The transcription factor Pf-POU3F4 regulates expression of the matrix protein genes Aspein and Prismalin-14 in pearl oyster (Pinctada fucata)

Abstract

Matrix proteins play key roles in shell formation in the pearl oyster, but little is known about how these proteins are regulated. Here, two POU domain family members, Pf-POU2F1 and Pf-POU3F4, were cloned and characterized. Functional domain analysis revealed that both them have conserved POUS and POUH domains; these domains are important for transcription factor function. The tissue distributions of Pf-POU2F1 and Pf-POU3F4 mRNAs in pearl oyster revealed different expression patterns, and the expression of Pf-POU3F4 mRNA was relatively high in the mantle. The promoters of the matrix protein genes Aspein and Prismalin-14 were cloned using genome-walking PCR. Relatively high transcriptional activities of these promoters were detected in HEK-293T cells. In transient co-transfection assays, Pf-POU3F4 greatly up-regulated the promoter activities of the Aspein and Prismalin-14 genes in a dose-dependent manner. Structural integrity of Pf-POU3F4 was essential for its activation function. One region of the Aspein gene promoter, -181 to -77 bp, and two binding sites in the Prismalin-14 gene promoter, -359 to -337 bp and -100 to -73 bp, were required for activation of Pf-POU3F4. An electrophoresis mobility shift assay demonstrated that Pf-POU3F4 directly bound these sites. Pf-POU3F4 knockdown led to a decrease in Aspein and Prismalin-14 gene expression. Furthermore, expression levels for the Pf-POU3F4 gene were similar to those of the Aspein and Prismalin-14 genes during five development stages. Taken together, these results suggest that the transcription factor Pf-POU3F4 regulates expression of the matrix protein genes Aspein and Prismalin-14 in pearl oyster. Database The nucleotide sequence data of Pf-POU2F1 is available in the GenBank databases under the accession number KM588196. The nucleotide sequence data of Pf-POU3F4 is available in the GenBank databases under the accession number KM519606. The nucleotide sequence data of Aspein gene promoter is available in the GenBank databases under the accession number KM519607. The nucleotide sequence data of Prismalin-14 gene promoter is available in the GenBank databases under the accession number KM519601. The matrix proteins Aspein and Prismalin-14 participate in the formation of the prismatic layer of the shell in pearl oyster. The transcription factor, Pf-POU3F4, directly binds one region of the Aspein gene promoter, -181 to -77 bp, and two binding sites in the Prismalin-14 gene promoter, -359 to -337 and -100 to -73 bp, to enhance their gene expressions.