Temporal expression and DNA hypomethylation profile of CD30 in ...

Temporal expression and DNA hypomethylation profile of CD30 in Marek’s disease virus-infected chicken spleens Kaiyang Li,1 Ling Lian,1 Ning Yang, and Lujiang Qu2 Department of Animal Genetics and Breeding, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China ABSTRACT Marek’s disease (MD) is a viral neoplastic disease of chickens caused by Marek’s disease virus (MDV), which is serious threat to worldwide poultry industry. Our previous studies showed that the CD30 gene was hypomethylated in MD lymphoma. In this study, we further analyzed differential expression patterns and methylation levels of the CD30 gene between MDV-infected and noninfected spleens at 4, 7, 14, 21, and 28 d postinfection (dpi). The results showed that the expression of CD30 in MDV-infected spleens was significantly lower than that in noninfected spleens at

4 dpi. The expression of CD30 did not present significant difference between MDV-infected and noninfected spleens at 7 and 14 dpi. However, an increased expression of CD30 was presented in MDV-infected spleens at both 21 and 28 dpi. Simultaneously, CD30 showed a lower DNA methylation level in MDV-infected spleens at 14, 21, and 28 dpi. The results indicated that CD30 gene was involved in the whole process of MD tumorigenesis and upregulated expression of CD30 in MDV-infected spleens might be attributed to the hypomethylation of promoter of CD30 gene.

Key words: chicken, Marek’s disease, CD30, gene expression, methylation 2015 Poultry Science 94:1165–1169 http://dx.doi.org/10.3382/ps/pev100

INTRODUCTION Marek’s disease (MD) in chickens, caused by Marek’s disease virus (MDV), is characterized by immune suppression, neurological disorders, and neoplastic T-cell lymphomas of multiple visceral organs. MDV is a highly cell-associated α-herpesvirus and spreads through cellto-cell contact (Biggs, 1975). Natural in vivo MDV infection was typically divided into four phases, including early cytolytic phase, latent phase, late cytolytic phase, and tumor transformation phase (Davison and Nair, 2004). After 24 h postinfection, the virus becomes detectable in primary lymphoid organs, including spleen, thymus, and the bursa of Fabricius. From 3 to 6 d postinfection (dpi), the virus enters cytolytic phase and CD4+T cells are activated. After 7 dpi, MDV enters latent phase. During this period, virus is mainly restricted to CD4+T cells, and viral genome is inactive without production of infectious progeny virus. Only a small subset of latently infected T cells can proliferate and disseminate to multiple organs to generate tumors (Calnek, 2001). During 10 to 14 dpi, the infection goes into late cytolytic phase, virus is reactivated, and begins to replicate. Depending on the virulence of virus strain and genetic susceptibil C 2015 Poultry Science Association Inc. Received November 15, 2014. Accepted February 16, 2015. 1 These authors contributed equally. 2 Corresponding author: [email protected]

ity of chickens, solid visceral tumors containing transformed CD4+T cells and immunologically active and inactive cells may develop within 2 to 6 wk postinfection (Carrozza et al., 1973; Johnson et al., 1975; Niikura et al., 1999). After 21 dpi, infected lymphocytes were neoplastically transformed and proliferate to form gross lymphomas in susceptible chickens (Calnek, 2001). Marek’s disease has been proved to be a valuable model to investigate general tumorigenesis and virus-induced lymphomagenesis as well as disease resistance (Osterrieder et al., 2006). As a member of a tumor necrosis factor receptor II family, tumor necrosis factor receptor superfamily member TNFRSF8 (CD30), was initially discovered in Hodgkin and Reed–Sternberg cells in Hodgkin’s disease. In many non-Hodgkin’s lymphomas, CD30 has also been proposed as a molecular marker defining subgroups of leukemias, lymphomas, and other tumors (Blazar et al., 2004; Gallardo et al., 2002). The CD30 antigen functions as a cytokine receptor and can combine with its ligand (CD30L). The interaction of CD30 and CD30L play a critical role in malignant lymphomas, including Hodgkin’s disease, large cell anaplastic lymphomas, and Burkitt lymphomas. In lymphoma cell lines, CD30–CD30L can induce apoptosis or enhance cell proliferation (Hubinger et al., 2004; Mori et al., 1999; Somada et al., 2012). In our previous study (Zhang et al., 2012), we found that the upregulated CD30 gene showed a significantly lower methylation level in MD lymphoma at 40 dpi than

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that in noninfected group. The previous results provided us a clue that CD30 gene probably experienced epigenetic modification in initiating MD lymphoma transformation, but only one time point (40 dpi) was detected. The temporal expression and DNA methylation analysis of CD30 in other MDV infection phases remains unclear. In this study, we investigated differential expression of CD30 between MDV-infected and noninfected spleens at 4, 7, 14, 21, and 28 dpi. Besides, the methylation status of CD30 promoter between MDVinfected spleens and noninfected spleens at 14, 21, and 28 dpi was analyzed.

MATERIALS AND METHODS Tissue Collection MDV challenge trial was conducted in our previous study (Lian et al., 2010). Briefly, 150-day-old specificpathogen free White Leghorn day-old chicks were divided into 2 groups randomly. One hundred chicks were infected intraperitoneally with 2,000 pfu MDV– GA strain as the experimental group and the rest 50 chicks was injected with the same dosage of diluents as control group. Each group was reared separately in the filtered-air, positive-pressure isolated room. Four spleens from noninfected chickens and 4 spleens from infected group were collected at 4, 7, 14, 21, 28 dpi and stored in RNA fixer. All the samples were preserved at −4◦ C overnight and transferred to −80◦ C until to be used. Animal experiments were approved by the Animal Care and Use Committee of China Agricultural University (Approval Identifier: XXCB-20090209) and the experiment was conducted according to regulations established by this committee.

Expression Analysis of CD30 mRNA Total RNA was isolated by trizol reagent (Invitrogen, United States) and dissolved in RNase-free water. RNA concentration was determined by Nanovue (GE Healthcare, United States). The first strand cDNA was synthesized from total RNA using Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Promega, Madison, WI) according to the manufacturer’s instructions. The specific primers for genes glyceraldehydes-3-phosphate-dehydrogenase and CD30 were designed by the Primer premier 5.0 software (Premier Biosoft, Palo Alto, CA). The differential expression of CD30 between MDV-infected and noninfected spleens was then analyzed by qPCR using Applied Biosystems Prism 7300 system (Foster City, CA). The qPCR reactions were performed in a volume of 15 μL with a SYBR Green 1 Mastermix (Applied Biosystems, Foster City, CA) by the following PCR conditions: 50◦ C for 2 min, 95◦ C for 10 min, 40 cycles of 95◦ C for 15s, and 60◦ C for 1 min, 95◦ C for 15s, 60◦ C for 30s, and 95◦ C for 15s. Two replicates were performed in each

reaction. The mRNA expression of CD30 was normalized against the housekeeping gene glyceraldehydes-3phosphate-dehydrogenase. The 2−ΔΔCt method was used to calculate relative gene expression level between 2 groups. All analyses were performed using Student’s t-test.

Bisulfite Treatment Genomic DNA from the spleens was extracted using traditional phenol/chloroform method. The quality of DNA was assessed by 0.8% agarose gel electrophoresis using 100 ng extracted DNA. The concentration was detected by Nanovue (GE Healthcare, United States), and then 1.5 μg genomic DNA from each sample was treated with sodium bisulfite using EZ DNA Methylation-Gold Kit (CapitalBio, Beijing, China) according to the manufacturer’s protocol.

Quantitative Methylation Analysis The Sequenom MassARRAY platform (CapitalBio Beijing, China), a novel approach based on a basespecific cleavage along with matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis, was used to analyze DNA methylation. PCR primers were designed by using Methprimer (www.urogene.org/methprimer/). Two amplicons were designed to cover the promoter region of CD30 gene. For each reverse primer, an additional T7 promoter tag (5 -cagtaatacgactcactatagggagaaggct-3 ) for in vivo transcription, and a 10-mer tag (5 -aggaagagag-3 ) on the forward primer for adjustment of melting temperature were added. PCR amplification was performed as follows: 94◦ C for 10 min, 10 cycles of 94◦ C for 45s, 62◦ C (−0.5◦ C/cycle) for 48s, 72◦ C for 1 min, and then 35 cycles of 94◦ C for 45s, 57◦ C for 48s, and 72◦ C for 1 min, finally incubation at 72◦ C for 3 min. Unincorporated dNTPs of 0.4 μL were dephosphorylated by adding 1.6 μL premix including 0.5 μL shrimp alkaline phosphate enzyme (MassCLEAVE Kit, Sequenom, San Diego, CA), and the mixture was incubated at 37◦ C for 20 min, and then at 85◦ C for 5 min. After shrimp alkaline phosphate treatment, 2 μL PCR products were used as the template for in vitro transcription and RNase A was used for the reverse reaction according to the manufacturer’s instructions. The samples were conditioned and spotted on a 384-pad Spectro CHIP (Sequenom) using a MassARRAY Nanodispenser RS1000 Arrayer (Samsung, Irvine, CA), followed by spectral acquisition on a MassARRAY analyzer compact matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis (Sequenom). Then spectra’s methylation ratios of each CpG site or an aggregate of multiple CpG sites were analyzed by EpiTyper software v1.0 (Sequenom).

HYPOMETHYLATION OF CD30 IN MAREK’S DISEASE LYMPHOMA

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spleens was significantly (P < 0.01) lower than that in noninfected spleens at 4 dpi. At 7 and 14 dpi, though there was no significant difference between the 2 groups, the expression of CD30 in MDV-infected spleens showed a higher trend than that in noninfected spleens (Figure 2). At 21 and 28 dpi, the expression of CD30 in MDV-infected spleens was higher (P < 0.05) than that in noninfected spleens (Figure 1).

Methylation Level of Each CpG Site in CD30 Promoter Region Figure 1. The differential expression of CD30 between MDVinfected spleens (experiment group) and noninfected spleens (control group). The expression of CD30 gene is presented relative to β -actin expression and normalized to a calibrator. Error bars represent SEM. ∗ indicated P < 0.05 and ∗∗ indicated P < 0.01.

RESULTS Differential Expression of CD30 Gene between MDV-Infected Spleens and Noninfected Spleens at Different MDV Infected Phases Quantitative real-time PCR was conducted to analyze differential expression of CD30 between MDVinfected spleens and noninfected spleens at 4, 7, 14, 21, and 28 dpi. The expression of CD30 in MDV-infected

The DNA methylation levels of CD30 gene between MDV-infected and noninfected spleens at 14, 21, and 28 dpi were analyzed. Two amplicons covering 825 bp around the core promoter region were designed. Fourteen out of the 19 covered CpG sites in the 2 amplicons were analyzed. The transcription-factor binding sites were identified by using MATINSPECTOR (Quandt et al., 1995). The core promoter motif of CD30 and AP-1 binding sites were located in the amplicons. CpG Site 3 and CpG Site 4 were located in the middle of the promoter region, and CpG Site 9 was in Ap-1 binding site. The methylation difference of each CpG site between MDV-infected and noninfected spleens were further compared. The methylation levels varied at different CpG sites. Overall methylation level in MDVinfected spleens was lower than that in noninfected spleens (Figure 2). At 14 dpi, CpG Site 13 showed the

Figure 2. Methylation level of each CpG site of CD30 promoter between MDV-infected and non-infected spleens. A. Methylation level of each CpG site between 2 groups at the 14 dpi. B. Methylation level of each CpG site between 2 groups at the 21 dpi. C. Methylation level of each CpG site between 2 groups at the 28 dpi. Error bars represent SEM. ∗ indicated P < 0.05 and ∗∗ indicated P < 0.01.

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lowest methylation level (3%), whereas the CpG Site 3 and 4 had the highest methylation level (73.25%). The CpG Sites 2 to 6 showed lower methylation levels in MDV-infected spleens than that in control group (Figure 2a). At 21 dpi, the methylation level of CpG Site 14 was lowest (5%), and CpG Site 17 was the highest (87.25%). The methylation levels of CpG Sites 17 and 19 were lower in MDV-infected spleens than that in control group (Figure 2b). At 28 dpi, CpG Site 14 was the lowest level (3%), and both Sites 3 and 4 were the highest level (81%). The methylation levels of CpG Sites 2, 8, 15, and 19 were lower in MDV-infected spleens than that in control group (Figure 2c).

DISCUSSION DNA methylation, a kind of epigenetic phenomenon, plays an important role in biological processes, such as proliferation, development, and differentiation. DNA hypomethylation was involved in carcinogenesis and considered as a typical characteristic in neoplasia (Laird et al., 1995). In mice, genome-wide hypomethylation was implicated in tumor formation (Gaudet et al., 2003). DNA methylation was essential for regulating gene expression in eukaryotes (Razin and Riggs, 1980). DNA hypomethylation can affect gene expression. Many evidences indicated that there was an inverse correlation between methylation level of a gene and its transcriptional activity (Felsenfeld and McGhee, 1982). Study on pancreatic cancer also indicated that gene hypomethylation was a frequent epigenetic event and was commonly associated with the overexpression of affected genes (Sato et al., 2003). It is very well-documented that the MDV can alter the expression and methylation levels of genes in both genome wide and individual gene level in the host (Luo et al., 2011; Luo et al., 2012; Yu et al., 2008a; Yu et al., 2008b). In our previous study (Zhang et al., 2012), MD lymphoma was collected at 40 dpi, when the tumors were completely formed. The results showed that overexpression and hypomethylation of CD30 appeared in MD lymphoma, which suggested that CD30 gene experienced epigenetic modification. In this study, we further determined expression patterns of CD30 gene between MDV-infected and noninfected spleens at 4, 7, 14, 21, 28 dpi. The results showed that the expression of CD30 was extremely decreased in MDVinfected spleens compared with noninfected spleens at 4 dpi, which could be explained by cytolysis of B cells. Generally, the early cytolytic infection lasts from 3 to 6 dpi and targets primarily on B lymphocytes with temporary profound immunosuppression (Calnek, 2001). The CD30 gene mainly expressed in B and T cells, and reduced number of B cells in MDV-infected spleens probably leaded to down-regulation of CD30 gene. On the contrary, the expression of CD30 was significantly higher in MDV-infected spleens than that in noninfected spleens at 21 and 28 dpi, which was also consistent with our previous study (Zhang et al., 2012). It

could be explained by the infiltration of neoplastic cells overexpressing CD30 into spleens. Since the higher expression level of CD30 in infected group was found at 21 and 28 dpi, we decided to detect the methylation level at 14, 21, and 28 dpi in order to discover the implication of methylation in the whole tumorigenesis phase. Our results showed that hypomethylation of CD30 had already started at 14 dpi, i.e., the initial phase for tumor cells infiltrating into the spleen. Overexpressed CD30 gene acted as a highly conservative oncogene among human, mice, and chickens (Burgess et al., 2004). It was reported that hypomethylation of CD30 promoter were relevant with pathogenesis of Hodgkin lymphoma and anaplastic large cell lymphomas (Watanabe et al., 2008). Fifteen predicted high-stringency AP-1 transcription factor binding sites are included in the promoter of CD30 gene in the chicken (Burgess et al., 2004). Meq, a basic leucine zipper protein encoded by MDV, is an oncogene and it can form heterodimer with C-Jun by binding to AP-1 site. The dimer upregulated the expression of CD30 gene (Levy et al., 2003; Levy et al., 2005; Burgess et al., 2004). The high level of DNA methylation has been proved to reduce the binding affinity of sequence with specific transcription factors (Jones and Laird, 1999). Therefore, the hypomethylation of CD30 promoter might result in high affinity to meq/CJun dimer which upregulated the expression of CD30 in Marek’s disease. Collectively, we found that hypomethylation of CD30 gene presented in the whole tumorigenesis phase from initial stage to late transformation phase. The high expression and hypomethylation level of CD30 could be an indicative for lymphomas formation after MDV infection.

ACKNOWLEDGMENTS This work was supported by Beijing innovation team attached to poultry industry technology system (China Agriculture Research System–Poultry Science and Technology in Peking), the National Natural Science Foundation of China (31320103905 and 31301957), modern agricultural industry technology system (CARS-41), National Scientific Supporting Projects of China (2012BAD39B04), Natural Science Foundation of Beijing, China (Grant No. 6132022 and 5154030), and the National High Technology Development Plan of China (2013AA102501, 2011AA100305).

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