Heme-Peroxidase 2 Modulated by POU2F1 and SOX5 is Involved in Pigmentation in Pacific Oyster (Crassostrea gigas).

Color polymorphism is frequently observed in molluscan shellfish, while the molecular regulation of shell pigmentation is not well understood. Peroxidase is a key enzyme involved in melanogenesis. Here, we identified a heme-peroxidase 2 gene (CgHPX2), and characterized the expression patterns and transcriptional regulation of CgHPX2 in the Pacific oyster Crassostrea gigas. Tissues expression analysis showed that CgHPX2 was a mantle-specific gene and primarily expressed in the edge mantle in black shell color oyster compared with white shell oyster. In situ hybridization showed that strong signals for CgHPX2 were detected in the both inner and outer surface of the outer fold of mantle in the black shell color oyster, whereas positive signals in white shell oyster were mainly localized in the outer surface of the outer fold of mantle. In the embryos and larvae, a high expression level of CgHPX2 was detected in the trochophore stage in both black and white shell color oysters. The temporal localization of CgHPX2 was mainly detected in the shell gland and edge mantle of trochophore and calcified shell larvae, respectively. In addition, a 2227 bp of 5' flanking region sequence of CgHPX2 was cloned, which contained a presumed core promoter region and many potential transcription factor binding sites. Further luciferase assay experiment confirmed that POU domain, class 2, transcription factor 1 (POU2F1), and SRY-box transcription factor 5 (SOX5) were involved in transcriptional regulation of CgHPX2 gene through binding to its specific promoter region. After CgPOU2F1 and CgSOX5 RNA interference, the CgHPX2 gene expression was significantly decreased. These results suggested that CgPOU2F1 and CgSOX5 might be two important transcription factors that positively regulated the expression of CgHPX2 gene, improving our understanding of the transcriptional regulation of molluscan shell pigmentation.