted June 24, 2005.
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