WANG Lu-lu,ZHAO Chun-fang,LIU Chang-jun,ZHANG Hao,LIAN Ling

1 National Engineering Laboratory for Animal Breeding and MOA Key Laboratory of Animal Genetics and Breeding,College of Animal Science and Technology,China Agricultural University,Beijing 100193,P.R.China

2 Division of Avian Infectious Diseases,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150001,P.R.China

Abstract Marek’s disease (MD),an immunosuppressive disease induced by Marek’s disease virus (MDV),provides an ideal model for studying diseases caused by a carcinogenic virus.CD79B is a B-cell antigen receptor complex-associated protein β-chain precursor which is involved in the activation,proliferation,differentiation of B-cell and the transmission of downstream signals.This study analyzed CD79B gene mRNA expression and methylation by two schemes #20 (5´ flanking to intron 1) and #27(intron 2 to intron 3),between MDV-infected tumorous spleens (TS) and non-infected spleens (NS).Results showed that average methylation levels of CpGs in #20 and #27 were higher in TS than in NS (P＜0.05),while,CD79B mRNA expression was lower in TS than in NS (P＜0.01).Six of 40 CpG sites showed significantly (P＜0.05) different methylation levels between TS and NS.Correlation analysis showed that the average methylation level rather than a single site methylation level in#20 affected (P＜0.05) mRNA expression.Collectively,it was found that the change of CD79B gene expression after MDV infection might be partly explained by modification of DNA methylation.

Keywords:chicken, Marek’s disease,Marek’s disease virus,DNA methylation,gene expression

1.Introduction

Marek’s disease (MD) induced by Marek’s disease virus(MDV) is an immunosuppressive disease that can cause visceral lymphoma,neurological symptoms,severe brain oedema and acute deaths (Osterriederet al.2006).MD has resulted in great economic loss to domestic poultry industry since its outbreak in 1970.Vaccination is an effective measure to prevent MD currently.However,vaccine cannot block the virus from replicating and spreading (Cuiet al.2016;Abd-Ellatieffet al.2018).Therefore,the effective way to fundamentally eliminate or mitigate the damage of MD is to elucidate the host resistance factors that are in turn utilized in chicken breeding.Moreover,MD provides an ideal medical model for studying diseases induced by virus (Osterriederet al.2006;McPherson and Delany 2016)due to its high similarity with Varicella zoster virus (VZV),human herpesvirus 6 (HHV-6) and Epstein-Barr virus (EBV).

Up to now,underling molecular mechanism in chicken resistant to MD have been reported,such as mRNA expression profile (Lianet al.2012b;Heidariet al.2016),non-coding RNA expression profile (Lianet al.2012a;Parnaset al.2014) and the competing endogenous RNAs network between mRNA and ncRNA (Wanget al.2020).Genes involved in MD resistance or susceptibility have been identified,such asmiR-155(Cullenet al.2011),miR-26aandNEK6(Liet al.2014),GH1(Liuet al.2001),SCA2(Liuet al.2001),major histocompatibility complex class II β-chain (Niikuraet al.2007),andCD79B(Meydanet al.2011).It was reported thatCD79Bexpression was transcriptionally coupled withGH1(Hoet al.2006).CD79Bshowed allele-specific expression in bursa and thymus tissues (Meydanet al.2011).A disulfide-linked heterodimer ofCD79BandCD79Aproteins and the membrane immunoglobulin (mIg) were covalently combined to form a BCR composite structure (Dal Portoet al.2004).CD79A/B contained immunoreceptor tyrosine-based activation motif(ITAM) within their cytoplasmic tails,which initiated signal transduction following BCR aggregation (Flaswinkel and Reth 1994).The function mutation caused by the loss of BCR complex components or downstream signaling molecule could inhibit B cell development and induce human immune deficiencies (Pieperet al.2013).

As an important component of the genetic basis for all aspects of phenotypic variations,gene expression was regulated bycis-andtrans-acting transcriptional components,alternative splicing,RNA stability,expression of regulatory RNAs and epigenetics (Cragget al.2002;Wrayet al.2003;Ghotbiet al.2009;Jianget al.2019).As an important epigenetic information,DNA methylation is related to various diseases,such as hypertension (Bogdarinaet al.2007),cancer (Liet al.2019),neurodegenerative disorders(Obeidet al.2009),and depression (Jianget al.2019).Luoet al.(2012) found that genes involved in responding to stimulus,cell adhesion,and immune system process harbored a higher methylation level in MD-susceptible (L72)birds than in MD-resistant (L63) birds,and the expression level ofCD4was negatively correlated with the methylation ofCD4promoter (Luoet al.2011).

A previous study showed thatCD79Bwere involved in chicken’s resistance/susceptibility mechanism of Marek’s disease (Meydanet al.2011).RNA-seq data also showed thatCD79Bdownregulated in the TS group (LFC=-2.69,TSvs.NS) (Youet al.2019).Given that methylation level can affect gene mRNA expression,the present study proposed a scientific hypothesis that deregulation ofCD79Bexpression would be resulted from abnormal methylation of its flanking or gene body region.To verify this,the mRNA expression and methylation level ofCD79Bwere detected and correlation analysis was conducted on them.It is expected that the present work can provide a basis for a more indepth analysis of the role ofCD79Bin the occurrence and development of MD tumors.

2.Materials and methods

2.1.Biological samples

The specific information of samples used in this work was described in previous study (Lianet al.2012b).Briefly,150 1-d-old specific-pathogen-free White Leghorn (BWEL)chicks were divided into two groups.A total of 100 chicks were infected intraperitoneally with 2 000 plaque-forming units (PFU) of the MDV-GA,and the remaining 50 birds were injected with the same volume of diluent (0.2 mL) as non-infected controls.The two groups were kept separately in different places.Chicken conditions were observed and recorded.After 31 days of infection,in the MDV infection group,individuals exhibited severe clinical conditions were euthanized,tumorous spleens (TS) of which were sampled.Meanwhile,non-infected spleens (NS) in the control group were sampled.Finally,four tumorous spleens (TS) and four non-infected spleens (NS) were used for this study,while another seven TS and five NS were used for mRNA-Seq to verify the accuracy of the results (Youet al.2019).The sample information used in each part of the experiment is in Table 1.All sample tissues were preserved in RNA fixer at 4°C overnight and transferred to -80°C for further study.

2.2.Extraction of genomic DNA (gDNA) and synthesis of cDNA

Genomic DNA of all samples was extracted by TIANamp Genomic DNA Kit (TIANGEN,China) according to the manufacturer’s instructions.Total RNA of all samples was extracted by Trizol (Invitrogen,USA) with the instructions of manufacturer.The RNA purity was detected by the NanoPhotometer®Spectrophotometer (IMPLEN,USA).RNA concentration was monitoredviaQubit®RNA Assay Kit in Qubit®2.0 Flurometer (Life Technologies,USA).The RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 System(Agilent Technologies,USA) was adopted to measure the RNA integrity.A total of 1.0 µg RNA per sample was used to synthase cDNA using FastKing RT Kit (TIANGEN,China)following the manufacturer’s instructions.The obtained genomic DNA and cDNA were stored at -20°C for further study.

2.3.Detection of CD79B mRNA expression

The expression ofCD79Bgene was detected by qRT-PCR withβ-actingene as the reference gene.The qRT-PCR was performed using SuperReal PreMix Plus (SYBR Green)Kit (TIANGEN,China) and in a BIO-RAD CFX96 System(Bio-Rad,USA) as follows:95°C for 10 min;40 cycles of 95°C for 10 s,58°C for 20 s,and 72°C for 24 s.The relative expression level ofCD79Bgene was calculated using 2-ΔΔCtmethod.The details of primer sequences are showed in Table 2.

2.4.Determination of DNA methylation frequency of CD79B

The potential CpG islands ofCD79B(version:Gallus_gallus-4.0;Entrez Gene ID:419940) were predicted by the online prediction website (http://www.ebi.ac.uk/Tools/seqstats/emboss_cpgplot/).The region where observed/expected ratio＞0.60,percent (C+G)＞50,and length＞100 was regarded as a CpG island.According to the prediction results,targeted CpG island sequences were used as a template to design primer for methylation level using the MassArray technology,and the detailed primers are displayed in Table 2.The methylation level ofCD79Bgene was measured using Massarray platform described previously (Coolenet al.2007).Briefly,1.0 µg of gDNA was treated with sodium bisulfite,where Cytosine (C)was converted to Uracil (U) in 100% unmethylated DNA and Cytosine (C) unchanged in 100% methylated DNA.PCR products were obtained by adding T7-promoter sequences in the PCR process.T7 RNA polymerase was used to transcribe PCR products into RNA fragmentsin vitrotranscription system,and then cleaved by RNase A to get small RNA fargments.The mass spectrum of each fragment was detected by Agena MassArray®flight mass spectrometry,and finally the data was calculated by EpiTYPER Software (Sequenom,USA) (Yinet al.2013) to obtain the level of methylation of each fragment.

Table 1 Information of 16 samples used for study

Table 2 Detailed information of primers used in this study

2.5.Statistical analyses

SPSS 16.0 Software was used for statistical analyses.The differences of CD79B gene expressions and DNA methylation levels between TS and NS were analyzed byt-test.Association between CD79B expressions and DNA methylation levels in TS and NS were analyzed by Pearson test.Each replicate sample served as an experimental unit.Results were considered statistically significant atP＜0.05.

3.Results

3.1.Detection of mRNA expression level and methylation level of CD79B in MDV-infected spleens

The relative mRNA expression ofCD79Bwas detected in NS and TS tissues by qRT-PCR (Fig.1).Result showed that the expression ofCD79Bin the MDV-infected spleensdecreased (P＜0.01) compared to that in the non-infected spleens.

Fig.1 The expression level of CD79B gene.NS represents the spleens from noninfected chickens;TS means the tumorous spleens from MDV-infected chickens.The representation of the data is the mean±standard deviation (n=4).＊＊ means P＜0.01.

To investigate the methylation level ofCD79Bgene,the entire sequences ofCD79Bgene and 650 bp in its 5´-flanking regions were submitted to the prediction website(http://www.ebi.ac.uk/Tools/seqstats/emboss_cpgplot/).Four CpG islands were identified and two schemes (#20 and#27) were designed to analyze the DNA methylation levels of these four islands (Fig.2-A and B).The result showed that the average methylation levels in both #27 and #20 were higher (P＜0.05) in TS than in NS (Table 3;Fig.3-A).In #27,among 15 detected CpG sites,sites of CpG_9,CpG_10,CpG_14 and CpG_15 exhibited hypermethylation level (P＜0.05) in TS rather than in NS (Table 3).In #20,among 18 detected CpG sites,CpG_11 and CpG_17 sites showed higher (P＜0.05) methylation levels in TS than in NS (Table 3).

Fig.2 Detailed description of methylation experiment.A,the genomic structure and CpG islands of the CD79B gene.B,the nucleotide sequence of two CpG islands.

Fig.3 The methylation level of CD79B gene.A,each site methylation level in #27 and #20 schemes of CD79B gene.TS means the tumorous spleens from MDV-infected chickens;NS represents the spleens from noninfected chickens.The value of each fraction is the mean±standard deviation (n=4).B,the result of transcription factor prediction.DEGs means differentially expressed genes in mRNA-Seq.

3.2.Association of CD79B mRNA expression with DNA methylation level

To evaluate the effect of methylation level onCD79Bgene expression,the correlation between methylation level ofCD79Band its mRNA expression was analyzed (Table 3).Six CpG sites with different methylation levels between TS and NShad no (P＞0.05) correlation withCD79BmRNA expression either in TS or NS.This suggested that not only differential but also non-differential methylated CpG sites might contribute to the regulation of mRNA expression.

Table 3 Distribution and level of variation in methylation frequency in the CD79B CpG island and their correlation with relative CD79B mRNA level in spleen tissues1)

This study further checked the correlation of average methylation levels of CpG sites in #27 and #20 with mRNA expression.It showed that the average methylation level of 18 CpG sites in #27 showed no correlation (r=0.58,P＞0.05)withCD79BmRNA expression in either group.There was significant (r=0.98,P＜0.05) correlation betweenCD79BmRNA expression with average methylation level of 15 CpG sites in #20 in TS group,rather than in NS group.

4.Discussion

Results of this study have verified the hypothesis that deregulation of CD79B expression would be resulted from abnormal methylation of its flanking or gene body region.This study found that average methylation level of 33 detected CpG sites was higher in TS than in NS,and six sites possessed significantly high methylation level in TS.Correlation analysis showed that the average methylation level of 15 CpG sites in #20 had significant correlation with mRNA expression ofCD79B.This study provided a basis for further exploring the role ofCD79Bin the prevention and treatment of MD.

Although larger samples can improve the reliability of results,this study could only use four samples from each group as implemented in previous studies (Jieet al.2013;Liet al.2015) due to sample shortage.Fortunately,from a previous RNA-seq study,another seven TS and five NS were used and theCD79BmRNA expression in the data was rechecked,which also showed lowerCD79Bexpression in TS (Youet al.2019).Spleen,the most important organ for antibacterial and antifungal immune reactivity,could detect viral integration into the host genome during cells’ early post infection.Thus,spleen may play an important role in virus-induced tumor.DNA methylation-associated silencing played a crucial role in tumorigenesis (Esteller 2008) and hypermethylation could lead to the inactivation of certain tumor suppressor genes (Kulis and Esteller 2010).CDH1andCDH13,involved in cell adhesion,were silenced by DNA hypermethylation,which induced invasion,metastasis and tumor progression (Katoh 2005;Kimet al.2005).In sporadic breast and ovarian cancer,hypermethylation of theBRCA1promoter was detected,which was related to double-stranded break repair,DNA repair and transcription(Catteau and Morris 2002).Similarly,hypermethylation level was detected inCD79Bin spleen tumor,which was involved in regulating surface BCR expression (Reichlinet al.2001) and activation of downstream pathways (Clarket al.1992;Johnsonet al.1995).Doerret al.(2005) reported that silencedCD79Bcould be reactivated and reexpressed by 5-Aza-2´-deoxycytidine,which indicated thatCD79Bshould be regulated by DNA methylation in Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma(cHL) and primary effusion lymphoma (PEL).In a previous unpublished study,MSB1 cell was treated with demethylation reagents 5-Aza-2´-deoxycytidine,the expression of mRNA was detected by microarray,and theCD79Bexpression in the treatment group was up-regulated relative to the control group(FC=29).This study found a significant correlation between average methylation in #20 andCD79BmRNA expression.Hence,it was speculated that the hypermethylation ofCD79Bgene inhibited the expression ofCD79B,blocked the downstream transmission of BCR signal and led to the occurrence and development of tumors.

It was found that the average methylation of CpG sites in #20 and #27 showed a significant difference between TS and NS,and the methylation level of six single CpG sites also possessed a significant difference between TS and NS.Interestingly,the part of the region in #20 located in 37-140 amino acids (aa) ofCD79B,which happened to locate in the immunoglobulin family (IgSF) domain (48-138 aa).IgSF belonged to adhesion molecules which were critical to most aspects of leukocyte function in the immune system,such as lymphocyte recirculation,leukocyte recruitment into inflammatory sites,antigen-specific recognition and wound healing (Cruseet al.2004).The average methylation level in #27 and six sites with differential methylation level showed no correlation betweenCD79BmRNA and methylation level.While in #20,there was significant correlation between average methylation of 15 CpG sites in TS andCD79BmRNA expression,but two CpG sites with different methylation level were not related to mRNA expression ofCD79B.To explain this phenomenon,sequences in #20 and #27 were submitted to online website(JASPAR and AnimalTFDB 3.0) to predict transcription factors.In total,412 and 395 genes were predicted in#20 and #27.Combining previous differentially expressed genes (DEGs) (Youet al.2019),FOXA1,PAX5,andKLF5in #20 andEBF1,FOXA1,KLF5,RUNX2,andPAX5in#27 were considered as potential transcript factors that may affectCD79Bexpression in addition to methylation level (Fig.3-B).

In normal cells,tumor-suppressor genes are usually unmethylated or in low methylation level,and the transcriptional silencing by hypermethylation of CpG island in these genes played a critical role in the tumorigenic process,which was a typical hallmark of cancer cells induced by tumor-suppressor inactivation (Walshet al.1998).As a gene related to immunity response and transmission of BCR signal,the CpG island ofCD79Bwas hypermethylated in spleen tissues after MDV infection,which may contribute to the change of gene expression and chromatin structure(Wanget al.2004).

5.Conclusion

This study investigated the methylation level inCD79Band found that 18 sites in #27 (ATG:-307-+191) and 15 sites in#20 (ATG:+1 754-+2 350) showed a higher methylation level in TS than in NS.Besides,four CpG sites in #27 and two CpG sites in #20 showed a significant difference between TS and NS.Moreover,CD79Bgene downregulated in TS.Combination analysis ofCD79BmRNA expression and DNA methylation level suggested that gene expression may be regulated by multiple CpG sites of the region rather than a single site.This study explained the mechanism of downregulation ofCD79Bin TS based on methylation level,which provided the basis for further study of the role ofCD79Bin Marek’s disease.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (31301957,31320103905),the Young Scientist Supporting Project,the project from Beijing Key Laboratory for Animal Genetic Improvement,the Program for Changjiang Scholars and Innovative Research Team in University,China (IRT_15R62),and the China Agriculture Research Systems of MOF and MARA(CARS-40).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Ethical approval

All animal handling procedures were conducted according to regulations and guidelines established by Animal Care and Use Committee of China Agricultural University (Approval ID:XXCB-20090209).