Spata4 promoter contains a functional RFX1 binding site

Bioinformatics analysis through combination of Mulan and multi-TF scanning [32], [33] showed that the Spata4 promoter contains conserved binging sites of several transcription factors, including c-jun (key component of AP-1 complex), RREB1, HSF1, SP1 and RFX1 (Figure 2A). To address which factor could regulate Spata4 promoter activity, we serially deleted their binding sites from the −1425/+128 reporter construct. Deletion of the RFX1 binding site resulted in 4.2-fold increase of luciferase activity, while deletion of SP1 binding site decreased the luciferase activity by 0.67-fold. Deletion of AP1, RREB1 and HSF1 binding sites had little effect (Figure 2B), compared to the luciferase activity of −1425/+128 construct, respectively. These results indicate that RFX1 is a negative modulator of mouse Spata4 promoter and SP1 binding sites which locate between −131, −32 might be essential for the basal transcriptional activity of Spata4.

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Figure 2

The Spata4 promoter contains a functional RFX1 binding site.

(A) Nucleotide sequences of the 5′-flanking region of the Mm/hg/rn Spata4 genes. The 5′-flanking sequences are numbered with respect to the transcription start site, Mulan combined with multiTF (http://multitf.dcode.org/) scanning revealed several conserved potential binding sites of different transcription factors. (B) Effect of deletion mutants on the relative luciferase activity of the Spata4 promoter region. TM4 cells were co-transfected with the −1425/+128 Spata4 promoter construct and each deletion mutant. The results are the means ± S.E.M. of three independent experiments performed in triplicate. ***P<0.001.

Deletion of RFX1 binding site revealed markedly up-regulation of Spata4 promoter activity, indicating that RFX1 may function as a significant transcription repressor of Spata4. The RFX1 binding sites in human, rat and mouse Spata4 promoters are highly conserved, and have high similarity with the consensus and other published RFX binding sequences (Figure 3A). Chromatin immunoprecipitation assays using two different RFX1 antibodies showed that RFX1 binds to the mouse Spata4 promoter in cultured cells. In addition, the sequences containing the GAPDH promoter are not precipitated by RFX1 antibodies (Figure 3B). These results indicated that the RFX1 binding site in the Spata4 promoter is functional.

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Figure 3

RFX1 binds to the Spata4 promoter through its DNA binding domain.

(A) Alignment of the conserved 14-bp sequences of human, rat and mouse Spata4 promoter with other RFX1 binding sequences shows high similarity to the RFX1 consensus binding sites. (B) Chromatin immunoprecipitation assay confirm that RFX1 could bind the conserved 14-bp sequence in mouse Sertoli cells. (C) DNA binding domain is essential for the binding of RFX1 to the conserved sequence of Spata4 promoter. Vector, pcDNA3.1 plasmid; WT-RFX1, pcDNA3.1-RFX1 plasmid; ΔDBD-RFX1, pcDNA3.1-RFX1 plasmid with deletion of the sequences encoding the DNA binding domain. **P<0.01. ***P<0.001.

Then, we constructed a mutant of mouse RFX1 protein which lacks the DNA-binding domain to further confirm the functional binding of RFX1 to the Spata4 promoter. Deletion of the DNA-binding domain abolished the regulation of Spata4 promoter activity by RFX1, demonstrating that the DNA-binding domain is essential for the binding of RFX1 to the Spata4 promoter (Figure 3C).

RFX1 regulates endogenous Spata4 expression in TM4 Sertoli cells

Spata4 is localized in the cytoplasm of TM4 cells (Figure S1 A). The mRNA level of Spata4 is dramatically increased on postnatal days 15 in mouse testes (Figure S1 B). Furthermore, the expression of Spata4 is abundant in primary Sertoli cells harvested from mouse testis (Figure S1 C). To investigate whether RFX1 regulates endogenous Spata4 expression, we transiently overexpressed different amounts of RFX1 in TM4 cells and detected its effect on the promoter activity and the mRNA levels of Spata4. Overexpression of RFX1 was confirmed by western blotting and immuno-fluorescent assay (Figure 4A). Not only the promoter activity but also the mRNA level of Spata4 was significantly decreased by RFX1 in a dose dependent manner (Figure 4 B and C).

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Figure 4

RFX1 regulates Spata4 mRNA expression in mouse Sertoli cells.

After transient transfection of pcDNA3.1-RFX1 for 36 h, the overexpressed RFX1 protein was detected by immunocytochemistry staining and Western blot (A), overexpression of RFX1 was shown to down-regulate the promoter activity (B) and Spata4 mRNA expression (C) and in a dose-dependent manner. The Spata4 mRNA expression was analyzed by quantitative PCR and normalized to GAPDH. **P<0.01. ***P<0.001.

Next, RFX1 expression was knocked down by RNA interfere in TM4 cells (Figure 5 A and B). The mRNA level of Spata4 was significantly up-regulated by RFX1 siRNA interference (Figure 5C). In addition, endogenous Spata4 gene expression could be markedly increased by RFX1 knockdown in a time-dependent manner (Figure 5 D and E).

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Figure 5

Knockdown of RFX1 could up-regulate endogenous Spata4 expression.

Treatment of mouse RFX1-RNAi-I, -II and -III for 48 h could efficiently knock down the endogenous RFX1 expression (A, B). (C) Spata4 mRNA expression level was elevated upon RFX1 knockdown. Control, cells treated with nonspecific siRNA. (D) RFX1 mRNA expression level was decreased by RFX1-RNAi-III treatment. (E) Spata4 mRNA expression level was significantly elevated by RFX1-RNAi-III treatment in a time-dependent manner. *P<0.05. ***P<0.001.

Plasmid constructs

The mouse genomic sequence of the 5′-flanking region of the Spata4 gene including the transcription start site was obtained from GenBank (accession no. NC_000074). DNA extracted from the whole blood cells from C57/BL6 mice with TIANamp Blood DNA Kit (TIANGEN Biotech., Beijing, China) was used as the template. Different truncations of mouse Spata4 promoter were cloned into the Kpn I and Xho I restriction sites of the pGL4.17 vector (Promega, Madison, WI). The expression plasmid for wild-type RFX1 and was constructed by cloning the corresponding sequence into Kpn I and Xho I restriction sites of pCDNA3.1 myc-His A vector (Invitrogen, Carlsbad, CA). Mutation PCR reactions were performed with PrimSTAR HS polymerase (Takara Bio Inc., Shiga, Japan), 5′-end of the purified PCR products was kinated and ligated using DNA Kination Kit (Takara) referring to the manufacturer's protocol. The primers used during the plasmid construction are shown in Table S1 and S2.

Cell culture

Mouse Sertoli cell TM4 were cultured in DMEM/F-12 (Thermo Scientific HyClone, Logan, UT), supplemented with 10% fetal bovine serum (Thermo Scientific HyClone), 100 U/ml penicillin and 100 μg/ml streptomycin, at 37°C in a humidified atmosphere containing 5% CO₂.

5′ Rapid amplification cDNA ends

Rapid Amplification of cDNA Ends (RACE) was performed using the SMART^TM RECE cDNA Amplification kit (Clontech, Shiga, Japan) following manufacturers' protocols, to determine the transcription start sites of mouse Spata4. Ploy A+ RNA was purified from testis total RNA of 2-months-old C57 BL/6J mice by Oligotex^TM mRNA Purification system (Qiagen Inc., Valencia, CA). 5′-RACE-Ready cDNA was synthesized using 5′-CDS (Clontech) primer and SMART II A oligo (Clontech). 5′ RACE PCR was performed with 5′-RACE UPM (Clontech) and Spata4 specific primer: 5′-GCAGGACAGAACGACTCAGGCGGG-3′, followed by a nested PCR using 5′- nested UPM (Clontech) and a nested Spata4-specific primer: 5′- TCCTGCGGGAACTGCTGGTGGTGTAG-3′. As a negative control, 5′-RACE Ready cDNA was amplified with 5′-RACE UPM only. The nested PCR products were then sub-cloned into pMD19-T vector (Takara) and a total of 60 positive clones were sequenced.

Bioinformatics analysis

Mulan (Multiple-sequence local alignment) analysis was carried out comparing the genomic sequences of about 2000 bp upstream Spata4 first exon of human, mouse and rat, combined multiTF (http://multitf.dcode.org/) scanning for conserved transcription factor binding sites (TFBS).

Cell transfection and luciferase assay

TM4 cells (1×10⁵ cells/ml) were seeded in 24-well plates in antibiotic-free culture medium the day before transfection. Each well of cells were transiently transfected with 0.8 μg of Spata4 reporter plasmids and 0.08 μg Renilla reporter plasmid (phRL-TK, Promega) as an internal control using Lipofectamine^TM 2000 (Invitrogen). Alternatively, cells were co-transfected with 0.4 μg Spata4 reporter plasmids, 0.04 μg phRL-TK plasmid and 0.4 μg total amounts of pcDNA3.1 and RFX1 expressing plasmids in a dose dependent manner. Cells were incubated with the DNA-Lipofectamine^TM 2000 complex for 6 h, then washed gently with phosphate-buffered saline (PBS) buffer, and cultured in fresh serum-supplemented media for an additional 18 h. Cell lysates were collected and used for measurement of the relative promoter activity of each fragment with the dual luciferase reporter assay system (Promega). pGL4.17 [luc2] vector contains no promoter region was used as a control.

RNAi knockdown experiment

Small interfering RNA knockdown experiments were performed with synthesized siRNA (Genechem, Shanghai, China). Target sequences of RFX1 for siRNA are shown as follow: RFX1-siRNA-I, 5′-AGAACACTGCACAGATCAA-3′; RFX1-siRNA-II, 5′-ACTGTGACAATGTGCTGTA-3′; RFX1-siRNA-III, 5′-TCATGGTAAACCTGCAGTT-3′. Non-specific siRNA was synthesized as forward: 5′- UUCUCCGAACGUGUCACGUtt-3′, reverse: 5′-ACGUGACACGUUCGGAGAAtt-3′. TM4 cells were used in RNAi knockdown experiments. Cells were transfected with the small interfering RNA using Lipofectamine RNAiMAX transfection reagent (Invitrogen) according to the manufacturer's instructions. The three different siRNA (I, II, and III) against RFX1 were tested, and RFX1-siRNA-III were used for further RNAi knockdown experiments.

Chromatin Immunoprecipitation (ChIP) Assay

ChIP was performed using the Magnetic Chromatin Immunoprecipitation Kit (Active Motif, Carlsbad, CA) as described in the manufacturer's protocol. Briefly, TM4 cells were washed once with phosphate buffered saline and then fixed in culture medium containing 1% formaldehyde at room temperature for 10 min. Cells were then collected and lysed to release the nucleus. The nucleus was digested with the enzyme mix at 37°C for 10 min to shear the chromatin into small segments. 2 μg of RFX1 (I-19) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), 2 μg of RFX1 (D-19) antibodies (Santa Cruz), or a negative control IgG (Abcam, Cambridge, UK) were used for immunoprecipitation of the sheared chromatin at 4°C for 4 h. The pulled-down chromatin was washed, reverse cross-linked and purified. PCR primers for mouse Spata4 promoter were forward: 5′-CACAGAGCCTTCTTGGAGG-3′ and reverse: 5′-GGGAACTGCTGGTGGTGTA-3′. PCR primers for mouse GAPDH promoter were forward: 5′-GCCCTTGAGCTAGGACTGGAT-3′ and reverse: 5′-CCTGGCACTGCACAAGAAGAT-3′.

Quantitative real-time polymerase chain reaction (RT-qPCR)

Total RNA was extracted from TM4 cells using Trizol reagent (Invitrogen) according to the manufacturer's protocol. cDNA was transcribed with RevaTra Ace reverse transcriptase (Toyobo, Osaka, Japan) using oligo (dT)₂₀ as primers. Each cDNA transcribed from 0.8 μg RNA was subsequently used for RT-qPCR analysis with SYBR Green Real-time PCR Mater Mix (Toyobo) in CFX96 Real-Time PCR Detection Systems (Bio-Rad, Hercules, CA). PCR primers for mouse Spata4 were forward: 5′-CAGATACAAGTCAAGAGGTTC-3′ and reverse: 5′-GTGTTCTCACAAGGATTTCCAC -3′. PCR primers for mouse RFX1 were forward: 5′-TGCTGCCCCTGCACCCTCACA-3′ and reverse: 5′-GGTGGGAACACCGGTCTGGCTT-3′. PCR primers for mouse GAPDH were forward: 5′-CATGGCCTTCCGTGTTCCTA-3′ and reverse: 5′-CCTGCTTCACCACCTTCTTGAT-3′. All the primers are intron-spanning. The specificity of primers was confirmed by the melting curves which performed on the amplified products. The thermal cycling conditions were as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturing at 95°C for 15 s, annealing at 60°C for 30 s and extension at 72°C for 30 s.

Western blotting

Cells were collected and lysed with the cell lysis buffer (Beyotime, Shanghai, China). Alternatively, nuclear protein was isolated with the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime). Protein concentrations were determined using the BCA Protein Assay Kit (Thermo Scientific). Protein samples were separated by electrophoresis through SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (Millipore, Schwalbach, Germany). The membranes were blocked at room temperature for 1.5 h in Tris-buffered saline containing 0.05% Tween 20 with 5% non-fat dry milk. The membranes were then incubated with primary antibodies specific for RFX1 (1500; Santa Cruz), Spata4 (11000; prepared by our laboratory), GAPDH (11000; Cwbiotech, Beijing, China) or β-tublin (11000; Cwbiotech) at 4°C overnight, followed by an incubation with horseradish peroxidase-conjugated anti-goat IgG (15000; Santa Cruz) or anti-rabbit IgG (110000) at room temperature for 1 h. The chemiluminescence reaction was performed using ECL reagent (Thermo Scientific).

Immunofluorescence staining

TM4 cells were seeded on a 6-well dish with a density of 1×10⁵ cells/ml and fixed with methylate-acetone (11) for 30 min. After blocking with 10% bovine serum albumin (BSA) for 1 hour, a primary antibody against RFX1 (1100; Santa Cruz) was added for 2 h, followed by an anti-goat IgG conjugated with FITC (1100; Santa Cruz) for 1 h. DAPI (0.1 mg/ml) was added to label the nucleus for 10 min. A negative control without the primary antibody was used to monitor the staining background. Images were obtained through Leica DX100 (Leica Microsystems Wetzlar GmbH, Germany).

CCK-8 assay

TM4 cells were suspended at a final concentration of 2×10⁴ cells/ml and seeded in 96-well plates, then transiently transfected with pcDNA3.1-vector, pcDNA3.1-RFX1, non-specific siRNA, siRNA-RFX1, respectively. After incubation for the indicated time as 0, 24 and 48 h, cell growth was determined by the CCK-8 assay (Beyotime). In brief, 10 μl CCK-8 solutions were added to each well and cultures were incubated at 37°C for 2 h. The absorbance at 450 nm was measured with a Benchmark microplate reader (Bio-Rad).