The Landscape Of Dynamic Accessible Chromatin In Response To Bombyx Mori Nucleopolyhedrovirus Infection

Baculoviruses are a large group of invertebrate-specific DNA viruses and one hallmark of the baculovirus infection cycle is the production of two different phenotypic virions:the budded virion(BV)and the occlusion-derived virion(ODV).During baculovirus infection,viral DNA is synthesized in the virogenic stroma(VS),and the host chromatin is marginalized as a result of VS expansion.Bombyx mori nucleopolyhedrovirus(BmNPV),a representative member of alphabaculoviruses,is a major pathogen of silkworm Bombyx mori,which can cause severe damage to the sericulture industry around the world.Thus,the study of epigenome regulation of host caused by NPV infection is not only helpful to understand the mechanism of baculovirus infection,but also to deeply understand the pathogenesis of BmNPV,providing theoretical basis for the prevention of BmNPV infection.Therefore,this study systematically studied the dynamic regulation of host chromatin in response to BmNPV infection by using ATAC-seq in combination with biochemical and cellular analysis.The conclusions were drawn as followings:1.BmNPV infection causes the marginalization of host chromatin,but does not affect its integrityWe used a commercial histone H3 antibody to analyze host chromatin distribution using immunofluorescence microscopy.Following VS expansion from 8 to 48 h p.i.,the host genome was gradually located at the lamin-labeled inside region of nuclear membrane.At the same time,the results of electron microscopy also showed that the host chromatin was marginalized after viral infection.TUNEL assay showed that no significant host chromatin fragmentation was detected between 8 and 72 h p.i.,suggesting that viral infection does not affect chromatin integrity.2.The dynamics changes of chromatin in response to BmNPV infection.As an emerging technique in recent years,ATAC-seq was applied in silkworm for the first time in this study.Data quality control indicated that the ATAC-seq data obtained in this study were of high quality and could be used for subsequent analysis.Preliminary analysis showed that the number of accessible regions of host chromatin significantly increased at 48 h p.i.,and the overall status of open chromatin changed with the progression of infection.3.Analysis of ATAC-seq and RNA-seq revealed that dynamically changing regions of chromatin accessibility were involved in host genes transcriptional regulation.The analysis of the differences in the chromatin accessibility revealed that there were a small number of different regions at the early stage of infection.At the late stages,chromatin accessibility changed dramatically,indicating that the chromatin location changed and its state was remodeling.The correlation analysis of transcriptome data showed that the promoter-TSS regions with differences at different infection phases significantly regulated the expression of host genes.These results indicated that the chromatin remodeling caused by viral infection was not a disorder change,but an orderly participation in the transcription regulation of genes.4.At 48 h p.i.,host chromatin is in a more open state and nucleosomes are disassembled.K-means clustering analysis revealed that host chromatin was in a more open state at 48 h p.i.DNase ?&TUNEL assay supported this conclusion.In addition,the fragment sizes were identified,and it was found that the BmN cells,8 and 24 h p.i.had similar nucleosome assembly states,while the nucleosomes were disassembled at 48 h p.i.Western blotting also showed a significant decrease in the expression of histone H3,confirming that host chromatin was more accessible and nucleosomes disassembled at the very late stage.5.Histone modifications are involved in host chromatin changes during BmNPV infectionConsidering the nucleosome assembly is also affected by histone modifications the relationships of chromatin accessibility and histone modifications were analyzed.The results suggested H3K4me3 and H3K27ac were involved in the formation of open chromatin regions.H3K27me3 participated in the maintenance of epigenetic repression state in cluster ? regions in the control,and this cluster was activated at the very late stage of BmNPV infection.Moreover,the levels of these modifications over the course of infection were examined by western blot analysis.The results indicated that,H3K4me3 and H3K27me3 were markedly down-regulated at the very late stage of BmNPV infection,but H3K27ac was up-regulated,suggesting that the increase of H3K27ac as well as the decrease of H3K27me3 might contribute to the elevated DNA accessibility.Spike-in ChIP-seq results suggested that H3K27ac exerted essential roles in viral-induced the regions of clusters ? and ? that gained accessibility at the very late stage of infection.