- DNA methylomes and transcriptomes analysis reveal implication of host DNA methylation machinery in BmNPV. - proliferation in Bombyx mori. - Background: Bombyx mori nucleopolyhedrosis virus (BmNPV) is a major pathogen that threatens the sustainability of the sericultural industry. - DNA methylation is a widespread gene regulation mode in epigenetics, which plays an important role in host immune response. - This study aims to explore the role of DNA methylation in BmNPV proliferation.. - The integrated analysis of transcriptomes and DNA methylomes in silkworm midguts infected with or without BmNPV showed that both the expression pattern of transcriptome and DNA methylation pattern are changed significantly upon BmNPV infection. - BS-PCR validated the hypermethylation of BGIBMGA014008, a structural maintenance of chromosomes protein gene in the BmNPV-infected midgut. - Conclusion: Our results indicate that DNA methylation plays positive roles in BmNPV proliferation and loss of DNMT activity could induce the apoptosis of infected cells to suppress BmNPV proliferation. - DNA methylation, as one of the important epigenetic regulations, occurs in both eukaryotes and prokaryotes.. - Recently, the roles of DNA methylation in host immune responses have. - Many studies have dem- onstrated that viruses, especially DNA tumor viruses, could induce aberrant DNA methylation of host genome to suppress the host immune responses and evade anti- viral immunity [4–6]. - 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - Full list of author information is available at the end of the article Huang et al. - Although comparing to mammals, the researches on DNA methylation in insects are relatively lagging be- hind, with the improvement and innovation of DNA methylated research methods, especially for the rapid progress in large-scale sequencing technology, some pro- gresses and achievements on DNA methylation in in- sects have been made in recent years. - Based on whole genome bisulfite sequencing (WGBS), insects such as Drosophila melanogaster, Tribolium castaneum, Bombyx mori and Nasonia Vitripennis [11–14] have proven that DNA methylation in insect genome does exist and the maintenance and regulation mechanism of DNA methy- lation system in insects may greatly differ from that of mammals and plants [15–17]. - The function of DNA methylation generally associates with the evolution, aging, memory and regulation of caste determination in honey bees and in ants [18–21].. - It is re- ported the genome wide-patterns of DNA methylation in the Aedes aegypti are disrupted with the infection of a virulent strain of Wolbachia [22]. - Our previous study found that 27 genes were shown to have both differential expression and differential methylation in the midgut and fat body of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) in- fected larvae, respectively, indicating that the BmCPV infection-induced expression changes of these genes could be mediated by variations in DNA methylation [24].. - Early study has found that in Autographa calfomnica nuclear polyhedrosis virus (AcNPV), the ac- tivity of the p10 gene promoter was severely affected when the 5′-CCGG-3′ site in this promoter was methyl- ated, suggesting that methylation of specific promoter sequences in an insect virus can affect viral gene expres- sion [27]. - DNA methylation in silkworm immune response and BmNPV-host interaction.. - By observing under the fluorescence microscope we observed that both the amount of cells with fluorescence and the fluorescence intensity of the treated group (Zeb/. - BmNPV) were significantly less than that of the control group (DMSO/BmNPV) (Fig. - More than clean reads were obtained from each sample and the summary of the sequenced data is shown in Additional file 1: Table S1. - To evaluate epigenetic changes in insect cells caused by BmNPV infection, genome-wide DNA methylation profiles of BmNPV-infected midgut (T group) and normal midgut (C group) were performed at single-base resolution. - Furthermore, we comparatively ana- lyzed the methylation level on different genomic functional regions between these two groups and the results revealed that in silkworm, DNA methylation was mostly targeted to gene bodies. - Differential DNA methylation regions (DMRs) upon BmNPV infection. - To explore the potential function of DNA methylation in response to BmNPV infection, we compared the DMR of the whole genome between BmNPV infected midgut and the control tissues. - The results revealed that 241 DMRs were obtained with 177 located in the gene region. - 1 Determination of the cytotoxicity of zebularine and the chemicals effect on BmNPV proliferation. - As a result, we found 26 DMGs showing differential mRNA levels in the midgut of the infected silkworm (Table 1), suggesting that the expression levels of these genes involved in BmNPV infection may altered via vari- ation in DNA methylation. - Analysis of the distribution of DNA methylation on these genes revealed that CG methy- lation of 25 genes presented in the gene body, predomin- antly in the introns of genes, accounted for about 61.54%. - Recent studies have shown that inhibition of DNA methylation by chemicals such as 5-aza-2′- deoxycytidine significantly induced antitumor immune responses in colon and ovarian cancers [9, 10] and re- sulted in cell cycle arrest, p53-dependent apoptosis, and IFN signaling activation of HPV-positive HNC cells [30].. - To explore whether the BmNPV infection can induce the variance on genomic DNA methylation of silkworm and whether DNA methylation could regulate the ex- pression of genes associated with BmNPV infection, we performed transcriptome analyses and whole-genome bi- sulfite sequencing between BmNPV-infected midguts and control midguts of silkworm larvae. - As a result, the characteristics of genomic DNA methylation in Bombyx mori are similar to previous studies on other insects and distinct from that of animal and plant. - DNA methylation in vertebrates distributes in the entire genome [31] while due to the low level of methy- lation in insects, DNA methylation in insects is scattered throughout the genome and is mainly concentrated in gene body (introns and exons). - In this study, as to Bombyx mori, it exhibits low levels of DNA methylation with about 0.185% and CG methylation is substantially enriched in gene bodies.. - The function of DNA methylation on gene expression is well established, but the underlying mechanisms of this function are poorly understood. - Color scale from green to blue represents DNA methylation level. - Color scale from blue to red represents DNA methylation difference between two groups. - b Distribution of DNA methylation levels of genes in T and Cgroup. - study, we found 26 DEGs showing differential methyl- ated levels in the midgut of BmNPV-infected silkworm larvae with 17 hyper-methylated and 9 hypo-methylated, which demonstrated that variance in DNA methylation may lead to expression changes of those genes associated with BmNPV infection.. - In addition, SMC4 ex- pression was significantly associated with tumor size, dedif- ferentiation, advanced stages and vascular invasion of the primary liver cancers [36]. - In our study, BGIBMGA014008 was identified up-regulation and hypermethylation in the midgut after BmNPV infection (Fig. - Nup188, one of the nucleoporins can regulate chromo- some segregation, promote chromosome alignment, con- fine the passage of membrane proteins and is thus crucial for the homeostasis of the different nuclear membranes [42, 43]. - In this study, both the mRNA transcript level of Nup188 (BGIBMGA002694) and DNA methylation level. - Recent studies have revealed that gene expression regulation by DNA methylation may play a critical role in arms races between viruses and their hosts [48]. - Studies on virus-driven dysregulation of host immune-related gene expression through DNA methylation present a novel viral mechanism to inhibit immune responses [51].. - There- fore, to explore whether DNA methylation could affect the JAK/STAT pathway, we detected the expression of three key genes involved in this pathway and we found that after inhibition of DNMT, two genes, which are negative regulators of the JAK/STAT signaling pathway [58], displayed the opposite expression pattern with up- regulation of socs6 and down-regulation of socs2. - 6), indicating that DNA methylation may have no obvious effects on JAK/STAT pathway.. - Apoptosis as an important immune response plays a critical role in the antiviral defense in insects [59].. - a The number of differentially methylated genes in different regions of the genome. - RichFactor is the ratio of the number of differentially expressed genes in this pathway term to the number of all genes in this pathway term. - Only the top 20 of the enriched pathway terms are displayed here. - In summary, based on parallel analyses of global gene expression and the cellular methylome upon BmNPV in- fection, our results suggest that alterations in DNA methylation status may have effects on the expression of genes associated with BmNPV infection. - Our results may provide a new idea and research direction for the molecular mechanism of the interaction between silkworm and viruses.. - Data represent the relative transcript levels of genes in the infected midguts compared to the control tissues from three independent samples. - The absorbance of the suspension was measured at 490 nm. - The percentages of metabol- ically active cells were compared with the percentage of control cells of the same culture plate. - The sequences of the primers were listed in Additional file 1: Table S4.. - Then these DNA fragments were treated twice with bisulfite using EZ DNA Methylation-Gold™ Kit (Zymo Research) before PCR amplification. - The unique best alignment reads are then com- pared to the normal genomic sequence and the methylation level of all cytosine in the reads is evaluated.. - In order to calculate the methylation level of the sequence, we divided the sequence into multiple bins with bin size is 10 kb. - (2) the proportion of the difference sites with P value less than 1e-05 was greater than 50% of the region (parameter: p.threshold = 1e-05;. - (3) the number of the sites was greater than 3, and the length was greater than 50 (param- eter: minlen = 50. - DNA methylation of individual CG sites were analyzed using Quma software (http://quma.cdb.riken.jp/).. - Summary of the sequenced reads. - Differential DNA methylation regions of silkworm midguts upon BmNPV infection. - DMRs: Differential DNA methylation regions. - This work was financially supported by the National Nature Science Foundation of China (No.31502016) for the design and execution of the study. - Comparative analyses of DNA methylation and sequence evolution using Nasonia genomes. - Hypermethylation of the interferon regulatory factor 5 promoter in Epstein-Barr virus-associated gastric carcinoma. - KSHV LANA inhibits TGF-beta signaling through epigenetic silencing of the TGF-beta type II receptor. - Inhibiting DNA methylation causes an interferon response in Cancer via dsRNA including endogenous retroviruses.. - Cytosine DNA methylation is found in Drosophila melanogaster but absent in Saccharomyces cerevisiae, Schizosaccharomyces pombe, and other yeast species. - Single base-resolution methylome of the silkworm reveals a sparse epigenomic map. - First evidence of DNA methylation in insect Tribolium castaneum: environmental regulation of DNA methylation within heterochromatin. - Whole-genome DNA methylation profile of the jewel wasp (Nasonia vitripennis). - DNA methylation in insects: on the brink of the epigenomic era. - Genome, transcriptome and methylome sequencing of a primitively eusocial wasp reveal a greatly reduced DNA methylation system in a social insect. - DNA methylation in silkworm genome may provide insights into epigenetic regulation of response to Bombyx mori cypovirus infection. - Genome-wide and caste-specific DNA methylomes of the ants Camponotus floridanus and Harpegnathos saltator. - DNA methylation in social insects: how epigenetics can control behavior and longevity. - Infection with a virulent strain of Wolbachia disrupts genome wide-patterns of cytosine methylation in the mosquito Aedes aegypti. - Conservation of DNA methylation in dipteran insects. - The progress and future of enhancing antiviral capacity by transgenic technology in the silkworm Bombyx mori. - The promoter of the late p10 gene in the insect nuclear polyhedrosis virus Autographa californica: activation by viral gene products and sensitivity to DNA methylation. - The genome of the stick insect Medauroidea extradentata is strongly methylated within genes and repetitive DNA. - Gradual transition from mosaic to global DNA methylation patterns during deuterostome evolution. - Genome-wide evolutionary analysis of eukaryotic DNA methylation. - General transcription factor binding at CpG islands in normal cells correlates with resistance to de novo DNA methylation in cancer cells. - Overexpression of the structural maintenance of chromosome 4 protein is associated with tumor de-differentiation, advanced stage and vascular invasion of primary liver cancer. - Functions of DNA methylation: islands, start sites, gene bodies and beyond. - Role of the host restriction factor APOBEC3 on papillomavirus evolution. - DNA Tumor Virus Regulation of Host DNA Methylation and Its Implications for Immune Evasion and. - Epigenome-wide differential DNA methylation between HIV-infected and uninfected individuals.. - Identification, gene expression and immune function of the novel Bm-STAT gene in virus-infected Bombyx mori. - Differentially expressed JAK-STAT signaling pathway genes and target microRNAs in the spleen of necrotic enteritis-afflicted chicken lines. - The role of JAK-STAT signaling pathway and its regulators in the fate of T helper cells. - Antiviral activity in the mulberry silkworm , Bombyx mori L. - Functional analysis of inhibitor of apoptosis 1 of the silkworm Bombyx mori
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