CgC/EBPβ promotes haemocyte proliferation in the Pacific oyster (Crassostrea gigas) by mediating transcriptional regulation of CgRunx-1.

The CCAAT/enhancer-binding protein beta (C/EBPβ) plays crucial roles in various biological processes, including cell proliferation, differentiation, energy metabolism, and signal transduction. In this study, the CgC/EBPβ with a conserved DNA-binding domain and a leucine zipper (BZIP) domain was identified in the Pacific oyster (Crassostrea gigas). CgC/EBPβ was highly expressed during the later developmental stages, especially in the stage of Umbo larva, which was 921.09-fold of that in the Morula stage (p < 0.05). Furthermore, CgC/EBPβ was highly expressed in haemocytes (360.80-fold of that in hepatopancreas) (p < 0.05), especially in granulocyte subpopulation (30.71-fold of that in agranulocytes (p < 0.0001). The mRNA expression level of CgC/EBPβ in haemocytes was significantly up-regulate at 12 h post Vibrio splendidus stimulation, and its protein was predominantly translocated into nucleus of haemocytes (5.01-fold increase compared with that in control group, p < 0.01). The CgC/EBPβ binding site was predicted in promoter of hematopoietic transcription factor CgRunx-1, and its enrichment in promoter region was significantly increased after V. splendidus stimulation. After the expression of CgC/EBPβ was knock-down by injection of dsCgC/EBPβ, the expressions of its downstream CgRunx-1, CgBMP-7, and cell cycle-related gene CgCDC45, as well as the granulocyte marker CgAATase were also markedly reduced, with expression levels declining to 0.26, 0.56, 0.58, and 0.39 times of that in control group, respectively (p < 0.05). Furthermore, the ratios of EdU + haemocytes and granulocytes in the CgC/EBPβ-RNAi group were significantly reduced, which was 0.31-fold and 0.27-fold of that in control group. In summary, CgC/EBPβ is predominantly expressed in the granulocyte of C. gigas, which drives the expression of hematopoietic transcription factor CgRunx-1 to promote the proliferation of haemocytes in response to V. splendidus stimulation.