Transcriptional regulation of the matrix protein Shematrin-2 during shell formation in pearl oyster

The molluscan shell is a fascinating biomineral consisting of a highly organized calcium carbonate composite. Biomineralization is elaborately controlled and involves several macromolecules, especially matrix proteins, but little is known about the regulatory mechanisms. The matrix protein Shematrin-2, expression of which peaks in the mantle tissues and in the shell components of the pearl oyster Pinctada fucata, has been suggested to be a key participant in biomineralization. Here, we expressed and purified Shematrin-2 from P. fucata and explored its function and transcriptional regulation. An in vitro functional assay revealed that Shematrin-2 binds the calcite, aragonite, and chitin components of the shell, decreases the rate of calcium carbonate deposition, and changes the morphology of the deposited crystal in the calcite crystallization system. Furthermore, we cloned the Shematrin-2 gene promoter, and analysis of its sequence revealed putative binding sites for the transcription factors CCAAT enhancer–binding proteins (Pf-C/EBPs) and nuclear factor-Y (NF-Y). Using transient co-transfection and reporter gene assays, we found that cloned and recombinantly expressed Pf-C/EBP-A and Pf-C/EBP-B greatly and dose-dependently up-regulate the promoter activity of the Shematrin-2 gene. Importantly, Pf-C/EBP-A and Pf-C/EBP-B knockdowns decreased Shematrin-2 gene expression and induced changes in the inner-surface structures in prismatic layers that were similar to those of antibody-based Shematrin-2 inhibition. Altogether, our data reveal that the transcription factors Pf-C/EBP-A and Pf-C/EBP-B up-regulate the expression of the matrix protein Shematrin-2 during shell formation in P. fucata, improving our understanding of the transcriptional regulation of molluscan shell development at the molecular level.

A vast array of organisms can convert inorganic ions into solid minerals by a process termed biomineralization. Due to their superior performance as materials and their medical benefits, products of biomineralization, such as teeth, bones, otoliths, spicules, shells, and pearls, have intrigued many investigators from life science, biophysics, and materials science (1). As the understanding of the mechanisms of shell and pearl formation may improve pearl quality or lead to the generation of new medical materials (2,3), these mechanisms have been extensively studied by researchers in various fields.
The pearl oyster Pinctada fucata, the shell of which is composed of an inner nacre and an outer prismatic layer, is a major seawater pearl shellfish found in the southeast of China. Both nacre and prism are mainly composed of calcium carbonate; less than 5% of their components are organic macromolecules, including matrix proteins, polysaccharides, and lipids (4). Matrix proteins, such as nacrein (5), Pif (6), the KRMP family (7), and the Shematrin family (8), have been proven to be the major components responsible for nucleation, orientation, morphology, and organization during the shell formation process in P. fucata (9). The Shematrin family, one of the two main families that participate in the shell formation process, is highly expressed in the mantle of P. fucata and other pearl oyster species (Pinctada maxima and Pinctada margaritifera) (10,11), which indicates their significant and conserved roles in shell formation. The Shematrin-2 protein, a member of the Shematrin family, is abundantly expressed at the edge of the mantle and is supposed to function as a framework protein in the formation of the prismatic layer of the shell (8). However, information on the function and transcriptional regulation of Shematrin-2 is limited. The characteristic structural organization of the shell is of interest; although the mechanisms of calcification that matrix proteins participate in have been sought by many investigators, little is known about how the upstream transcription factors regulate the downstream matrix protein genes (12)(13)(14)(15).
The C/EBP 4 family consists of six isoforms (␣, ␤, ␥, ␦, ⑀, and ), and the isoforms are structurally similar, with a highly conserved basic leucine zipper domain at the C terminus, which facilitates dimerization and DNA binding (16,17). However, C/EBP isoforms are functionally distinct, as their transcriptional activation domains are less well conserved. This difference gives rise to a wide range of responses in which C/EBP isoforms have been implicated, including cell proliferation and differentiation, the immune response, the cell cycle, and cancer development, hematopoiesis, and osteoclast formation (16, 18 -23). In mammals, the C/EBP isoforms have been reported to participate in various biological processes, especially in the formation of teeth and bones. Previous studies have shown that C/EBP␣ can active the transcription of the amelogenin gene, which is essential for enamel biomineralization (24,25). In addition, C/EBP␤ and C/EBP␦ participate in bone formation by activating the transcription of osteocalcin (26). Although homologous genes of C/EBP have been identified in some mollusk species (27,28), their function in biomineralization has not been further explored.
In this study, we expressed and purified the Shematrin-2 protein for the first time and explored its function during shell formation in P. fucata. In addition to the cloning and identification of C/EBP transcription factors, we further explored the upstream transcription mechanisms of Shematrin-2 in P. fucata. Our results provided the foundation for understanding the transcriptional regulation of matrix proteins and shed light on the mechanisms of shell formation at the molecular level.

The distribution, expression, and purification of Shematrin-2
The quantitative PCR results showed that the relative expression level of Shematrin-2 in mantle tissues was maintained at an extremely high level compared with the expression of three other matrix proteins, namely, ACCBP, MIS60, and N19 (Fig.  1A), which was in accordance with the results from transcriptome sequencing (8). The high expression level in mantle tissues indicated the significant role that Shematrin-2 plays dur-ing the process of shell formation. However, its function has not been identified yet due to difficulties in protein expression. In our study, a soluble MBP label was added to the N terminus of Shematrin-2 to enhance the amount of soluble protein expressed. The SDS-PAGE results showed that the purified Shematrin-2-MBP recombinant protein and the MBP label protein appeared as clear and distinct bands on the gel (Fig. 1B).
Next, to explore the exact distribution of Shematrin-2 in the shell layers, polyclonal antibodies raised against recombinant Shematrin-2 were used for the immunodetection of native Shematrin-2 in EDTA-soluble matrix and EDTA-insoluble matrix (EISM) extracts of separated nacre and prisms and in the extrapallial fluid. The Western blotting results showed that Shematrin-2 was present in the EISM of the nacreous and prismatic layers of the shell and in the extrapallial fluid (Fig. 1C). According to previous reports on matrix proteins (29,30), EISM proteins are conventionally believed to be responsible for the construction of the structural organic framework of the shell and for the physical properties of the shell during calcification. Moreover, the extrapallial fluid is considered the place where shell biomineralization occurs, and some of the matrix proteins play direct regulatory roles in the extrapallial cavity (30 -32). Therefore, it is logical to assume that Shematrin-2 may be not only a structural matrix protein but also a regulatory factor affecting crystalline deposition in shell biomineralization.

The effects of Shematrin-2 on shell formation in vitro
To confirm the hypothesis that Shematrin-2 plays an important role in calcium carbonate crystallization, we conducted a series of experiments. First, we investigated the binding properties of Shematrin-2 with the main components of the shell by the crystal-binding assay. As shown in Fig. S1, compared with the soluble MBP-labeled protein, Shematrin-2-MBP recombinant protein was found to be bound to calcite, aragonite, and Transcriptional regulation of matrix protein Shematrin-2 chitin after three washes with Milli-Q water and 200 mM NaCl (Fig. S1, lanes 3, 6, and 9), and the binding of the protein to chitin was weaker than its binding to aragonite and calcite. In addition, a precipitation experiment was performed to analyze the effect of Shematrin-2 on the rate of calcium carbonate precipitation. Compared with the control, 2 g/ml of Shematrin-2-MBP recombinant protein significantly slowed the process of precipitation 2 min after the reaction, and this effect occurred in a concentration-dependent manner ( Fig. 2A).
To further characterize the role that Shematrin-2 plays in the growth of calcium carbonate crystals, we performed calcite and aragonite crystallization experiments in the presence of 10 and 40 g/ml of Shematrin-2-MBP recombinant protein in vitro; 40 g/ml of MBP protein was used as a negative control. In the calcite crystallization system, the precipitated crystals were all typical calcite rhombohedra in the control groups with or without 40 g/ml of MBP (Fig. 2, B and C), and the Raman spectra showed that the crystals were calcite (Fig. 2, F and G). With 10 g/ml of Shematrin-2-MBP recombinant protein, the morphologies of the deposited crystals were modified: the edges of the crystals were no longer regular, and irregular depositions, similar to the multinucleation site superpositions, were observed ( Fig.  2D). As the concentration of the recombinant protein increased, there was increased formation of irregular crystals and a decrease in the number of crystals that formed (Fig.  2E). However, analysis of the Raman spectra showed that in the presence of 10 and 40 g/ml of Shematrin-2-MBP recombinant protein, the crystals were still calcite (Fig. 2, G and H). These results suggested that the Shematrin-2 protein could regulate the morphology of calcite crystallization rather than crystal formation. In contrast to calcite, the different concentrations of the Shematrin-2-MBP recombinant protein had little effect on aragonite formation (Fig.  S2).

The effects of Shematrin-2 on shell formation in vivo
Due to the abundance of Shematrin-2 in the extrapallial fluid, where shell biomineralization occurs, an in vivo antibody inhibition assay was performed to explore the specific physiological role of Shematrin-2 in pearl oysters. The affinity-purified polyclonal antibody against Shematrin-2 (anti-Shematrin-2) was injected into the extrapallial space of P. fucata to disrupt the physiological functions of Shematrin-2. In contrast to the negative group that was treated with preimmune rabbit serum (Fig.  2J), the surface of the prismatic layer in the low-dosage anti-Shematrin-2-injected group demonstrated incomplete calcite crystallization with obvious holes (Fig. 2K). An abnormal phenomenon turned out to be more significant in the high-dosage group (Fig. 2L), where the regular polygon and the organic framework disappeared. Interestingly, no significant differences in the nacreous layer were observed after antibody injection, which was in agreement with the results of the aragonite crystallization assay in vitro. These results suggest that the prismatic layer was perturbed to some extent when the function of Shematrin-2 was inhibited, which agrees well with the observed functional experiments in vitro.

Cloning and transcriptional activities of the Shematrin-2, ACCBP, MSI60, and N19 gene promoters
To further explore the regulation mechanism, the promoters of the matrix protein genes Shematrin-2, ACCBP, MSI60, and N19 were cloned and identified (Fig. 3A). The sequences of the promoters of Shematrin-2, ACCBP, MSI60, and N19 were submitted to the GenBank TM database under the accession numbers KM519602, KM519603, KM519604, and KM519605, respectively. The cloned promoter region of Shematrin-2 was a 1616-bp fragment, and intron 1 was 384 bp. The lengths of the ACCBP, MSI60, and N19 promoters that were cloned were 2283, 2233, and 2020 bp, respectively. The intron lengths of MSI60 and N19 were 924 and 1685 bp, respectively.
To detect the transcriptional activities of the above four promoters, the obtained gene promoters were subcloned into the pGL4.10 luciferase vector individually, and then, the combined plasmids were transiently transfected into HEK293T cells. The dual luciferase reporter system results showed that the relative luciferase activity of the Shematrin-2 gene promoter was obviously higher than that of the other three gene promoters and ϳ100 times higher than that of the pGL4.10 control group (Fig.  3B). The results demonstrate that the promoter of Shematrin-2 has a relatively high transcriptional activity in HEK293T cells, which was consistent with the high expression of Shematrin-2 in mantle tissues (Fig. 1B).
To confirm our classification and to further understand the evolutionary position of Pf-C/EBP-A, Pf-C/EBP-B, and Pf-C/ EBP-␥, a phylogenetic tree was constructed. The results showed that Pf-C/EBP-A, Pf-C/EBP-B, and Pf-C/EBP-␥ were clustered with their homologs in Crassostrea gigas, which has a close relationship with pearl oyster (Fig. 3C). Similarly, the results of the phylogenetic tree for Pf-NF-YA, Pf-NF-YB, and Pf-NF-YC showed that the three proteins clustered in different branches, and they each clustered with homologs from other species (Fig. 3D). Furthermore, Pf-NF-YA and Pf-NF-YC also clustered with their homologs in C. gigas.

The expression patterns of Pf-C/EBPs and Pf-NF-Ys among different tissues and during embryonic and larval development
The mRNA expression levels of three Pf-C/EBPs members and three Pf-NF-Ys members among seven tissues in pearl oyster were measured by real-time PCR. The results showed that Transcriptional regulation of matrix protein Shematrin-2 the tissue distribution patterns of the six genes were totally different (Fig. S9), which suggests that they might be involved in different physiological activities of P. fucata. In addition, the relative expression levels of the six genes in mantle tissues were compared, as mantle tissues play a key role in shell formation. The results showed that Pf-C/EBP-A and Pf-C/EBP-B had higher expression levels in mantle tissues (Fig. 3E) than the other four genes, which implied that Pf-C/EBP-A and Pf-C/ EBP-B were more likely to participate in the process of shell formation.
The development of embryos and larvae is accompanied by the formation of shells in P. fucata; prodissoconch I is formed during the early D-shape stage, and the morphology of the juvenile stage is almost the same as that of the adult pearl oyster. Therefore, the genes that showed higher expression in the D-shape stage and juvenile stage were more likely to play prominent roles in the process of shell formation. In this study, the mRNA expression levels of the six transcription factor genes were also determined in the oosperm, trochophore stage, D-shape stage, umbonal stage, and juvenile stage (Fig. S10). The data in Fig. 3F showed the relative expression levels of the six genes in the D-shape stage and juvenile stage. Intriguingly, in the D-shape stage and juvenile stage, the expression levels of Pf-C/EBP-A and Pf-C/EBP-B were significantly higher than those of the other four genes. Altogether, these findings support the hypothesis that transcription factors Pf-C/EBP-A and Pf-C/EBP-B play a role in shell formation.

Analysis of the regulatory effects of Pf-C/EBP-A and Pf-C/EBP-B on Shematrin-2 in vitro
To further explore whether Pf-C/EBPs and/or Pf-NF-Y participate in the transcriptional regulation of Shematrin-2, cotransfection studies followed by dual-luciferase assays and Western blotting experiments were performed. As shown in Fig. 4, Pf-C/EBP-A and Pf-C/EBP-B significantly enhanced the promoter activities of Shematrin-2, and the activating effects were apparently dose-dependent, whereas Pf-C/EBP-␥ had no effects on the promoter activity of the Shematrin-2 gene. Similarly, the three members of PF-NF-Y, alone or in combination, had no obvious effect on the promoter activity of the Shematrin-2 gene. These results suggested that the transcriptional factors Pf-C/EBP-A and Pf-C/EBP-B considerably enhanced the promoter activities of the Shematrin-2 gene.

Regulation effect of Pf-C/EBP-A and Pf-C/EBP-B on Shematrin-2 during shell formation in vivo
Shells were slightly notched to investigate the functions and the correlation of Pf-C/EBP-A, Pf-C/EBP-B, and Shematrin-2 during shell formation. As shown in Fig. 5, in the process of shell regeneration, the expression patterns of Pf-C/EBP-A, Pf-C/EBP-B, and Shematrin-2 were extremely consistent; the expression level increased after shell notching, reached its highest point at 24 h after shell injury, and then decreased slowly. In addition, the statistical analysis results showed that the expression of the matrix protein Shematrin-2 was significantly correlated with Pf-C/EBP-A and Pf-C/EBP-B (Table 1). The expression patterns of KRMP and nacrein were in agreement with previous results (Fig. S11) (41), which excluded artificial errors. These results demonstrate that Pf-C/EBP-A and Pf-C/EBP-B may play essential roles in shell regeneration by regulating Shematrin-2.
To further elucidate the regulatory function of Pf-C/EBP-A and Pf-C/EBP-B on Shematrin-2 during shell formation in vivo, we performed a knockdown experiment of Pf-C/EBP-A and Pf-C/EBP-B individually by RNAi. Compared with the group injected with Milli-Q water, the expression levels of Pf-C/EBP-A and Pf-C/EBP-B were decreased by nearly 50% after dsRNA injection (Fig. 6, A and B). No significant variation in the Pf-C/EBP-A and Pf-C/EBP-B expression levels was observed in the negative control group injected with GFP dsRNA. In addition, the expression levels of Shematrin-2 were suppressed by nearly 70 and 80% after the injection of Pf-C/EBP-A and Pf-C/EBP-B dsRNA, respectively. In contrast, the expression levels of Shematrin-2 only slightly decreased in the group injected with GFP dsRNA (Fig. 6, A  and B). Altogether, these data support the hypothesis that Pf-C/ EBP-A and Pf-C/EBP-B regulate the expression of the matrix protein gene Shematrin-2 in pearl oysters.
The inner surface structures of both the prismatic and nacreous layers of the groups treated with GFP, Pf-C/EBP-A, and Pf-C/EBP-B dsRNA were also observed by SEM. In the GFP dsRNA-injected group, the surfaces of the prismatic and nacreous layers were normal, which was the same result seen in the untreated pearl oysters (Fig. 6, C and D). The normal, stair-like surface of the nacreous layer was not obvious in either the Pf-C/ EBP-A dsRNA-or the Pf-C/EBP-B dsRNA-injected groups, and the organic framework was also disrupted (Fig. 6D). The Pf-C/EBP-A dsRNA-injected group showed abnormal formation of the organic framework in the prismatic layer, where the lacuna became larger and some tiny holes appeared. In the Pf-C/EBP-B dsRNA-injected group, the changes in the prismatic layer were more obvious; the organic framework gradually disappeared, and an irregular polygon emerged (Fig. 6C). The effect of knocking down the transcription factors Pf-C/EBP-A and Pf-C/EBP-B on calcite growth during shell mineralization was similar to that of blocking Shematrin-2 in the extrapallial fluid. All the results indicated that Pf-C/EBP-A and Pf-C/ EBP-B could control the formation of the organic framework of the prismatic and nacreous layers by regulating Shematrin-2 during shell formation in vivo.

Discussion
According to previous sequence information (GenBank accession no. AB244420.1), the full-length of Shematrin-2 sequence has been acquired, which contains glycine-and tyrosine-rich domains, 41.2% of the amino acids is glycine and 8.1% of which is tyrosine. The 6 repetitive sequence elements are GGGYG, they are arranged in tandem with highly conserved repeats, making Shematrin-2 one of the most repetitive molluscan shell proteins. Although the mechanisms of calcifica-tion that matrix proteins participate in have been sought by many investigators, information on the function of Shematrin-2 is limited, as the difficulty in expressing this protein in vitro. Here, we identify the dual roles of Shematrin-2 wherein it acts as a structural matrix protein and as a regulatory factor that affects crystalline deposition. In addition, we demonstrate that the role Shematrin-2 plays during shell formation is regulated by transcription factors Pf-C/EBP-A and Pf-C/EBP-B.   (Table S2)

Transcriptional regulation of matrix protein Shematrin-2
Currently, dozens of matrix proteins have been identified, and their roles in mineralization have been characterized. It is well known that multifunctionality is a common characteristic of many shell matrix proteins, for example, MSI60 (42), nacrein (5), and Prisilkin-39 (30); we infer that Shematrin-2 shares this characteristic. The extracted matrix proteins can be classified as soluble proteins and insoluble proteins after the decalcification treatment by EDTA. Even though the insoluble matrix proteins were generally thought to be structural proteins that are responsible for the formation of the organic framework, some of these proteins have been reported to participate in the regulation of shell biomineralization (30,43). Here, we observed that the matrix protein Shematrin-2 is present in the EISM of shell nacreous and prismatic layers and in the extrapallial fluid where shell biomineralization occurs (Fig. 1C), suggesting that Shematrin-2 might act as both a structural protein and regulatory factor. To confirm this hypothesis, the biomineralization function of this protein was tested both in vivo and in vitro. In the calcium carbonate crystallization assay in vitro (Fig. 2, B-I), the recombination protein Shematrin-2-MBP influenced the morphology of calcite crystallization, instead of aragonite crystallization, in a concentration-dependent manner, and it significantly inhibited the rate of calcium carbonate precipitation ( Fig. 2A). Consistent with the above results, the in vivo observation showed that Shematrin-2 is essential for the integrity of the prismatic layer. Hence, we infer that Shematrin-2 is one of the matrix proteins that acts as both a component and a regulator of calcite growth. In addition, crystal binding experiments (Fig. S1) further revealed that the Shematrin-2 recombinant protein could bind calcite, aragonite, and chitin. Thus, the spe-cific role of Shematrin-2 in aragonite formation needs to be further investigated as the regulation of crystal growth is subtle and complex.
What is the regulatory role of Shematrin-2 during shell formation? Our study first demonstrated that Pf-C/EBP-A and Pf-C/EBP-B, the transcription factor C/EBP homologs, are involved in transcriptional regulation of the matrix protein gene Shematrin-2 in the pearl oyster. In the present study, three members of the C/EBP family, namely, Pf-C/EBP-A, Pf-C/ EBP-B, and Pf-C/EBP␥, and three subunits of NF-Ys, namely, Pf-NF-YA, Pf-NF-YB, and Pf-NF-YC, were cloned and characterized. These transcription factors also play important roles in the formation of biomineralization products such as bones or teeth in mammals (24 -26). Our results demonstrate that in contrast to the other four transcription factors, Pf-C/EBP-A and Pf-C/EBP-B could bind the promoter of the Shematrin-2 gene to enhance the transcriptional activity, and the activating effects were dose-dependent at both mRNA and protein levels (Fig. 4). These data were in agreement with the significantly higher expression levels of Pf-C/EBP-A and Pf-C/EBP-B genes in the mantle tissue and during the D-shape and juvenile stages of P. fucata development than those of the other four transcription factors (Fig. 3, E and F). Furthermore, RNAi experiments showed that Pf-C/EBP-A or Pf-C/EBP-B knockdown lead to a decrease in Shematrin-2 gene expression (Fig. 6, A and B). These results presented above are direct evidence that Pf-C/ EBP-A and Pf-C/EBP-B could regulate the transcriptional expression level of the matrix protein Shematrin-2. In addition, the correlation assays show that Pf-C/EBP-A and Pf-C/EBP-B have close relationships with Shematrin-2 during shell regeneration, which further validates our model of the regulatory mechanism. Intriguingly, the expression pattern of Shematrin-2 in the shell regeneration process of P. fucata was similar to that in the scallop Chlamys farreri (44), which suggests that the role that Shematrin-2 plays in biomineralization among different species might be similar. However, the SEM images of Pf-C/EBP-A and Pf-C/EBP-B knockdown demonstrate that these two transcription factors are related to the formation of the prismatic and nacreous layers. As the C/EBP family members are involved in multiple signaling pathways and can regulate various factors in mammals, for example, NF-B (45), EAK (46), NF-Y (24), HOX and MEIS (23), we speculate that Pf-C/ EBP-A and Pf-C/EBP-B, together with other transcription factors, might synergistically regulate some other matrix proteins, such as Aspein, Prismalin-14, and Pearlin. The Aspein and Prismalin-14 matrix proteins participate in the formation of the prismatic layer, whereas Pearlin functions in the formation of the nacreous layer (47)(48)(49). Thus, our experiments demonstrate the pivotal roles of Pf-C/EBP-A and Pf-C/EBP-B in the transcriptional regulation of Shematrin-2 and some other factors, which are critical upstream factors in biomineralization.

Conclusions
In summary, in this study, we report the first systematic exploration of the function and regulatory mechanisms of Shematrin-2. We found that Shematrin-2 has dual roles in shell biomineralization and provide evidence of the important roles that Pf-C/EBP-A and Pf-C/EBP-B play in regulating the tran-

Transcriptional regulation of matrix protein Shematrin-2
scription of Shematrin-2 in vitro and in vivo. In addition, further investigations are needed to understand the regulatory mechanism of Pf-C/EBP-A and Pf-C/EBP-B and to elucidate the regulatory network among different transcription factors and matrix proteins in P. fucata.

Ethics statement
This study was approved by the Animal Ethics Committee of Tsinghua University, China.

Experimental animals
The pearl oysters, P. fucata, were purchased from the Zhanjiang Pearl Farm (Guangdong Province, China) and maintained in an aerated artificial seawater tank for 5 days prior to experimentation.

Preparation of polyclonal antibodies against Shematrin-2
Total RNA samples were extracted from the mantle tissue of P. fucata using TRIzol (Invitrogen, USA), and RNA quality was determined by agarose gel electrophoresis and a NanoDrop spectrophotometer (Thermo Scientific, USA). Full-length cDNAs were synthesized using SMART MMLV reverse transcriptase (Clontech, Japan). The open reading frame (ORF) of Shematrin-2 (GenBank accession no. AB244420.1), without the signal peptide coding sequence, was amplified by the primers SH2-PET-F and SH2-PET-R (Table S1) using high-fidelity KOD polymerase (Toyobo, Japan) and then subcloned into the Transcriptional regulation of matrix protein Shematrin-2 expression vector pET28a. Recombinant Shematrin-2 with a His 6 tag at the C terminus was overexpressed in Escherichia coli BL21 (DE3) at 16°C for 12 h after 0.4 M isopropyl 1-thio-␤-Dgalactopyranoside induction. The expressed recombinant Shematrin-2 protein was purified using a HisTrap HP column (GE Healthcare, USA) under denaturing conditions (4 M urea) according to the manufacturer's instructions. Polyclonal antibodies against Shematrin-2 were prepared by immunizing New Zealand rabbits following standard immunization procedures and purified using an Antibody Purification Kit (Protein A) (Abcam, UK) according to the manufacturer's instructions.

Detection of Shematrin-2 in shell extracts and extrapallial fluid
EDTA-soluble and EDTA-insoluble matrices from different shell layers were prepared as previously described (50). The extrapallial fluid was extracted as previously described (51) and then concentrated by ultrafiltration (Millipore, 3-kDa cut-off, Germany). The protein concentrations were determined by using a Pierce TM BCA Protein Assay Kit (Thermo Scientific, USA). Detection of Shematrin-2 in different EDTA shell extracts and in the extrapallial fluid was conducted by Western blotting as described previously (52). Purified anti-Shematrin-2 polyclonal antibodies were used at dilutions of 1:2000, and peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology) was used at 1:5000. The final detection was performed using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Germany) and exposure to X-ray film.

Expression and purification of recombinant Shematrin-2 proteins
To obtain purified, soluble recombinant Shematrin-2 protein, the Shematrin-2-Pmal-C5X construct (the coding sequence of Shematrin-2 was constructed into the vector Pmal-C5X) was transformed into BL21 (DE3) competent E. coli cells. An individual colony was inoculated in LB medium overnight, and the starting cultures were diluted 100-fold with LB medium for further growth until the A 600 of the cultures reached ϳ0.6. Then, 0.4 M isopropyl 1-thio-␤-D-galactopyranoside was added to the cultures to induce expression. After incubation for over 12 h, the bacterial cells were harvested on ice and resuspended in PBS (200 mM NaCl, pH 7.4) containing 20 mM imidazole. The cells were then lysed by lysosome treatment and sonication on ice. The suspensions were then centrifuged at 12,000 ϫ g for 45 min at 4°C. The supernatants were collected and filtered through a 0.22-m filter (Pall, USA) followed by purification on a HisTrap HP column (GE Healthcare, USA) by AKTA (Sanyo, Japan). The collected soluble recombinant Shematrin-2-MBP protein was concentrated using an ultrafiltration device and desalted by HiTrap desalting (GE Healthcare). The purified Shematrin-2-MBP protein was dissolved in 20 mM Tris-HCl (pH 7.4).

Chitin-and calcium carbonate crystal-binding assay
The chitin-binding assay was conducted as described by Inoue et al. (53), and the calcium carbonate crystal-binding assay was performed as described by Suzuki et al. (6) with some modifications. The recombinant Shematrin-2 protein and the MBP tag protein (ϳ50 g each) were added to 20 mg of chitin (Wako), calcite (Sigma), or aragonite (Alfa Aesar) that had been previously equilibrated with 0.5% ammonium bicarbonate and mixed in a shaker at 4°C overnight. After removal of the solution by centrifugation, the mixture was washed with Milli-Q water and 200 mM NaCl (20 mM Tris, pH 7.4) three times each, and the supernatant was collected after the third wash. The final insoluble residue was boiled in 30 l of 2% (w/v) SDS containing 20 (w/v) 2-mercaptoethanol for 10 min along with the supernatant collected above. Each wash or supernatant was subjected to SDS-PAGE on a 10% gel under reducing conditions. After electrophoresis, the gel was stained with Coomassie Brilliant Blue.

Calcium carbonate precipitation assay
The experiment was examined according to the method of Suzuki et al. (6) with some modifications. Briefly, sample solution (10 l) was mixed with 100 l of NaHCO 3 (100 mM, pH 8.5). Then, after the addition of 100 l of CaCl 2 (100 mM) to the mixed solution, the formation of CaCO 3 precipitates was monitored by recording the changes in the turbidity every 30 s for 6 min based on the absorbance at 570 nm measured using a spectrophotometer (Bio-Rad 680). The protein solution buffer and the same concentration of MBP were chosen as negative controls.

In vitro calcium carbonate crystallization assay
Two types of crystallizing solutions were used to investigate the effect of Shematrin-2 on the morphology and polymorphism of calcium carbonate crystals. For calcite crystallization, saturated calcium bicarbonate solution was prepared as described by Xu et al. (54) with some modifications. CO 2 gas was bubbled through a mixture of calcium carbonate (0.1 g/ml) and Milli-Q deionized water for 4 h, the excess solid CaCO 3 was removed by filtration of a 0.22-m filter, and the bubbling of the CO 2 gas was maintained for 2 h. For the aragonite crystallization, 50 mM magnesium chloride was added to the above solution. Based on the designed experiments, different concentrations of protein were added to the two different crystal systems on a siliconized glass slide. After a 48-h crystallization in a sealed box, the solution was removed with Milli-Q water washing, and the crystals were air-dried for identification of the induced crystals using Raman spectroscopy analysis and for element analysis of crystals by using an FEI Sirion 2000 SEM equipped with an energy-dispersive X-ray spectroscopy system.

Sequence analysis and phylogenetic tree construction of NF-Y and C/EBP transcription factors
The conserved domains of Pf-NF-YA, Pf-NF-YB, Pf-NF-YC, Pf-C/EBP-A, Pf-C/EBP-B, and Pf-C/EBP-␥ were predicted from the deduced amino acid sequences using the online tool SMART (http://smart.embl-heidelberg.de/index2.cgi). 6 The ClustalX program was used to align amino acid sequences. Based on multiple sequence alignments, phylogenetic trees were constructed using the neighbor-joining method in MEGA6 with 10,000 bootstrap replicates.

Gene expressions of the NF-Y and C/EBP transcription factors during larval development
The relative gene expression levels of Pf-NF-YA, Pf-NF-YB, Pf-NF-YC, Pf-C/EBP-A, Pf-C/EBP-B, and Pf-C/EBP-␥ were determined by real-time PCR on a StepOnePlus TM Real-time PCR system (ABI, USA). Larval culture and sample collection of five different developmental stages, the oosperm, trochophore stage, D-shape stage, umbonal stage, and juvenile stage, were modeled on a previous study (55). cDNA templates were prepared using a PrimeScript TM RT Reagent Kit with gDNA Eraser (TaKaRa, Japan) according to the manufacturer's instructions. The real-time PCR was performed using SYBR Premix Ex Taq TM (Tli RNaseH Plus) (TaKaRa, Japan), and the expression levels were calculated using the 2 Ϫ⌬⌬Ct method. The 18S rRNA was used as an internal control (56). The primers used for realtime PCR are listed in Table S1.

Tissue distributions detected by real-time PCR
Tissue distributions of Pf-NF-YA, Pf-NF-YB, Pf-NF-YC, Pf-C/ EBP-A, Pf-C/EBP-B, and Pf-C/EBP-␥ mRNA among seven tissues, the adductor muscle, gonad, viscus, mantle pallial, mantle edge, gill, and foot, which were collected from three healthy pearl oysters, were also detected by real-time PCR. cDNA template preparation and real-time PCR were performed as described above. In addition, gene expression in the mantle tissue of these six transcription factors and four matrix proteins, Shematrin-2, MSI60, ACCBP, and N19, were also detected. GAPDH was used as an internal control. The primers used for real-time PCR are listed in Table S1.

Cloning of the promoter regions of four matrix protein genes
Genomic DNA was isolated from the adductor muscle of a pearl oyster using a TIANamp Marine Animals DNA Kit (Tiangen, China), and the quality of the DNA was determined by agarose gel electrophoresis and using a Nanodrop spectrophotometer. To obtain the promoter regions of the Shematrin-2 gene, ACCBP gene, MSI60 gene, and N19 gene, the genomewalking PCR was performed using the Genome Walking Kit as described by the manufacturer (TaKaRa, Japan). Three primers, namely, SH2-GW1R, SH2-GW2R, and SH2-GW3R, for Shematrin-2 gene promoter genome-walking PCR were designed based on the previously reported sequence scaffold 89285.1 (57). Three primers, namely, AC-GW1R, AC-GW2R, and AC-GW3R, for the ACCBP gene promoter genome-walking PCR were designed based on the previously reported sequence scaffold 13073.1 (57). The final sequences of the Shematrin-2 gene and the ACCBP gene promoter were amplified by two pairs of primers, namely, SH2-F and SH2-R, AC-F and AC-R, respectively, and were confirmed using KOD polymerase. Similarly, to obtain the promoter regions of the MSI60 gene and N19 gene, the partial gene promoter was first amplified by a pair of primers, namely, M60 -1F and M60 -1R, N19 -1F and N19 -1R, respectively. The primers of the MSI60 gene and N19 gene were designed based on the previously reported sequence scaffold 523.1 and sequence scaffold 16924.1, respectively (57). Then, a pair of primers, namely, M60-NF and M60-NR, was used to amplify the sequence of the first intron region of the MSI60 gene. For N19 gene, based on the sequence of the partial promoter and the first intron region, N19-GW1R, N19-GW2R, and N19-GW3R were designed for the first genome-walking PCR to amplify the longer promoter of the N19 gene. After the first genome-walking PCR, based on the obtained sequence, N19-GW4R, N19-GW5R, and N19-GW6R were designed for the second genome-walking PCR. Finally, two pairs of primers, namely, M60-F and M60-R, N19-F and N19-R, were used to verify the MSI60 gene and the N19 gene promoters by KOD polymerase, respectively. In addition, 5Ј-RACE was performed to verify the sequence of the first exon of the N19 gene using a SMARTer TM RACE cDNA Amplification Kit (Clontech, Japan) according to the manufacturer's instructions. All primers used are listed in Table S1. The transcription start sites of the promoters were predicted using the online BDGP software (http://www.fruitfly.org/seq_tools/promoter.html) 6 (58).

Cell culture and transfection
The HEK293T cells (China Infrastructure of Cell Line Resources, Beijing, China) used in our experiments were cultured in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal bovine serum at 37°C in a humidified 5% CO 2 incubator. The transfection was conducted in accordance with the manufacturer's instructions for VigoFect (Vigorous, Beijing, China). The detailed procedures were similar to those of our previous study (14).

Dual luciferase assay and Western blotting
Luciferase reporter analysis was performed 36 h after transfection. The transfected cells were collected in passive lysis buffer (Promega) after being rapidly washed with PBS at room temperature. The relative activity of the promoter was detected by using the dual luciferase assay system (Promega) and a Varioskan TM Flash multimode reader (Thermo Scientific). To determine the expression of Pf-C/EBP-A and Pf-C/EBP-B or their truncated forms at the protein level, the transfected cells were lysed by passive lysis buffer 36 h after transfection (Promega), and Western blotting was performed to analyze the cell lysate as described previously (52). Primary mouse anti-myc and secondary peroxidase-conjugated goat anti-mouse IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Shell notching
Shell-notching assays were performed as described by Mount (59), with some modifications. A V-shaped notch was cut on the shell margin without disturbing the mantle tissue, so that the prismatic layer and the margin of the nacreous layer were damaged. Animals were divided into eight groups with five animals each and sacrificed at 0, 6, 12, 24, 36, 48, 72, or 96 h after notching. Mantle tissues of the same group with areas of ϳ0.5 cm 2 from the notch were pooled and stored in liquid nitrogen. Mantle tissues from five oysters without shell notching were collected in the same way and used as controls. The experiment was repeated three times.

Silencing of the Pf-C/EBP transcription factors
The RNAi assay in vivo was conducted as described by Suzuki et al. (6), with some modifications. The synthesis and purification of the Pf-C/EBP-A, Pf-C/EBP-B, and GFP dsRNAs were performed using a T7 RiboMAX TM Express RNAi System Kit (Promega) according to the manufacturer's instructions. For GFP dsRNA synthesis, the vector pEGFP-C1 (Clontech) was used as the template. The primers used above are listed in Table  S1. The dsRNA was diluted to 80 g/200 l using Milli-Q water (Merck Millipore, Billerica, MA). The dsRNA, together with PBS and GFP, was then injected into the adductor muscle of 2-year-old individuals with a shell length of 5-6 cm. Each treatment group contained nine individuals. Two days after treatment, total RNA samples were extracted from the mantle tissue. Preparation of the cDNA templates and determination of relative gene expression levels by real-time PCR were performed as previously described (14).

In vivo antibody inhibition assay
The purified antibody against Shematrin-2 (anti-Shematrin-2) was injected into the extrapallial space through the zone of the mantle tissue outside the pallial line at 0.5 g (low dosage) and 1.5 g (high dosage) per gram of wet weight per day. Each group contained 5 specimens, which were sacrificed 5 days after antibody injection. The preimmune rabbit serum of 1.5 g/g of wet weight per day was used as a control. The col-lected shells were extensively washed with Milli-Q water, airdried, gold-coated, and observed by SEM.

Statistical analysis
Statistical Package for the Social Sciences version 18.0 software (SPSS Inc., Chicago, IL) was used for the statistical analysis. Values are the mean Ϯ S.D. of three independent experiments and were analyzed by Student's t test to identify the differences between groups. A p value Ͻ0.05 was considered statistically significant. Spearman's rank correlation was used to analyze the correlations between Pf-C/EBP-A, Pf-C/EBP-B, and Shematrin-2 during shell notching. p values Ͻ0.05 were considered statistically significant.