Pf-Sp8/9, a novel member of the specificity protein family in Pinctada fucata, potentially participates in biomineralization

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Abstract

Specificity protein (Sp) belong to a transcription factor family that contains nine subgroups with essential functions in development, including skeletogenesis, tooth development, neural tube closure, and limb formation. In molluscs, functions of the Sp protein family members have not been reported in detail. In this study, we report the first Sp protein-encoding gene in Pinctada fucata. We named the translated protein Pf-Sp8/9, based on the phylogenetic development tree constructed using Sp protein sequences from six model organisms, which showed that it was a Sp8/9 homolog. Alignment of the Pf-Sp8/9 sequence with the amino acid sequences of related proteins showed that Pf-Sp8/9 had conserved domains, including three DNA-binding motifs. The tissue distribution showed that while Pf-Sp8/9 mRNA expression was detected in all tested tissues, it was particularly high in the mantle. The luciferase reporter assay results showed that Pf-Sp8/9 had the ability to activate the transcription of a number of matrix proteins. The expression pattern of Pf-Sp8/9 during P. fucata pearl sac development was similar to that of some genes that encode matrix proteins, suggesting Pf-Sp8/9 may be involved in mantle-related physiological activities and biomineralization.

Introduction

Specificity proteins (Sp) are members of the classical zinc-finger transcription factor family. All Sp proteins have a highly conserved DNA-binding domain with three continuous zinc-finger domains in their C-terminus. Sp1 was the first transcription factor to be identified and cloned in mammals (Kadonaga et al., 1987) and, as a result, Sp1 has been well studied. Sp1 binds to GC sequences in the transcriptional regulatory regions of genes encoding proteins that participate in various physiological processes, such as cell growth, differentiation, apoptosis, carcinogenesis, and embryonic and early postnatal development (Safe et al., 2014, Vizcaino et al., 2015). Other mammalian Sp proteins have been identified and cloned according to their conserved zinc-finger domains, and further studies have described their essential functions. Inactivation of Sp3 was reported to inhibit the formation of bone and teeth, and hematopoiesis (Bouwman et al., 2000, Gollner et al., 2001a, Van Loo et al., 2003), and inactivation of Sp7 was shown to have similar effects (Nakashima et al., 2002). Sp4 was found to be important for neonatal survival and reproduction (Gollner et al., 2001b, Supp et al., 1996); Sp5 was reported to affect tail growth and development of the midbrain and hindbrain in zebrafish (Tallafuss et al., 2001, Zhao et al., 2003). Sp6 showed a dynamic expression pattern during mouse embryogenesis, and suggesting the Sp6 protein may promote cell proliferation (Nakamura et al., 2004); and Sp8 affected the growth of limbs and tail, as well as closure of the neuropore in mice (Bell et al., 2003). Sp8/9 homolog identified in beetle had a function similar to the mouse Sp8 (Beermann et al., 2004, Schaeper et al., 2009, Treichel et al., 2003); mouse Sp9 also affected growth of limbs and was shown to be regulated by the fibroblast growth factor Fgf10 (Kawakami et al., 2004). However, studies on Sp family members in invertebrates are limited. Although the functions of Sp protein family members have not been reported in detail in molluscs, Sp4 and Sp8 cDNA and translated protein sequences have been reported in Crassostrea gigas by high-throughput sequencing (GenBank: EKC38194.1, EKC19755.1, EKC30539.1, and EKC30538.1) (Zhang et al., 2012).

Few transcription factors have been identified in molluscs, unlike in mammals and other model species; therefore, the transcriptional regulatory network has not yet been elucidated in molluscs. Studies on the Pinctada fucata biomineralization mechanism are important to understand the process of biomineralization and will be of great value for the pearl production industry. We have studied transcriptional regulation in P. fucata for many years and recently identified two transcription factors, Pf-YY-1 and Pf-AP-1, as potential regulators of biomineralization (Zheng et al., 2015a, Zheng et al., 2015b). Other transcriptional factors related to biomineralization have also been reported (Zhao et al., 2014).

In this study, we cloned and characterized the complete Pf-Sp8/9 cDNA sequence from P. fucata. We analyzed the Pf-Sp8/9 mRNA expression levels in various tissues and investigated the involvement of Pf-Sp8/9 in physiological processes to predict its function. Alignment of the predicted Pf-Sp8/9 amino acid sequence with related sequences in model animals and phylogenetic analysis revealed that Pf-Sp8/9 belonged to the Sp8ā€“9 subgroup and had the conserved structure of this group. Transcription activation analysis showed that Pf-Sp8/9 could activate the promoters of a number of matrix proteins. The tissue distribution analysis revealed high expression of Pf-Sp8/9 mRNA in the mantle, which is similar to the expression patterns of genes encoding other biomineral proteins during pearl sac development.

Section snippets

Animals

Live adult P. fucata pearl oysters (age, approximately 2Ā years; shell length, 55ā€“65Ā mm; and wet weight, 45ā€“55Ā g) were purchased from the Guofa Pearl Farm (Beihai, Guangxi Province, China) and maintained in 3% saline aerated artificial seawater for 3Ā days before use.

Pearl sac selection

Artificial nuclei were implanted into the oysters near the gonad. Pearl sacs were then isolated carefully from tissues every 5Ā days for 30Ā days after implantation and stored in liquid nitrogen until used. Ten oysters were used in each

Prediction of transcription factor binding sites in the promoters of matrix proteins

We predicted transcription factor binding sites in the Pearlin, KRMP, and Pif (GenBank KJ028207) promoters using TESS and identified multiple putative Sp family binding sites that might be responsible for transcriptional activity (Fig. 1). Considering the importance of Sp family members in transcriptional regulation, we attempted to identify genes encoding Sp protein homologs in P. fucata.

Cloning and characterization of a Sp homolog in P. fucata

We identified a mRNA fragment of a potential P. fucata Sp homolog in a P. fucata mantle EST library (Fang

Discussion

Many matrix proteins have been characterized and studied in P. fucata; however, transcriptional regulation of these proteins has not been investigated. Research into the biomineralization transcriptional regulatory network in P. fucata is important for managing the pearl production industry.

Sp proteins are important transcription factors that participate in multiple physiological processes and have dual transcription regulatory abilities depending on the cell or physiological status (Suske, 1999

Conclusions

We identified the first Sp protein family member in P. fucata, and its alignment with Sp proteins from other organisms showed that it was a Sp8/9 homolog; therefore, we named it Pf-Sp8/9. The predicted Pf-Sp8/9 protein sequence contained multiple domains that were conserved in Sp8/9 proteins in model organisms. Pf-Sp8/9 expression was high in the mantle compared with other tissues, and was upregulated rapidly during the late stage of pearl sac development, which seemed to be synchronized with

Conflict of interest

The authors declare no conflicts of interest.

Funding

This work was supported by the National Natural Science Foundation of China [grant numbers 31172382, 31372502, and 31572594].

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