The Regulatory Mechanism Of DNA Methylation In Transplantation Immunity Of Pearl Oyster Pinctada Fucata Martensii

DNA methylation is a conserved epigenetic modification that plays an important role in regulating gene expression and maintaining genomic stability.Previous studies have shown that DNA methylation is involved in the regulation of transplantation immunity in Pinctada fucata martensii,but the mechanism of regulation is not clear.In this study,P.f.martensii was used as the research object,the effects of DNA methylation on the expression of genes related to transplantation immunity were analyzed by Whole-genome bisulfite sequencing(WGBS)and transcriptome(RNAseq)techniques.The effects of DNA methylation modification on transcription factor binding were analyzed by transposase accessible chromatin using sequencing(ATACseq)and DNA pull down.The regulatory genes and regulatory mechanisms of DNA methyl-binding protein(MBD)were analyzed by DNA affinity purification sequencing(DAP-seq),RNA interference,CO-IP and GST pull down.The results are as follows:1.Regulation of DNA methylation on transplantation immunity-related genes:(1)WGBS was used to analyze the differentially methylated regions(DMRs)induced by transplantation.A total of 131094 DMRs were obtained,corresponding to 13474 genes.These genes were mainly enriched in Spliceosome,Proteasome,m TOR signaling pathway,Cell cycle,RIG-I-like receptor signaling pathway and et al.A total of 5041 differentially expressed genes(DEGs)were obtained by transcriptome analysis.DEGs were mainly enriched in Proteasome,DNA replication,Mismatch repair,Base excision repair,cell cycle and other signaling pathways.Combined analysis of WGBS and RNAseq showed that DNA methylation of upstream 2kb,gene body and downstream 2kb was positively correlated with gene expression level.A total of 2767 DEGs with methylation differences were obtained.These genes were mainly enriched in Proteasome,Base excision repair,cell cycle,DNA replication,Mismatch repair and other signaling pathways,and most of the genes were significantly up-regulated at 24 h,indicating that DNA methylation is involved in the regulation of transplantation immunity by affecting these genes.2.The effect of DNA methylation modification on transcription factor binding:(1)A total of 306 transcription factors were enriched in chromatin accessible regions in P.f.martensii by ATAC-seq technology.Combined with WGBS analysis,20 transcription factors(TFs)such as REST and PAX9 tended to bind to the methylated motifs,and 286 TFs such as NRF1 and KLF4 tended to bind to non-methylated motifs.Combined analysis of WGBS,ATAC-seq and RNA-seq showed that 813 genes,including 306 motifs,were enriched in the unique open chromatin regions(OCRs),DEGs and DMRs of pearl oysters in the control(Con)group.Among them,the TFs regulated by PAX1,JUNB(var.2),Crem,E2F4 and PAX9 were highly expressed in Con,and the proportion of DNA methylation modification was also high.The specific OCRs,DEGs and DMRs of pearl oyster were enriched to 331 genes at 24 h after transplantation,including 304 motifs.The TFs regulated by HIF1 A,ARNT:: HIF1 A,E2F4,Rarg(var.2)and JUNB(var.2)were highly expressed at 24 h,and the proportion of DNA methylation modification was also high.(2)According to the results of WGBS,ATAC-seq and RNA-seq correlation analysis,the methylation difference sites were screened,the methylation probes and non-methylation probes were designed,and the DNA pull down experiment was carried out to analyze the effect of DNA methylation modification on transcription factor binding.The results showed that X-box binding protein 1(XBP1)could bind to the methylation modified probe,and 50.91%-51.98% of the binding motif of XBP1 contained methylation modification.ELF3 can bind to non-methylated probes,and 63.62%-65.99% of the ELF3 binding motif is not methylated,further indicating that DNA methylation affects transcription factor binding.The results of dual luciferase assay showed that the promoter methylation modification regions of PmSR and PmCFH genes contained promoter activity regulatory elements.3.The regulatory role and mechanism of PmMBD2 protein in transplantation immunity: The genome of P.f.martensii contains one PmMBD2.Compared with mammal MBD2,mollusk PmMBD2 lacks more than 140 amino acids at the N-terminus,but the key amino acids that bind to methylated DNA do not change,indicating that it may have the ability to bind to methylated DNA.Promoter activity analysis showed that DNA methylation modification affected the promoter activity of PmMBD2.Subcellular localization showed that PmMBD2 was mainly located in the nucleus.A total of 83 PmMBD2 binding proteins such as TEF-1,histone H2 B,H4,HDAC11 and Jumonji(Jmjd)were obtained by GST pull down and CO-IP.Bimolecular fluorescence complementation experiments confirmed that PmMBD2 could interact with Jmjd,TEF-1 and HDAC11.DAP-seq results showed that there were 7597 target genes of PmMBD2,which were mainly enriched in ECM-receptor interaction,Fc epsilon RI signaling pathway,PI3K-Akt signaling pathway and other signaling pathways.Combined analysis of DAP-seq and WGBS found that 96.81 % of the PmMBD2 binding sequence contained methylation modification.Microscale thermophoresis(MST)results showed that PmMBD2 could bind to both methylated and unmethylated probes.RNA interference was used to reduce PmMBD2,and 4663 DEGs were found,mainly enriched in Fc epsilon RI signaling pathway,Gap junction,Gn RH signaling pathway and other signaling pathways.Combined with DAP-seq and transcriptome analysis of hemocytes before and after transplantation,506 PmMBD2 targeted regulatory genes were found to be involved in the immune process of transplantation.The detection of serum immune indexes showed that the decrease of PmMBD2 expression could significantly increase the activity of superoxide dismutase(SOD),catalase(CAT),phenoloxidase(PO).4.The role and mechanism of PmMBD4 protein in transplantation immunity: The genome of P.f.martensii contains one PmMBD4 gene,which has MBD domain.Promoter activity analysis showed that the DNA methylation modification region affected the promoter activity of PmMBD4,and subcellular localization showed that PmMBD4 mainly existed in the nucleus.DAP-seq results showed that PmMBD4 had a total of 8662 target genes,mainly enriched in m TOR signaling pathway,T cell receptor signaling pathway,TNF signaling pathway,MAPK signaling pathway and other signaling pathways.DAP-seq and WGBS combined analysis found that all of the PmMBD4 binding sequence contained methylation modification.A total of 4501 DEGs were obtained by RNA interference to reduce PmMBD4,which were mainly enriched in NF-kappa B signaling pathway,Lysosome,Toll-like receptor signaling pathway and other signaling pathways.Combined with DAP-seq and transcriptome analysis of hemocyte before and after transplantation,522 PmMBD4 targeted regulatory genes were found to be involved in the immune process of transplantation.Serum indicators showed that PmMBD4 silencing significantly reduced the activities of SOD,CAT and inducible nitric oxide synthase(i NOS),and increased the activity of PO.5.The effect of DNA methylation modification on alternative splicing: Correlation analysis of WGBS and full-length transcriptome data before and after transplantation showed that the methylation level of included exons in P.f.martensii was higher than the skipped exons,and the genes with high methylation level are longer,the genes with high alternative splicing are longer,and the genes with methylation modification and alternative splicing are longer than those without methylation and alternative splicing.DNA pull down experiments were performed using the gene body region of DNA polymerase delta catalytic subunit(DNA pol-delta).The results showed that the proteins bound by the methylation probe included RNA binding protein,SWI / SNF complex,histone binding protein RBBP7,integrin-linked protein kinase subtype X1,etc.The unmethylated probes bind to transcription factor A,E2F3,ELF-3,elongation factor EF-1,histone lysine N-methyltransferase 2A,Jmjd and so on,indicating that DNA methylation modification can regulate the transcription and alternative splicing process of gene ontology by affecting these proteins.