Molecular Characterization and Functional Analysis of Two Steroidogenic Genes TSPO and SMAD4 in Yellow Catfish

Abstract

The steroid hormones are required for gonadal development in fish. The present study was undertaken to characterize the cDNA and promoter sequences of TSPO and SMAD4 genes in yellow catfish Pelteobagrus fulvidraco, explored the mRNA tissue expression and deciphered their promoter regions. Yellow catfish TSPO and SMAD4 shared the similar domains to the corresponding genes from other vertebrates. The TSPO and SMAD4 mRNAs were widely expressed in the detected tissues, but at different levels. Several transcription factors were predicted, such as Sp, GATA, AP1, SOX1, SRY, STAT, HNF4α, PPARγ, Pu.1 and FOXL2. PPARγ overexpression increased but STAT3 overexpression reduced TSPO promoter activity, and FOXL2 overexpression inhibited the promoter activity of TSPO and SMAD4. The site mutation and EMSA analysis indicated that TSPO promoter possessed STAT3 and FOXL2 sites. Overall, our provided the novel understanding into the transcriptionally regulatory mechanisms of TSPO and SMAD4 in fish.

Keywords: Pelteobagrus fulvidraco; molecular characterization; promoter; steroidogenesis-related genes; transcriptional regulation.

Figure 1

Quantitative PCR (Q-PCR) analysis for mRNA expression of TSPO and SMAD4 across heart (H), liver (L), brain (B), spleen (S), kidney (K), muscle (M), fat (F), intestine (I), testis (T) and ovary (O) of P. fulvidraco. Data (mean ± SEM, n = 3 replicates. For each replicate, 4 fish were sampled) were expressed relative to expression of reference gene (β-actin and ubce). Bars that share different letters indicate significant differences among various tissues (p < 0.05).

Figure 2

Nucleotide sequences and putative regulatory elements of the TSPO promoter in P. fulvidraco. Numbers are relative to the transcription start site (+1). The putative regulatory elements are indicated in bold letters below the underlined sequence.

Figure 3

Nucleotide sequences and putative regulatory elements of the SMAD4 promoter in P. fulvidraco. Numbers are relative to the transcription start site (+1). The putative regulatory elements are indicated in bold letters below the underlined sequence.

Figure 4

5′ unidirectional deletion analysis of the TSPO and SMAD4 promoters for yellow catfish. Schematic diagram of truncated promoters is shown at left panel. The corresponding luciferase reporter assay results are shown at right panel. A series of plasmids containing 5′ unidirectional deletions of the TSPO (pGL3 −2015/+205, −1558/+205, −1076/+205, −504/+205) and SMAD4 (pGL3 −1506/+89, −999/+89, −559/+89) promoter regions fused in frame to the luciferase gene were transfected into HEK293T cells. Values represent the ratio between firefly and Renilla luciferase activities, normalized to the control plasmid pGl3 −504/+205 (A) and pGl3 −559/+89 (B), respectively. Results are shown as mean ± standard error of mean (SEM) (n = 3). Hash symbol (#) means significant differences between two groups (p < 0.05).

Figure 5

Overexpression analysis of 5′ unidirectional deletion assays of the TSPO and SMAD4 promoters of yellow catfish. (A) PPARγ overexpression; (B) STAT3 overexpression and (C,D) FOXL2 overexpression. Values represent the ratio between firefly and Renilla luciferase activities, normalized to the control. Results are shown as mean ± standard error of mean (SEM) (n = 3). Hash symbol (#) means significant differences between two groups (p < 0.05). Asterisk (*) indicate significant differences between different treatments with the same plasmid (p < 0.05).

Figure 6

Assays of predicted PPARγ, STAT3 and FOXL2 binding sites after site-directed mutagenesis. (A) Site mutagenesis of PPARγ on −2015/+205 TSPO promoter. (B) Site mutagenesis of STAT3 on −2015/+205 TSPO promoter. (C) Site mutagenesis of FOXL2 on −2015/+205 TSPO promoter. (D) Site mutagenesis of FOXL2 on −1506/+89 SMAD4 promoter. Values are presented as mean ± SEM (n = 3). Hash symbol (#) means significant differences between two groups (p < 0.05). Asterisk (*) indicate significant differences between different treatments with the same plasmid (p < 0.05).

Figure 7

EMSA analysis of predicted SREs. (A) −734/−748 binding site of TSPO (TSPO-PPARγ); (B) −1507/−1516 binding site of TSPO (TSPO-STAT3); (C) −1650/−1663 binding site of TSPO (TSPO- FOXL2); (D) −777/−789 binding site of SAMD4 (SAMD4- FOXL2).