Whole-genome bisulfite sequencing reveals the function of DNA methylation in the allotransplantation immunity of pearl oysters.

Introduction: In the pearl culture industry, a major challenge is the overactive immunological response in pearl oysters resulting from allotransplantation, leading to shell-bead rejection and death. To better understand the molecular mechanisms of postoperative recovery and the regulatory role of DNA methylation in gene expression, we analyzed the changes in DNA methylation levels after allotransplantation in pearl oyster Pinctada fucata martensii, and elucidated the regulatory function of DNA methylation in promoter activity of nicotinic acetylcholine receptor (nAChR) gene. Methods: We constructed nine DNA methylomes at different time points after allotransplantation and used bisulfite genomic sequencing PCR technology (BSP) to verify the methylation status in the promoter of nAChR. We performed Dual luciferase assays to determine the effect of the dense methylation region in the promoter on transcriptional activity and used DNA pull-down and mass spectrometry analysis to assess the capability of transcription factor binding with the dense methylation region. Result: The DNA methylomes reveal that CG-type methylation is predominant, with a trend opposite to non-CG-type methylation. Promoters, particularly CpG island-rich regions, were less frequently methylated than gene function elements. We identified 5,679 to 7,945 differentially methylated genes (DMGs) in the gene body, and 2,146 to 3,385 DMGs in the promoter at each time point compared to the pre-grafting group. Gene ontology and pathway enrichment analyses showed that these DMGs were mainly associated with "cellular process", "Membrane", "Epstein-Barr virus infection", "Notch signaling pathway", "Fanconi anemia pathway", and "Nucleotide excision repair". Our study also found that the DNA methylation patterns of the promoter region of nAChR gene were consistent with the DNA methylomics data. We further demonstrated that the dense methylation region in the promoter of nAChR affects transcriptional activity, and that the methylation status in the promoter modulates the binding of different transcription factors, particularly transcriptional repressors. Conclusion: These findings enhance our understanding of the immune response and regulation mechanism induced by DNA methylation in pearl oysters after allotransplantation.

Immunomodulatory effects of decitabine in pearl oyster Pinctada fucata martensii.

Decitabine (DAC), an inhibitor of DNA methyltransferase, is typically used to reverse DNA methylation and is considered an epigenetic modifying drug. DNA methylation is crucial to the regulation of gene expression without altering genetic information. Our previous research showed that the DNA methylation levels of many immune-related genes changed after the pre-grafting condition in pearl production. In the present study, we evaluated the DNA methylation level and analyzed transcriptome, enzyme, and antimicrobial activities after DAC treatment to evaluate the effect of DAC on DNA methylation and immune system of pearl oyster Pinctada fucata martensii. Results showed that DAC significantly decreased the level of global DNA methylation in the hemocytes of the pearl oysters. Transcriptome analysis obtained 577 differentially expressed genes (DEGs) between the control and DAC treatment group. The DEGs were mainly enriched in the following pathways: "Relaxin signaling pathway," "Cytosolic DNA-sensing pathway," "Platelet activation," and "Peroxisome," and related genes were overexpressed after DAC treatment. DAC treatment resulted in a substantial increase in the levels of serum superoxide dismutase, interleukin-17, phenol oxidase, tumor necrosis factor, and antimicrobial activity, compared with the control. These results suggested that DAC can alter DNA methylation level, activate immune-related genes, and improve the level of humoral immunity in pearl oysters, thereby increasing our understanding of the mechanism underlying DNA methylation in immune regulation.

Exploring crucial molecular events in pearl oyster after pre-grafting conditioning by genome-wide bisulfite sequencing for DNA methylation analysis.

Pre-grafting condition is an important method to promote recovery from transplant surgery during pearl production. In the present study, we constructed two DNA methylomes from pearl oysters with and without conditioning to investigate the molecular mechanism of the pearl oyster Pinctada fucata martensii underlying the pre-grafting condition. A total of 4,594,997 and 4,930,813 methyl CG in the control (Con) and pre-grafting group (PT) were detected, resulting in the whole genome methylation profile and methylation pattern in P. f. martensii. Results reveal that the promoter, especially the CpG island-rich region, was more infrequently methylated than the gene function elements in P. f. martensii. A total of 51,957 differently methylated regions (DMRs) between Con and PT were obtained, including 3789 DMR in the promoter and 16,021 in the gene body. Based on gene ontology and pathway enrichment analyses, these DMRs were mainly related to "cellular process", "metabolic process", "Epstein-Barr virus infection", and "Fanconi anemia pathway". The methylation site in the promoter region may be associated with the promoter activity and transcription factor binding. These results help our understanding of the mechanism of pre-grafting condition, thereby providing key information in guiding to improve the conditioning methods for enhanced pearl oyster survival rate after transplantation.