The DNA cytosine-5-methyltransferase 3 (DNMT3) involved in regulation of CgIL-17 expression in the immune response of oyster Crassostrea gigas.

DNA methyltransferase, a key enzyme mediating DNA methylation, is involved in numerous processes including genomic imprinting, X chromosome inactivation, transposable element suppression, and immune defense in vertebrates. In the present study, a DNA cytosine-5-methyltransferase 3 was identified from oyster Crassostrea gigas (designed as Cg DNMT3). There were a PWWP domain, a PHD domain and a DNA-methylase domain in the deduced amino acid sequences of Cg DNMT3, and the conserved motifs I, IV, VI, Ⅷ, IX and X were identified in its C-terminal catalytic DNA-methylase domain. The mRNA transcripts of Cg DNMT3 were detected in haemocytes, mantle, gill, adductor muscle, digestive gland and labial palp, with higher expression level in haemocytes (6.54 folds of those in gill, p < 0.01). The expression level of Cg DNMT3 mRNA in haemocytes increased significantly after LPS primed (2.87 folds of that in control group, p < 0.05) in vitro or Vibrio splendidus challenging (1.94 folds of that in control group, p < 0.05) in vivo. Immunocytochemical analysis revealed that Cg DNMT3 protein was distributed mainly in cytoplasm and partial in nucleus of oyster haemocytes. After Cg DNMT3 was transfected and expressed in HEK293T cells, the DNA 5-methylcytosine (5-mc) level in the transfected group was significantly increased, which was 1.22 folds (p < 0.05) of the pcDNA-3.1 group. The expressions of oyster Cg IL17-1, Cg IL17-2 and Cg IL17-5 in haemocytes increased (13.05 folds, 4.78 folds and 9.41 folds of that in control group, respectively) at 12 h after V. splendidus challenging, but the increase were significantly inhibited when the oysters were pre-treated with DNA methyltransferase inhibitor 5-Azacytidine, which were 9 folds, 1.93 folds and 3.22 folds of that in control group, respectively. These results collectively suggested that Cg DNMT3 was a conserved member of DNA methyltransferase 3 family in oyster, and participated in regulating the expression of cytokines during immune response. • Cg DNMT3 is a conserved member of DNMT3 family in oysters. • Cg DNMT3 is constitutively expressed in adult tissues with slightly higher level in haemocyets. • LPS stimulation and Vibrio splendidus challenge up-regulated the expression of Cg DNMT3. • Cg DNMT3 was involved in the regulation of Cg IL17 expressions during immune response. [ABSTRACT FROM AUTHOR]