NEUROGENIN3 REGULATES SOHLH1 DURING SPERMATOGNESIS IN MICE.
Spermatogonial stem cells (SSCs) are tissue-specific stem cells in testis, and differentiation of SSCs contributes to spermatogenesis and male fertility. However, little is known about factors regulating spermatogonial differentiation. Neurogenin3 (Ngn3), a class B basic helix-loop-helix (bHLH) transcription factor, recognized for its role in promoting the development of neurons in the hypothalamus, specifically expresses in undifferentiated spermatogonia and is a critical differentiation factor of mammalian stem and progenitor spermatogonia. Sohlh1 (spermatogenesis and oogenesis specific basic helix-loop-helix 1) is regarded as a key factor in spermatogenesis, which promotes the expression of Kit, the marker of differentiated spermatogonia. In this study, we firstly confirmed that the expressionlevelofNgn3 is decreased in Sohlh1-/- mice by real-time PCR and western blot, and revealed the associationbetweenNgn3and Sohlh1.
We then showed that Ngn3 could bind to the promoter of Sohlh1andincrease the transcription of Sohlh1 by ChIP and luciferase reporter gene assay. Lastly, we demonstrated that the protein of Ngn3 and Sohlh1 could bind together in wild type testis by immune precipitation. In conclusion, our study showed the association between Ngn3 and Sohlh1 for the first time. Ngn3 may regulate spermatogenesis by binding with Sohlh1, which may be the direct target of Ngn3 in differentiation of SSC.
Key-words: Ngn3; Sohlh1; spermatogenesis; transcription factor.
The basic helix-loop-helix (bHLH) transcription factors of the Neurogenin (Ngn) family are positive regulators of neurogenesis (Kageyama and Nakanishi, 1997) and inhibitors of gliogenesis(Nieto et al., 2001; Sun et al., 2001). Basic-helix-loop-helix (bHLH) transcriptional regulators control both the determination and the differentiation of specific cell types in a variety of tissues, including muscle and nerve(Garrell and Campuzano, 1991). The three neurogenins (Ngn1, 2, and 3) form a subfamily of atonal-like sequences, which control early determination steps and then activate neuro D in each of the regions where they are expressed (Ma et al., 1996).
Ngn3 is also expressed in testis, specifically in As, Apr, and Aalspermatogonia (Yoshida et al., 2004). Spermatogonia are adult germline stem cells that can both self-renew and differentiate into spermatocytes. Differentiation of spermatogonia contributes to spermatogenesis and male fertility. Usually A1 to A4, Intermediate, and Type B spermatogoniaarenamed differentiated spermatogonia, whereas As, Apr, and Aalspermatogonia are undifferentiated spermatogonia. As spermatogoniais thought as spermatogonial stem cells (SSC) while Apr and Aal cells show virtually very few differentiation characteristics. Apr and Aalspermatogonia in seminiferous epithelium are actually differentiating cells in the sense that they are irreversibly committed to take further developmental steps in the direction of spermatocytes, which are also called spermatogonial progenitors(Phillips et al., 2010).
Little is known about factors regulating spermatogonial differentiation, especially the differentiation of undifferentiated spermatogonia. Ngn3 has been well- known as a marker of undifferentiated spermatogonia, which delineates the earliest stages of spermatogenesis in the mouse testis(Kaucher et al., 2012; Yoshida et al., 2004) has demonstrated that Ngn3 is a critical downstream effector for STAT3-regulated differentiation of mammalian stem and progenitor spermatogonia. However, the downstream effector of Ngn3 is still unclear.
Sohlh1(spermatogenesis and oogenesis specific basic helix-loop-helix 1) is regarded as a key factor in spermatogenesis. Loss of Sohlh1 causes male infertility by disrupting spermatogonial differentiation into spermatocytes. Ballow et al. (2006) reported that Ngn3 expression decreased in Sohlh1knockout mice, indicating that there may be association between Ngn3 and Sohlh1.
In this study, we analyzed the association between Ngn3 and Sohlh1 in testis by ChIP and other experiments.
MATERIALS AND METHODS
Materials: Human embryonic kidney cells (HEK293T) and C57BL/6J mice were supplied by the Laboratory Animal Centre of China Medical University, China. Sohlh1 knockout mouse was a gift of Professor Wei Yan (Department of Cell Biology and Physics, University of Nevada, Reno, USA). All animals were housed in a barrier facility under 12 hour light and 12 hour dark conditions with free access to food and water. All procedures were approved by the IACUC of China Medical University, and all experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
Real-Time Polymerase Chain Reaction: RNA was extracted from 7-daytestes by using TRIzol reagent (Invitrogen) according to the manufacturer's instructions, and RNA concentration and purity (as determined by 260/280-nm absorbance reading ratios) were measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific).For each sample, 1g of RNA was reverse transcribed with oligo(d)T priming by using a Prime Script RT Master Mix Perfect RealTime Kit (TAKARA). The cDNA was amplified with the SYBR Premix Ex TaqTMII kit (TAKARA) on a LightCycler480 (Roche). Actb served as the reference gene, and samples without cDNA or without oligonucleotides were used as negative controls. The control Actb fragment was amplified using the following primer sequences: Actb forward, 5'- GACGGCCAGGTCATCACTAT-3'; and Actb reverse, 5'-ATGCCACAGGATTCCATACC-3'. The Ngn3 primers were as follows: Ngn3-forward: 5'- AGTGCTCAGTTCCAATTCCAC-3'; and Ngn3-reverse: 5'-CGGCTTCTTCGCTTTTTGCTG-3'.
The 25 l PCR included 5 l 10 x diluted cDNA, 12.5 l SYBR Premix EX TaqTMII,2l of primers, and 5.5l H2O. The reaction was carried out for 45 cycles of 5 s at 95 C, 20 s at 60 C. The threshold cycle (Ct) data were determined from the default threshold settings, and all measurements were performed at least three times.
Western blot analysis: 7-day testis total protein was extracted through the use of Protein Extraction Kit (Thermo, USA), then 30 g of total cell extract was separated on SDS-polyacrylamide gel, transferred to a PVDF membrane (Millipore, Tokyo, Japan), and incubated overnight at 4 C with the specific antibody (NGN3, 1:1000; Abcam; GAPDH, 1:4000; Santa Cruz). Bound primary antibodies were detected with peroxidase-coupled anti-mouse or anti-rabbit secondary antibodies, and immunoreactivity was visualized by chemiluminescence (Western Lightning, PerkinElmer Life Sciences, Japan) according to the manufacturer's instructions. Digital images were captured after 30 sec of exposure by using a ChemiDoc XRS molecular imaging system (Bio-Rad Laboratories).Densitometric analysis of bands was conducted using Quantity One software (Bio- Rad Laboratories), and expression levels of NGN3 protein were compared between samples based on the normalized ratio of NGN3 to GAPDH.
Chromatin immune precipitation: Testes of 7-day mice were used for Chromatin immunoprecipitation (ChIP).After tearing, tissues were fixed with 1% formaldehyde in PBS for 10 min at 37C, to cross-link protein-DNA complexes. After nuclear protein extraction, thesamples were sonicated to fragment genomic DNA to the size ~1 kb.Then ChIP assay was performed with an EZ-ChIP - Chromatin Immunoprecipitation Kit (Millipore) according to the manufacturer's instructions. Protein-DNA complexes were immune precipitated overnight in the presence of the specific anti-NGN3 antibody (Abcam) or anti- SOHLH1 antibody (Abcam). Purified DNA was used for PCR analysis. Genomic regions of Sohlh1 or Ngn3promoter containing the E-boxes binding sites were amplified by PCR using specific primers:Sohlh1-E-box F: 5'-ACTAGAGACGGGTTACTT-3'Sohlh1-E-box R: 5'- TGGAGGGAAGCAACAAGC-3'; Ngn3-E-box F: 5'-CCCCAAACCTCCTTCATG-3', Ngn3-E-box R: 5'- CCTGGCACGCTTTATCTG-3'. Primer efficiency was examined by genomic DNA before ChIP analysis.
Ngn3 expression vector construction: The gene encoding mouse Ngn3was amplified from cDNA of mouse testis. Specific primers were: Ngn3 CDS-F: 5'- ATGGCGCCTCATCCCTTG-3', Ngn3 CDS-R: 5'- CAAGAAGTCTGAGAACACCA-3'. The PCR products were ligated into pUCm-T vector (Beyotime, China) first, and then subcloned into pEGFP-N1 with BglII and KpnI. The final expression vector was sequenced to verify construction.
Mouse Sohoh1promoter luciferase reporter constructs: We analysed the upstream region of Sohlh1(GenBank Gene ID: 227631) -2000 bp to +500 bp using Berkeley Drosophila Genome Project Neural Network Promoter Prediction software (http:// www.fruitfly.org/seq_tools/promoter.html ), and designed seven pairs of PCR primers based on the results (shown in Table 1):
Table 1. PCR primers for construction of Sohlh1promoter Luc plasmid
Primer name###Sequence###Product size
###F: 5'-CGGGGTACC AGCATTCAAGTCTACGAGCCTAT-3'###2399bp
###R: 5'- GGAAGATCT TGGAGGGAAGCAACAAGC-3'###( -2357~+42)
###F: 5'-CGGGGTACC GGAGGTGGCCTTGTTGTGT-3'###2069bp
###R: 5'- GGAAGATCT TGGAGGGAAGCAACAAGC-3'###(-2027~+42)
###R: 5'- GGAAGATCT TGGAGGGAAGCAACAAGC-3'###(-1644~+42)
###R: 5'- GGAAGATCT TGGAGGGAAGCAACAAGC-3'###(-1304~+42)
###R: 5'- GGAAGATCT TGGAGGGAAGCAACAAGC-3'###(-993~+42)
###R: 5'- GGAAGATCT TGGAGGGAAGCAACAAGC-3'###(-652~+42)
###R: 5'- GGAAGATCT TGGAGGGAAGCAACAAGC-3'###(-304~+42)
The KpnI and BglII digested sequences (underlined in Table 1) were designed in the primers, and the PCR products were subcloned into pGL3-basic with KpnI and BglII. All constructs were confirmed by sequencing.
Cell culture, transfection and luciferase assays: The ability of Ngn3binding the Sohlh1promoter region and regulate transcription was assessed using a dual firefly and Renilla luciferase assay. HEK293T cells were grown in DMEM containing 10% heat inactivated fetal bovine serum (Gibco, Carlsbad, CA, USA), at 37C in 5% CO2. Cells were plated on 96 well plates prior to performing transient transfection.24 h after plating, HEK293T cells were cotransfected with 100 ng ofNgn3 expression vector and pGL3-basic vector containing a 2399-bp portion of the Sohlh1promoter region upstream of the firefly luciferase gene (pGL3-sohlh1A Luc) plasmid, using Lipofectamine-2000 reagent (Invitrogen) in Opti- MEM(Minimum Essential Media) (Invitrogen). The pEGFP-N1, pGL-3 basic, and 20ng of Renilla luciferase plasmid (pRL-TKVector; Promega) were used as control.
After 4 h, Opti-MEM was replaced by DMEM supplemented with a 1% penicillin-streptomycin solution (Invitrogen) and a 1% fetal bovine serum (Invitrogen).48 h after transfection, cells were lysed by 1x passive lysis buffer (Promega) and luciferase activity was assayed with the dualluciferase assay system kit (Promega) according to the manufacturer's instructions (Promega, Madison, WI, USA). The ratio of reporter activity (firefly) to internal control activity (Renilla) was used to determine a relative luciferase unit (RLU) for comparison between treatments. Each transfection was performed in triplicate and experiments were carried out a total of three times. Different lengths of Sohlh1promoter region were subcloned into pGL3-basic vector as shown above, and they were cotransfected with Ngn3 expression vector to determine the core region of Sohlh1promoter. The luciferase assays were performed as above.
Immunoprecipitation: The immunoprecipitation (IP) assays were performed using Ngn3 and Sohlh1 antibodies in wild type and Sohlh1KO testes. 7weekold male mice were used in immunoprecipitation for they were adult. The testes were lysed in modified RIPA buffer (1% NP-40, 0.25% deoxycholate, protease inhibitor cocktail) after sonic. Prior to IP, the lysates were precleared by incubating with Protein A or G agarose beads (Beyotime, China) for 2h at 4C. To immune precipitate the protein of interest, the total protein was incubated with the antibody-conjugated beads overnight at 4C. The Beads were washed twice in IP lysis buffer and once in PBS and boiled in 2xloading buffer (0.1M Tris-HCl, pH7.4, 50% Glycerol, 10% SDS, 5% 2-Mercaptoethanol, 0.5% Bromophenol Blue) before SDS-PAGE.
Statistical analysis: All experimental data were analysed using the SPSS 17.0 software (SPSS, Chicago, IL, USA). The differences in the mean value between groups were compared using one-way analysis of variance. Paired comparisons of the mean value between groups were performed using a two-sample t-test. A value of P less than 0.05 was considered statistically significant.
Expression of Ngn3 decreased in Sohlh1-/-mice: (Ballow et al., 2006)compared gene expression in Sohlh1-/- and wild type mice with Affymetrix 430 2.0 microarray chip, in which the microarray chip result showed that Ngn3 is significantly down-regulated in Sohlh1-/- testes. Firstly, we use real-time PCR and western blot to confirm the microarray result. Real-time PCR showedthat the Ngn3 mRNA was decreased in Sohlh1-/- mice compared to wild type C57BL/6 miceand western blot showed that the protein of Ngn3 was also decreased in Sohlh1-/- mice, which is consistent with the real-time PCR data (Figure 1).
Ngn3bind to the promoter region of Sohlh1: The expression of Ngn3 decreased in Sohlh1 knockout mice, which would be usually thought that Sohlh1 regulates the expression of Ngn3. Therefore, we usedSohlh1antibody to perform ChIP, but the result showed that Sohlh1 did not bind to the promoter region of Ngn3. The cells expressing Ngn3 were in the prior stage of the ones expressing Sohlh1, thus Ngn3 may lie in upstream of Sohlh1, and regulate the expression of Sohlh1. Then ChIP was performed with rabbit anti mouse Ngn3antibody. 7- day mouse testes were used in Chromatin immunoprecipitation for their high rate of spermatogonia. The DNA sequences of Sohlh1promoter(-2500~+500) were analyzed by a Neural Network Promoter Prediction software(a product of Berkeley Drosophila Genome Project, http://www.fruitfly.org/seq_tools/promoter.html). The prediction was showed in Figure 2A, in which the most possible start site of transcription is T (shown in larger font), -55bp upstream of sohlh1 gene.
DNA collected by ChIP was further analysed by PCR. The specific primers used for ChIP analysis covered the -304~+42 region of Sohlh1 promoter, and there were 5 E- boxes in this area (Figure2B). The ChIP result indicated that Ngn3bind to the promoter region of Sohlh1in vivo (Figure 2C).
Ngn3increased the transcription of Sohlh1in vitro: To determine whether Ngn3could activate the Sohlh1 promoter, we performed reporter gene assay using HEK293T cell line for its high transfection efficiency. The -2357~+42 region of Sohlh1promoter was subcloned into pGL3-basic vector (named Sohlh1-A-Luc). The Ngn3 expression vector and Sohlh1-A-Luc vector were cotrasfected into HEK293T cells. As shown in Figure 3, Ngn3enhanced the Sohlh1 reporter activity in HEK293T cells. These data suggest that Ngn3 could increase the expression of Sohlh1in vitro.
To further define the function of Ngn3 in the activation of the Sohlh1 promoter, we generated several deletion promoter constructs (A, B, C, D, E, F, and G) in the pGL3 luciferase reporter vector and performed reporter analysis in HEK293T cells. The Sohlh1-A to G promoter, were all activated by co-expression of Ngn3. There is no difference between the 7 promoters. These results suggest that -304~+42 promoter region of Sohlh1may be critical forNgn3 binding.
Interaction of NGN3 protein andSOHLH1 in testis: Since we revealed that Ngn3 could bind to the promoter of Sohlh1and could promote the transcription of Sohlh1 in vitro, we then take a step forward to perform IP to identify whether NGN3 could bind withSOHLH1 protein to regulates permatognesis in testis. As shown in Figure 4, SOHLH1 was detected in NGN3 complexes immune precipitated from 7week-old mice testis extracts, and NGN3 was also confirmed in the reciprocal SOHLH1 immunoprecipitation. We also performed the same experiments using Sohlh1-/- testes, but no IP bands were detected (data not shown). Taken together, our data indicate that NGN3andSOHLH1 protein formed a complex, which may function together.
Spermatogenesis is a complex process that generates millions of spermatozoa per day in mammals, in which SSCs play a key role and can self-renew to maintain the amount of spermatogonia and can differentiate into spermatogonial progenitors(Song and Wilkinson, 2014). Several transcription factors have been identified that can promotes permatogonial differentiation (DMRT1, NGN3, SOHLH1, SOHLH2, SOX3, and STAT3), some of which may affect the fate of SSCs either to differentiate or to promote later spermatogonial differentiation steps. Many of these transcription factors regulate each other and act on common targets, suggesting they integrate to form complex transcriptional networks in self-renewing and differentiating spermatogonia. In this study, we identified that Ngn3could bind to the promoter of Sohlh1, and promote the transcription of Sohlh1.
Ngn3, recognized for its role in promoting the development of cells in pancreas and(Gu et al., 2002; Heremans et al., 2002) neurons in the hypothalamus (Pelling et al., 2011; Simon-Areces et al., 2011). In testis, Ngn3expressed in a sub-set of undifferentiated spermatogonia (Yoshida et al., 2004), was thought to be one of the transcription factors that promote the conversion of spermatogonial progenitors into differentiating A-spermatogonia. Ngn3-positive spermatogonia distributed at a low frequency throughout the cell cycle and with the highest frequency at stages VII-VIII(Yoshida et al., 2004). This is compatible to the distribution of As, Apr, and Aalspermatogonia, which transform into A1 spermatogonia at seminiferous tubule stages VII-VIII(Chiarini-Garcia and Russell, 2001; Tegelenbosch and de Rooij, 1993). Kaucher et al., (2012) found that depletion of Ngn3led to an increase in the number of SSCs without measurably increasing cell proliferation, but by the blockade of SSC differentiation.
However, the molecular circuitry downstream of Ngn3 that drives SSC differentiation is unknown. As a member of the bHLH family, Ngn3could bind E box elements within promoters of target genes to regulate transcription (Davis et al., 1990; Murre et al., 1989). Studies of the pancreatic endocrine celllineage revealed a variety of genes regulated byNgn3, including neurogenic differentiation 1 (Neurod1) (Gradwohl et al., 2000; Heremans et al., 2002), transcription factor LIM/homeodomain (Isl1) (Gradwohl et al., 2000), paired box gene 4 (Pax4)(Gradwohl et al., 2000; Heremans et al., 2002), and paired box gene 6 (Pax6) (Gradwohl et al., 2000; Heremans et al., 2002). Scanning of microarray gene expression database for mouse undifferentiated spermatogonia indicates that these genes above are not expressed (Oatley et al., 2007).
Thus, the majority of downstream targets of Ngn3 for transcriptional regulation in the male germline are either distinct from those in pancreatic precursors or express later in germ cell development when transition to differentiating spermatogonia occurs.
Sohlh1 seems to be a possible downstream target of Ngn3.In males, distribution of SOHLH1 protein in the mouse seminiferous epithelium of adult testes was analyzed by immunohistochemistry. SOHLH1 is initially detected in Stage IV Aalspermatogonia and strongly expressed in Aal, A1, A2, A3, A4, Intermediate and Type B spermatogonia. Both Ngn3 and Sohlh1 were expressed in Aalspermatogonia, which was the key point of undifferentiated spermatogonia transition to differentiated spermatogonia. Loss of Sohlh1, undifferentiated spermatogonia failed in differentiating to spermatocytes.Sohlh1 directly stimulates Kit transcription in postnatal spermatogonia, thus activates the signalling involved in spermatogonia differentiation and spermatogenetic progression(Barrios et al., 2012).
To summarize, we propose that Ngn3 and Sohlh1may interact and regulate spermatogenesis for the following three reasons: i, cells which expressing Ngn3 is overlapped with cells expressing Sohlh1;ii, both Ngn3 and Sohlh1 regulate the differentiation of spermatogonia; iii, microarray array(Ballow et al., 2006) and our real- time PCR, western blot results show that Ngn3 expression decreased while Sohlh1 is absent. Our data strongly indicated there are associations between Ngn3 and Sohlh1.We used 7-day wild type and Sohlh1-/- mice in these experiments to evaluate the expression level of Ngn3, in which most cells in 7-dayseminiferous tubules were spermatogonia that haven't yet differentiated into spermatocytes and 7-d Sohlh1-/-testes were similar to wild type testis in histology, which indicated that the amount of spermatogonia in Sohlh1-/- testis was approximately equal to wild type testis. Thus, we think the decrease of Ngn3 in testis is due to the reduced expression, but not the amount of spermatogonia.
We analysed the relationship between Ngn3and Sohlh1 by ChIP, the results show that Ngn3could bind to the promoter of Sohlh1;Ngn3 may regulate the transcription of Sohlh1. To determine whether Ngn3 could activate Sohlh1 transcription, we performed luciferase reporter gene assay. We co-transfected Ngn3 expressing vector and Sohlh1 promoter luciferase reporter vectors into HEK293T cells, in contrast to pEGFP-N1 plasmid, the Ngn3 expressing vector increase the luciferase of Sohlh1 promoters. It indicated the protein of Ngn3could promote the transcription of Sohlh1. Forther more, the co-IP results show the protein interaction of NGN3 and SOHLH1. Before our report, there is only one paper about the relation between Ngn3 and Sohlh1, it is written by Ballow et al., (2006) in 2006, in their study the microarray array result show that the Ngn3 decrease in Sohlh1-/- mouse. Then some articlescited this paper, guessingNgn3 may be regulated by Sohlh1, but there is no other experiment result supporting that idea.
Our study showed the transcription regulation relationship between Ngn3 and Sohlh1 by ChIP and luciferase reporter gene assay for the first time, and proved that NGN3 protein may regulate spermatogenesis by forming complex with SOHLH1.Since Kaucher et al. (2012) reported that GDNF could negatively regulate Ngn3 by STAT3; and Barrios et al.(Barrios et al., 2012)identified that Kit was the target of Sohlh1 during spermatogenesis; Together with our results, it could demonstrate that GDNF regulate SSC differentiation through STAT3-Ngn3-Sohlh1-Kit pathway. Our study shows that Sohlh1 is the direct target of Ngn3 in differentiation of SSC.
Our results support the idea that Ngn3 is a key role in differentiation of SSC, and it is consistent with the role of Ngn3 in differentiation of pancreatic precursors. In the developing pancreas, Nng3 functions as a pro- endocrine factor, which is sufficient to force undifferentiated pancreatic epithelial cells to become islet cells (Gu et al., 2002). But the signal pathway may be different in different organs, as mentioned above, the targets of Ngn3 in pancreas, such as Neurod1, Isl1, Pax4, Pax6, were not expressed in testis; and it has been report that GDNF enhances Ngn3 gene expression and beta-cell proliferation in the developing mouse pancreas (Mwangi et al., 2010), while GDNF inhibits Ngn3 in testis(Kaucher et al., 2012).
Matson et al.(2010) reported DMRT1promotes spermatogonial differentiation, and the downstream target is Sohlh1. Chromatin immunoprecipitation studies show DMRT1 binding to the Sohlh1 promoter. The bound region shares similarity to the DMRT1 known DNA binding consensus site. Moreover, Dmrt1 is unlikely to be the only regulatorofSohlh1, as Dmrt1 conditional knockouts retain KIT expression and complete spermatogenesis. Sohlh1 mutants do not express KIT in adult spermatogonia, and meiotic-like cells eventually die. It is therefore likely that a sufficient quantity of SOHLH1 proteins remain in the Dmrt1 mutant(Murphy et al., 2010). Our result raised another direct regulator of Sohlh1, which may be the main regulator in differentiation of SSC.Recently,Ngn3 was reported function in promoting meiosis by up-regulating Stra8, an important gene in spermatogenesis(Tang et al., 2014). SOHLH1 and SOHLH2 could heterodimerize with each other in vivo, as well as homodimerize.
SOHLH proteins affect spermatogonial development by directly regulating Gfra1, Sox3 and Kit gene expression. SOHLH1 and SOHLH2 suppress genes involved in SSC maintenance, and induce genes important for spermatogonial differentiation (Suzuki et al., 2012).It is possible that many spermatogonial differentiation transcription factors regulate each other, and there may be a large and complex gene net of the transcription factors that promote SSCs and spermatogonial differentiation, even associated with self-renew of SSCs. In the future, further studies will be performed to understand the regulation net above.
Acknowledgment: This research was supported by Liaoning SandT project, No. 2013408001.
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