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Transcriptomic analysis of overexpressed SOX4 and SOX8 in TM4 Sertoli cells with emphasis on cell-to-cell interactions
Biochemical and Biophysical Research Communications 512 (2019) 678e683
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Transcriptomic analysis of overexpressed SOX4 and SOX8 in TM4 Sertoli cells with emphasis on cell-to-cell interactions Pauline Roumaud, Luc J. Martin* Biology Department, Universit e de Moncton, Moncton, New-Brunswick, E1A 3E9, Canada
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 March 2019 Accepted 16 March 2019 Available online 25 March 2019
Sertoli cells are localized in seminiferous tubules within the testis. They are the first testicular cells to differentiate during male sex determination. In the adult, Sertoli cells provide nutrients to germ cells, control factors for spermatogenesis and protection by establishing the blood-testis barrier (BTB). This BTB is composed of tight junctions, basal ectoplasmic specializations, adherent junctions and gap junctions. The transcription factor SOX8 is necessary for the maintenance of spermatogenesis during adult life whereas SOX4 is involved in developmental processes. These factors are highly expressed in Sertoli cells. However, few of their target genes in adult Sertoli cells are known. Hence, we compared the transcriptomes of TM4 Sertoli cells overexpressing or not SOX4 or SOX8 using RNA-Seq followed by pathways and networks analyses. We found that SOX4 overexpression leads to downregulated genes enriched for cell junction organization and positive regulation of cell-to-cell adhesion. Upregulated genes in response to SOX8 overexpression were enriched for Sertoli cell development and differentiation. However, downregulated genes were enriched for cell-to-cell adhesion, tight junction interactions, gap junctions’ assembly, as well as extracellular matrix binding. Hence, our results confirm that SOX8 is an important mediator of Sertoli cell maturation, whereas SOX4 and SOX8 influence gene expression related to regulation of blood-testis barrier assembly. In addition, TM4 cells can be considered as a useful model to better define the regulatory mechanisms of SOX4 or SOX8 on gene transcription in Sertoli cells. © 2019 Elsevier Inc. All rights reserved.
Keywords: Sertoli cells SOX4 SOX8 Transcriptomics RNA-Seq Cell-to-cell interactions
1. Introduction In the testis, Sertoli cells are somatic cells localized in seminiferous tubules and support spermatogenesis by providing protection, nutrients and proliferation or differentiation factors. Between Sertoli cells, the blood-testis barrier (BTB) is composed of adherent and tight junctions and allows the protection of germ cells from autoimmune reactions. Sertoli cells synthesize several molecules such as nutrient transport proteins (transferrin, clusterin), differentiation factors (TGFb, IGF1) or proliferation factors (activin). These supporting cells are regulated by testosterone, FSH, retinoic acid or vitamin D (for review [1,2]). Important functions of Sertoli cells are under the control of SOX transcription factors. The Sox genes encode transcription factors with a high mobility group (HMG)-type DNA binding domain consisting of three a-helices and allowing to bind the minor groove
de Moncton, 18 avenue * Corresponding author. Biology Department, Universite Antonine Maillet, Moncton, New-Brunswick, E1A 3E9, Canada. E-mail address: [email protected] (L.J. Martin). https://doi.org/10.1016/j.bbrc.2019.03.096 0006-291X/© 2019 Elsevier Inc. All rights reserved.
of DNA and bend it [3]. Twenty members of the SOX family have been identified and this family is subdivided into 8 groups (A-H) based on their identity within and outside the HMG box [4]. In the embryo, Sertoli cells play a critical role in sex determination, as well as the differentiation and development of testes. Indeed, the expression of Sex-determining Region of the chromosome Y (SRY) by 10.5 dpc in mice and by 41 dpc in humans, leads to the differentiation of progenitor Sertoli cells. SRY activates the expression of SRY-related high-mobility-group (HMG) box 9 (Sox9), a member of the SoxE group of SOX transcription factors. In mouse, we have confirmed that SOX4 and SOX8 transcription factors are highly expressed within the adult Sertoli cells [5]. SOX4 belongs to the SoxC group and have a transactivation domain (TAD) located in its C-terminal region [6]. Sox4/ mice die at 14.5 dpc due to cardiac defects [7]. Several SOX4 target genes have already been identified, notably in C2C12 myoblasts [8,9], in lung cancer cell lines [10] and in neural or mesenchymal progenitors [11]. In Sox4/ foetal mice testes, Zhao et al. identified genes potentially regulated by SOX4, such as Nanos2, Dmnmt3l, Cripto, Nanog and p15INK4b [12]. Interestingly, they found that SOX4 downregulates Sox9
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expression directly even though considered a transcriptional activator. As SOX9 and SOX10, SOX8 belongs to the SoxE group. These transcription factors are involved in many developmental processes such as neurogenesis, chondrogenesis and male gonadogenesis (for review [13]). Interestingly, Sox8/ mice are viable and fertile with a significant weight reduction [14]. However, these mice become progressively infertile, suggesting an implication of SOX8 in the maintenance of spermatogenesis within the adult testis [15]. In humans, SOX8 mutations can lead to 46 XY sex-reversal, male infertility and female primary ovary insufficiency [16]. In this study, we aim to identify the potential target genes for SOX4 and SOX8 through overexpression in TM4 Sertoli cells, followed by transcriptomic RNA-Seq analyses and pathways and networks enrichments investigations. Our results confirmed that SOX4 regulates cell-to-cell adhesion, while SOX8 modulates the expression of genes involved in Sertoli cell development and differentiation as well as cell-to-cell interaction. 2. Material and methods
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counts and mean fit type [22]. 2.3. Pathways and networks analyses From the total gene list of differentially expressed genes, we performed a gene set enrichment analysis using the GSEA software (version 3.0) from the Broad Institute [23]. A ranking of the differential expression results was determined by score calculation based on fold changes and p-values of DESeq2 results (sign(logFC) *-log10(pvalue)) and compared to all pathways, released 2018-1101 (http://download.baderlab.org/EM_Genesets/), or curated gene ontology biological process annotations, released 2018-11-01 [24,25], using a modified Kolmogorov-Smirnov statistic. Differentially expressed genes were also analysed using the gProfiler gene group functional profiling platform for biological process, molecular function, biological pathways and reactome annotations using the Benjamini-Hochberg FDR significance threshold [26]. In addition, differentially expressed genes were analysed using the GeneMania platform (version 3.5.0) with biological process and molecular function network weighting [27].
2.1. Cell culture and transfection 2.4. Real-time quantitative PCR Mouse TM4 Sertoli cells were obtained from American Type Culture Collection (Manassas, VA, USA) and cultured as described previously [17]. The SOX8 expression plasmid was obtained by amplification of cDNA using the forward primer containing a HindIII cloning site, 50 -GCA AGC TTA TGC TGG ACA TGA GTG AGG CCC GCG CC-30 , and the reverse primer containing a XhoI cloning site, 50 CGC TCG AGG GGT CGG GTC AGG GTG GTG TAC AC-30 , followed by cloning into pCDNA6-V5His. SOX4 expression vector has been described previously [18] and was provided by Dr. James Wells (Cincinnati Children's Hospital Research Foundation, Cincinnati, OH). TM4 cells were transfected with an empty vector (control), with SOX4 or SOX8 expression plasmid using polyethylenimine according to the previously described method [17] with a 3:1 ratio (polyethylenimine to DNA). After a 48 h incubation, positively transfected cells were selected with blasticidin (Bioshop, Burlington, ON, CAN) at 15 mg/ml for 48 h. 2.2. RNA extraction and RNA sequencing analyses After 96 h of transfection, total RNA was isolated with E.Z.N.A. Total RNA kit (Omega Bio-Tek, Inc., Norcross, GA). RNA quality was determined by electrophoresis and characterisation of the 18S and 28S ribosomal RNA bands. Libraries were constructed using the TruSeq Stranded mRNA LT (Illumina) for cDNA synthesis and polyA enrichment. Quality and quantification of libraries were evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Mississauga, ON, CAN) and KAPA SYBR® FAST qPCR Kit (KAPA Bio Systems, Wilmington, MA, USA), respectively. Transcriptomic sequencing was performed on an Illumina HiSeq 2000 in single reads layout with approximately 45 million reads per sample and 100 bp of sequencing length. Libraries and next generation sequencing were completed by the McGill University and Genome Quebec Innovation Centre (Montreal, QC, CAN). RNA-Seq analyses were performed using the Galaxy web-based platform (https:// usegalaxy.org). Single-end raw reads in FASTQ format were trimmed using the Trimmomatic (version 0.36.5) sliding window with a minimal average quality of 20 and Illuminaclip for TruSeq3 adapters [19]. Trimmed reads were aligned to reference mouse genome (mm10) using the HISAT2 alignment program (version 2.1.0) [20]. Transcripts assemblies and quantifications were performed with the StringTie assembler (version 1.3.4) for NCBI RefSeq curated reference transcripts from mm10 [21]. Differential gene expression was assessed using DESeq2 (version 2.11.40.2) for gene
The qScript cDNA SuperMix (Quantabio, Beverly, MA, USA) was used to synthesize cDNA from total mRNAs. Quantitative real-time polymerase chain reaction (qPCR) was performed using PerfeCTa SYBR® Green FastMix (Quantabio, Beverly, MA, USA) on a CFX Connect™ Real-Time PCR Detection System (Bio-Rad). The primers (Integrated DNA Technologies, Coralville, Iowa) used are described in Supplementary Table S1. Primers specificities were assessed by melt curve analysis. Gene expression levels were evaluated by the DD threshold cycle (Ct) method after confirmation that amplification efficiencies were between 90% and 110% for all primer pairs. The reference gene Rpl19 mRNAs was used as control for total mRNA recovery. Experiments were repeated four times and quantified as technical triplicates. 2.5. Statistical analyses Significant expression differences between control and SOX4 or SOX8 overexpressed conditions in qPCR were characterized using nonparametric Mann-Whitney tests. For all statistical analyses, P < 0.05 was considered significant. Statistical analyses were performed using the GraphPad Prism 6 software package (GraphPad Software Inc, La Jolla, CA). 3. Results To determine if over-expressions of Sox4 or Sox8 have a modulatory effect on gene expression from Sertoli cells, we transfected the TM4 cell line with Sox4 or Sox8 expression plasmids, followed by total RNA extraction and high throughput RNA sequencing. Following single-end reads trimming, alignments to reference genome (mm10) and differential gene expression analysis, the quality of the obtained data was assessed by performing a M (log ratio) and A (mean average) plot of RNA-Seq data comparing TM4 cells transfected with pCDNA6 to TM4 cells transfected with Sox4 or Sox8 expression plasmids (Fig. 1). The Sox4 transcript was significantly higher in Sox4 overexpressed cells by 1.8 folds, whereas Sox8 transcript was increased by 127 folds in Sox8 overexpressed cells compared to control. Less than 13.3% of genes being significantly upregulated or downregulated by SOX4 or SOX8 in TM4 Sertoli cells (P < 0.05) were common (Fig. 2a and b). Results for gene differential expression were also filtered for FDR <0.05 and divided between genes being upregulated by SOX4 or SOX8 (Tables S2 and S3) or
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Fig. 1. Overview of RNA-Seq data from TM4 Sertoli cells following over-expressions of SOX4 or SOX8. MA plot of RNA-Seq data comparing overexpressed SOX4 (A) or overexpressed SOX8 (B) to control samples. The x-axis shows mean expression for each gene and the y-axis shows log2 fold change (overexpressed SOX vs control), as estimated in DESeq2.
downregulated by SOX4 or SOX8 (Tables S4 and S5). A total of 24 genes were upregulated and 16 downregulated by Sox4 overexpression, whereas 15 genes were upregulated and 15 downregulated by Sox8 over-expression in TM4 Sertoli cells. During gene set enrichment analysis using the GSEA software [23], a ranking of the differential expression results was first compared to curated gene ontology biological process annotations. Sox4 overexpression resulted in gene enrichment of downregulated genes for regulation of interleukin-6 production (p < 0.05; FDR < 0.1) and regulation of cell-cell adhesion (p < 0.002; FDR < 0.29). For Sox8 overexpression in Sertoli cells, upregulated genes were enriched for multiple biological processes, including Sertoli cell differentiation and development (p < 0.017; FDR < 0.24). Downregulated genes in response to Sox8 overexpression were enriched for numerous biological processes, including cellsubstrate adhesion, cell-cell junction organization and sterol biosynthetic process (p < 0.01; FDR < 0.135). To illustrate the propensities of SOX4 and SOX8 in downregulating genes encoding components of cell-cell junctions, ranked differential expression results were compared to pathway databases and filtered for junctions and adhesion functions (Fig. 2c and d). Gene lists were also analysed for functional interpretation using the web server gProfiler [26] (Fig. S1-S4). Downregulated genes by Sox4 overexpression in Sertoli cells were enriched for cell junction organization (p ¼ 0.038), cell-cell adhesion (p ¼ 0.020) and positive regulation of cell-cell adhesion (p ¼ 0.018). For Sox8 overexpression in Sertoli cells, downregulated genes were enriched for positive regulation of cell adhesion molecule production (p ¼ 0.017), cellmatrix adhesion (p ¼ 0.018), positive regulation of cell-substrate adhesion (p ¼ 0.014) and extra-cellular matrix (ECM)-receptor interaction (p ¼ 0.034). According to the GeneMania algorithm, genes downregulated by Sox4 overexpression in TM4 cells (p < 0.05) are related to sex differentiation (FDR ¼ 0.007), development of primary female sexual characteristics (FDR ¼ 0.019), female sex differentiation (FDR ¼ 0.021), ovulation cycle (FDR ¼ 0.032) and gonad development (FDR ¼ 0.034). Genes downregulated by Sox8 overexpression were associated to extracellular matrix organization (FDR ¼ 9.72E06) and positive regulation of cell adhesion (FDR ¼ 0.037). Supporting the accuracy of RNA-Seq data, we confirmed a selection of up and down-regulated genes expressions by real-time quantitative PCR (Fig. 3). Among genes being modulated by Sox8 overexpression in TM4 Sertoli cells, some are related to the extracellular matrix with genes coding for collagen fibers, such as
Col11a2 (p ¼ 0.002) and Col5a3 (p ¼ 0.026), and have been confirmed by real-time PCR quantification (Fig. 3 i, j). Oppositely, increased Car6 expression in response to Sox4 overexpression was confirmed (Fig. 3b). 4. Discussion 4.1. SOX8 may participate in the elaboration of the extracellular matrix around Sertoli cells In this study we used the TM4 Sertoli cell line which has been isolated from 11 to 13 day mouse testes [28]. These cells are nontumorigenic and can respond to FSH and retinoic acid stimulations. Furthermore, TM4 Sertoli cells express cell-to-cell junction proteins such as GJA1 [17] and tight junction proteins such as Claudin-11 or Tight Junction Protein-1 [29] which are necessary for Sertoli cells function. These cells also produce ECM components, including collagen, matrix metalloproteinase, laminin 1 and tissue inhibitor of metalloproteinase [29]. Outside Sertoli cells, the ECM allows the formation of the basement membrane and participates in cell polarization. In this study, we have shown that SOX8 overexpression downregulates the expression of genes encoding ECM components such as collagen (Col1a1, Col1a2, Col3a1, Col5a2, Col5a3, Col11a1 and Col11a2). Interestingly, Col11a2 is an important target gene of Sox9 during chondrogenesis [30]. Fbln2, being downregulated by SOX8, encodes an extracellular matrix protein with calcium binding and epidermal growth factor domains. Although the expression and function of Fbln2 in Sertoli cells is unclear, its expression at E11.5 in XY gonads [31] suggests that it may regulate testicular growth and/or morphogenesis. Thus, the downregulation of cell-to-cell adhesion by SOX8 overexpression, and to a lesser extent SOX4, could be relevant for the elaboration of the ECM by Sertoli cells. 4.2. SOX4 and SOX8 may regulate cell-to-cell interactions between Sertoli cells Using TM4 Sertoli cells, our study confirms the importance of SOX4 and SOX8 in gene regulation related to cell-to-cell interactions. Indeed, SOX4 and SOX8 downregulated the expression of the adherent junction Cdh13 and of the tight junction Cldn12. Although non-significant (P ¼ 0.124), overexpression of SOX8 resulted in a decrease of Cldn12 expression by 73.8%, supporting
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Fig. 2. Significantly upregulated or downregulated genes following SOX4 or SOX8 overexpression in TM4 Sertoli cells are involved in cell-to-cell interactions. Venn diagrams comparing significantly upregulated (A) or downregulated (B) genes following SOX4 and SOX8 overexpressions in TM4 Sertoli cells show the number of common (overlapping circles) and unique transcripts (non-overlapping circles). Ranked differential expression results for SOX4 (C) or SOX8 (D) were compared to pathway databases and filtered for junctions and adhesion functions using the GSEA 3.0 software and leading-edge analysis.
previous results showing a 1.4 fold induction of this gene in Sox8/ mouse testis [32]. Oppositely, Cldn3 expression was increased by 2.0 folds following SOX8 overexpression in TM4 cell, confirming previously reported implication of SOX8 in transcriptional regulation of Cldn3 in mouse testis [33]. Although the overexpression of SOX4 and SOX8 do not lead to major changes in gene expression related to cell-to-cell interactions, our transcriptomic study demonstrates a significant enrichment of downregulated genes for this biological process. Interestingly, SOX8 overexpression resulted in increased expression of Gata4 by 6.1 folds in TM4 Sertoli cells. Depletion of the transcription factor GATA4 in Sertoli cells was associated with altered expression of genes involved in tight and adherent junctions’ formation, including Cldn12, and extracellular matrix reorganization [34]. Thus, the regulation of genes responsible for cellto-cell interactions by SOX8 may be mediated, in part, by an
alteration of Gata4 expression. Our current study indicates that SOX4 and SOX8 overexpressions downregulate Gja1 expression. Importantly, such inhibitory effect may be reversed by cooperation between SOX transcription factors and cJUN [35]. Hence, protein-protein interactions and post-translational modifications may alter the activity of SOX factors, leading to a transcriptional regulation of target genes different from that observed during overexpression. 4.3. SOX8 may be implicated in Sertoli cells differentiation and development Our study provides new insights on SOX8-dependent transcriptional regulation participating in Sertoli cell differentiation and development. Indeed, Sox8 overexpression in TM4 Sertoli cells upregulated genes involved in Sertoli cell differentiation and
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Fig. 3. Quantitative real-time PCR for genes modulated by SOX4 or SOX8 over-expressions in TM4 Sertoli cells. Following transfections of TM4 Sertoli cells using a control, SOX4 or SOX8 expression plasmid, cells were selected with blasticidin (15 mg/ml for 48 h), followed by RNA extraction, reverse transcription and quantitative real-time PCR assays. Results are normalized using Rpl19 reference gene expression and presented relative to control (CTL) expression. Statistics were performed using nonparametric Mann-Whitney tests. Black bars correspond to control and grey bars to SOX4 or SOX8 overexpressed conditions.
development such as Atrx, Sox9, Akap9 and Safb2. Among the other genes being modulated and having an influence on the proliferation and differentiation of Sertoli cells, Smad5 transcript variant 1 was downregulated whereas the transcript variant 2 was upregulated by SOX4. Although weaker than in spermatogonia, Smad5 transcripts have been detected in mouse Sertoli cells from birth to adulthood [36] and may regulate cell proliferation [37]. Although Axl transcript variant 1 is downregulated by SOX4, the transcript variant 3 is rather upregulated by SOX8. This receptor tyrosine kinase is essential for male fertility and is expressed in Sertoli cells during postnatal development [38]. Normally, Fst is expressed in somatic cells of XX gonad and acts downstream of WNT4 during ovary development [39]. SOX8 and SOX4 overexpressions decreases the expression of Fst transcript variant 1 by 97.1% and 53.7% respectively, whereas the variant 3 was increased by 13.5 folds and 14.0% in TM4 Sertoli cells. The expression of Fst was reported to be increased in XY Sox8/ mice [40]. Moreover, Amh expression was increased by 3.1 folds in response to SOX8 overexpression in TM4 Sertoli cells, which supports previous findings in Sox8/ mice where Amh was decreased [40]. SOX8 is known to synergize with SF1 to activate Amh expression [41]. Overall, our results confirm that SOX4 and SOX8 influence gene expression related to regulation of blood-testis barrier assembly and are important mediators of Sertoli cell development and
differentiation. Further studies will be needed to characterize DNA recruitments of SOX4 and SOX8 and their regulatory mechanisms to modulate target genes expressions in Sertoli cells. Disclosure The authors declare that there is no conflict of interest that would prejudice their impartiality. Acknowledgements Current work was funded by the New Brunswick Innovation Foundation (#CV2017 to L.J.M.) and the Natural Sciences and Engineering Research Council (NSERC) of Canada (#386557 to L.J.M). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.03.096. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.03.096.
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Transcriptomic analysis of overexpressed SOX4 and SOX8 in TM4 Sertoli cells with emphasis on cell-to-cell interactions Pauline Roumaud, Luc J. Martin* Biology Department, Universit e de Moncton, Moncton, New-Brunswick, E1A 3E9, Canada
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 March 2019 Accepted 16 March 2019 Available online 25 March 2019
Sertoli cells are localized in seminiferous tubules within the testis. They are the first testicular cells to differentiate during male sex determination. In the adult, Sertoli cells provide nutrients to germ cells, control factors for spermatogenesis and protection by establishing the blood-testis barrier (BTB). This BTB is composed of tight junctions, basal ectoplasmic specializations, adherent junctions and gap junctions. The transcription factor SOX8 is necessary for the maintenance of spermatogenesis during adult life whereas SOX4 is involved in developmental processes. These factors are highly expressed in Sertoli cells. However, few of their target genes in adult Sertoli cells are known. Hence, we compared the transcriptomes of TM4 Sertoli cells overexpressing or not SOX4 or SOX8 using RNA-Seq followed by pathways and networks analyses. We found that SOX4 overexpression leads to downregulated genes enriched for cell junction organization and positive regulation of cell-to-cell adhesion. Upregulated genes in response to SOX8 overexpression were enriched for Sertoli cell development and differentiation. However, downregulated genes were enriched for cell-to-cell adhesion, tight junction interactions, gap junctions’ assembly, as well as extracellular matrix binding. Hence, our results confirm that SOX8 is an important mediator of Sertoli cell maturation, whereas SOX4 and SOX8 influence gene expression related to regulation of blood-testis barrier assembly. In addition, TM4 cells can be considered as a useful model to better define the regulatory mechanisms of SOX4 or SOX8 on gene transcription in Sertoli cells. © 2019 Elsevier Inc. All rights reserved.
Keywords: Sertoli cells SOX4 SOX8 Transcriptomics RNA-Seq Cell-to-cell interactions
1. Introduction In the testis, Sertoli cells are somatic cells localized in seminiferous tubules and support spermatogenesis by providing protection, nutrients and proliferation or differentiation factors. Between Sertoli cells, the blood-testis barrier (BTB) is composed of adherent and tight junctions and allows the protection of germ cells from autoimmune reactions. Sertoli cells synthesize several molecules such as nutrient transport proteins (transferrin, clusterin), differentiation factors (TGFb, IGF1) or proliferation factors (activin). These supporting cells are regulated by testosterone, FSH, retinoic acid or vitamin D (for review [1,2]). Important functions of Sertoli cells are under the control of SOX transcription factors. The Sox genes encode transcription factors with a high mobility group (HMG)-type DNA binding domain consisting of three a-helices and allowing to bind the minor groove
de Moncton, 18 avenue * Corresponding author. Biology Department, Universite Antonine Maillet, Moncton, New-Brunswick, E1A 3E9, Canada. E-mail address: [email protected] (L.J. Martin). https://doi.org/10.1016/j.bbrc.2019.03.096 0006-291X/© 2019 Elsevier Inc. All rights reserved.
of DNA and bend it [3]. Twenty members of the SOX family have been identified and this family is subdivided into 8 groups (A-H) based on their identity within and outside the HMG box [4]. In the embryo, Sertoli cells play a critical role in sex determination, as well as the differentiation and development of testes. Indeed, the expression of Sex-determining Region of the chromosome Y (SRY) by 10.5 dpc in mice and by 41 dpc in humans, leads to the differentiation of progenitor Sertoli cells. SRY activates the expression of SRY-related high-mobility-group (HMG) box 9 (Sox9), a member of the SoxE group of SOX transcription factors. In mouse, we have confirmed that SOX4 and SOX8 transcription factors are highly expressed within the adult Sertoli cells [5]. SOX4 belongs to the SoxC group and have a transactivation domain (TAD) located in its C-terminal region [6]. Sox4/ mice die at 14.5 dpc due to cardiac defects [7]. Several SOX4 target genes have already been identified, notably in C2C12 myoblasts [8,9], in lung cancer cell lines [10] and in neural or mesenchymal progenitors [11]. In Sox4/ foetal mice testes, Zhao et al. identified genes potentially regulated by SOX4, such as Nanos2, Dmnmt3l, Cripto, Nanog and p15INK4b [12]. Interestingly, they found that SOX4 downregulates Sox9
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expression directly even though considered a transcriptional activator. As SOX9 and SOX10, SOX8 belongs to the SoxE group. These transcription factors are involved in many developmental processes such as neurogenesis, chondrogenesis and male gonadogenesis (for review [13]). Interestingly, Sox8/ mice are viable and fertile with a significant weight reduction [14]. However, these mice become progressively infertile, suggesting an implication of SOX8 in the maintenance of spermatogenesis within the adult testis [15]. In humans, SOX8 mutations can lead to 46 XY sex-reversal, male infertility and female primary ovary insufficiency [16]. In this study, we aim to identify the potential target genes for SOX4 and SOX8 through overexpression in TM4 Sertoli cells, followed by transcriptomic RNA-Seq analyses and pathways and networks enrichments investigations. Our results confirmed that SOX4 regulates cell-to-cell adhesion, while SOX8 modulates the expression of genes involved in Sertoli cell development and differentiation as well as cell-to-cell interaction. 2. Material and methods
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counts and mean fit type [22]. 2.3. Pathways and networks analyses From the total gene list of differentially expressed genes, we performed a gene set enrichment analysis using the GSEA software (version 3.0) from the Broad Institute [23]. A ranking of the differential expression results was determined by score calculation based on fold changes and p-values of DESeq2 results (sign(logFC) *-log10(pvalue)) and compared to all pathways, released 2018-1101 (http://download.baderlab.org/EM_Genesets/), or curated gene ontology biological process annotations, released 2018-11-01 [24,25], using a modified Kolmogorov-Smirnov statistic. Differentially expressed genes were also analysed using the gProfiler gene group functional profiling platform for biological process, molecular function, biological pathways and reactome annotations using the Benjamini-Hochberg FDR significance threshold [26]. In addition, differentially expressed genes were analysed using the GeneMania platform (version 3.5.0) with biological process and molecular function network weighting [27].
2.1. Cell culture and transfection 2.4. Real-time quantitative PCR Mouse TM4 Sertoli cells were obtained from American Type Culture Collection (Manassas, VA, USA) and cultured as described previously [17]. The SOX8 expression plasmid was obtained by amplification of cDNA using the forward primer containing a HindIII cloning site, 50 -GCA AGC TTA TGC TGG ACA TGA GTG AGG CCC GCG CC-30 , and the reverse primer containing a XhoI cloning site, 50 CGC TCG AGG GGT CGG GTC AGG GTG GTG TAC AC-30 , followed by cloning into pCDNA6-V5His. SOX4 expression vector has been described previously [18] and was provided by Dr. James Wells (Cincinnati Children's Hospital Research Foundation, Cincinnati, OH). TM4 cells were transfected with an empty vector (control), with SOX4 or SOX8 expression plasmid using polyethylenimine according to the previously described method [17] with a 3:1 ratio (polyethylenimine to DNA). After a 48 h incubation, positively transfected cells were selected with blasticidin (Bioshop, Burlington, ON, CAN) at 15 mg/ml for 48 h. 2.2. RNA extraction and RNA sequencing analyses After 96 h of transfection, total RNA was isolated with E.Z.N.A. Total RNA kit (Omega Bio-Tek, Inc., Norcross, GA). RNA quality was determined by electrophoresis and characterisation of the 18S and 28S ribosomal RNA bands. Libraries were constructed using the TruSeq Stranded mRNA LT (Illumina) for cDNA synthesis and polyA enrichment. Quality and quantification of libraries were evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Mississauga, ON, CAN) and KAPA SYBR® FAST qPCR Kit (KAPA Bio Systems, Wilmington, MA, USA), respectively. Transcriptomic sequencing was performed on an Illumina HiSeq 2000 in single reads layout with approximately 45 million reads per sample and 100 bp of sequencing length. Libraries and next generation sequencing were completed by the McGill University and Genome Quebec Innovation Centre (Montreal, QC, CAN). RNA-Seq analyses were performed using the Galaxy web-based platform (https:// usegalaxy.org). Single-end raw reads in FASTQ format were trimmed using the Trimmomatic (version 0.36.5) sliding window with a minimal average quality of 20 and Illuminaclip for TruSeq3 adapters [19]. Trimmed reads were aligned to reference mouse genome (mm10) using the HISAT2 alignment program (version 2.1.0) [20]. Transcripts assemblies and quantifications were performed with the StringTie assembler (version 1.3.4) for NCBI RefSeq curated reference transcripts from mm10 [21]. Differential gene expression was assessed using DESeq2 (version 2.11.40.2) for gene
The qScript cDNA SuperMix (Quantabio, Beverly, MA, USA) was used to synthesize cDNA from total mRNAs. Quantitative real-time polymerase chain reaction (qPCR) was performed using PerfeCTa SYBR® Green FastMix (Quantabio, Beverly, MA, USA) on a CFX Connect™ Real-Time PCR Detection System (Bio-Rad). The primers (Integrated DNA Technologies, Coralville, Iowa) used are described in Supplementary Table S1. Primers specificities were assessed by melt curve analysis. Gene expression levels were evaluated by the DD threshold cycle (Ct) method after confirmation that amplification efficiencies were between 90% and 110% for all primer pairs. The reference gene Rpl19 mRNAs was used as control for total mRNA recovery. Experiments were repeated four times and quantified as technical triplicates. 2.5. Statistical analyses Significant expression differences between control and SOX4 or SOX8 overexpressed conditions in qPCR were characterized using nonparametric Mann-Whitney tests. For all statistical analyses, P < 0.05 was considered significant. Statistical analyses were performed using the GraphPad Prism 6 software package (GraphPad Software Inc, La Jolla, CA). 3. Results To determine if over-expressions of Sox4 or Sox8 have a modulatory effect on gene expression from Sertoli cells, we transfected the TM4 cell line with Sox4 or Sox8 expression plasmids, followed by total RNA extraction and high throughput RNA sequencing. Following single-end reads trimming, alignments to reference genome (mm10) and differential gene expression analysis, the quality of the obtained data was assessed by performing a M (log ratio) and A (mean average) plot of RNA-Seq data comparing TM4 cells transfected with pCDNA6 to TM4 cells transfected with Sox4 or Sox8 expression plasmids (Fig. 1). The Sox4 transcript was significantly higher in Sox4 overexpressed cells by 1.8 folds, whereas Sox8 transcript was increased by 127 folds in Sox8 overexpressed cells compared to control. Less than 13.3% of genes being significantly upregulated or downregulated by SOX4 or SOX8 in TM4 Sertoli cells (P < 0.05) were common (Fig. 2a and b). Results for gene differential expression were also filtered for FDR <0.05 and divided between genes being upregulated by SOX4 or SOX8 (Tables S2 and S3) or
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Fig. 1. Overview of RNA-Seq data from TM4 Sertoli cells following over-expressions of SOX4 or SOX8. MA plot of RNA-Seq data comparing overexpressed SOX4 (A) or overexpressed SOX8 (B) to control samples. The x-axis shows mean expression for each gene and the y-axis shows log2 fold change (overexpressed SOX vs control), as estimated in DESeq2.
downregulated by SOX4 or SOX8 (Tables S4 and S5). A total of 24 genes were upregulated and 16 downregulated by Sox4 overexpression, whereas 15 genes were upregulated and 15 downregulated by Sox8 over-expression in TM4 Sertoli cells. During gene set enrichment analysis using the GSEA software [23], a ranking of the differential expression results was first compared to curated gene ontology biological process annotations. Sox4 overexpression resulted in gene enrichment of downregulated genes for regulation of interleukin-6 production (p < 0.05; FDR < 0.1) and regulation of cell-cell adhesion (p < 0.002; FDR < 0.29). For Sox8 overexpression in Sertoli cells, upregulated genes were enriched for multiple biological processes, including Sertoli cell differentiation and development (p < 0.017; FDR < 0.24). Downregulated genes in response to Sox8 overexpression were enriched for numerous biological processes, including cellsubstrate adhesion, cell-cell junction organization and sterol biosynthetic process (p < 0.01; FDR < 0.135). To illustrate the propensities of SOX4 and SOX8 in downregulating genes encoding components of cell-cell junctions, ranked differential expression results were compared to pathway databases and filtered for junctions and adhesion functions (Fig. 2c and d). Gene lists were also analysed for functional interpretation using the web server gProfiler [26] (Fig. S1-S4). Downregulated genes by Sox4 overexpression in Sertoli cells were enriched for cell junction organization (p ¼ 0.038), cell-cell adhesion (p ¼ 0.020) and positive regulation of cell-cell adhesion (p ¼ 0.018). For Sox8 overexpression in Sertoli cells, downregulated genes were enriched for positive regulation of cell adhesion molecule production (p ¼ 0.017), cellmatrix adhesion (p ¼ 0.018), positive regulation of cell-substrate adhesion (p ¼ 0.014) and extra-cellular matrix (ECM)-receptor interaction (p ¼ 0.034). According to the GeneMania algorithm, genes downregulated by Sox4 overexpression in TM4 cells (p < 0.05) are related to sex differentiation (FDR ¼ 0.007), development of primary female sexual characteristics (FDR ¼ 0.019), female sex differentiation (FDR ¼ 0.021), ovulation cycle (FDR ¼ 0.032) and gonad development (FDR ¼ 0.034). Genes downregulated by Sox8 overexpression were associated to extracellular matrix organization (FDR ¼ 9.72E06) and positive regulation of cell adhesion (FDR ¼ 0.037). Supporting the accuracy of RNA-Seq data, we confirmed a selection of up and down-regulated genes expressions by real-time quantitative PCR (Fig. 3). Among genes being modulated by Sox8 overexpression in TM4 Sertoli cells, some are related to the extracellular matrix with genes coding for collagen fibers, such as
Col11a2 (p ¼ 0.002) and Col5a3 (p ¼ 0.026), and have been confirmed by real-time PCR quantification (Fig. 3 i, j). Oppositely, increased Car6 expression in response to Sox4 overexpression was confirmed (Fig. 3b). 4. Discussion 4.1. SOX8 may participate in the elaboration of the extracellular matrix around Sertoli cells In this study we used the TM4 Sertoli cell line which has been isolated from 11 to 13 day mouse testes [28]. These cells are nontumorigenic and can respond to FSH and retinoic acid stimulations. Furthermore, TM4 Sertoli cells express cell-to-cell junction proteins such as GJA1 [17] and tight junction proteins such as Claudin-11 or Tight Junction Protein-1 [29] which are necessary for Sertoli cells function. These cells also produce ECM components, including collagen, matrix metalloproteinase, laminin 1 and tissue inhibitor of metalloproteinase [29]. Outside Sertoli cells, the ECM allows the formation of the basement membrane and participates in cell polarization. In this study, we have shown that SOX8 overexpression downregulates the expression of genes encoding ECM components such as collagen (Col1a1, Col1a2, Col3a1, Col5a2, Col5a3, Col11a1 and Col11a2). Interestingly, Col11a2 is an important target gene of Sox9 during chondrogenesis [30]. Fbln2, being downregulated by SOX8, encodes an extracellular matrix protein with calcium binding and epidermal growth factor domains. Although the expression and function of Fbln2 in Sertoli cells is unclear, its expression at E11.5 in XY gonads [31] suggests that it may regulate testicular growth and/or morphogenesis. Thus, the downregulation of cell-to-cell adhesion by SOX8 overexpression, and to a lesser extent SOX4, could be relevant for the elaboration of the ECM by Sertoli cells. 4.2. SOX4 and SOX8 may regulate cell-to-cell interactions between Sertoli cells Using TM4 Sertoli cells, our study confirms the importance of SOX4 and SOX8 in gene regulation related to cell-to-cell interactions. Indeed, SOX4 and SOX8 downregulated the expression of the adherent junction Cdh13 and of the tight junction Cldn12. Although non-significant (P ¼ 0.124), overexpression of SOX8 resulted in a decrease of Cldn12 expression by 73.8%, supporting
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Fig. 2. Significantly upregulated or downregulated genes following SOX4 or SOX8 overexpression in TM4 Sertoli cells are involved in cell-to-cell interactions. Venn diagrams comparing significantly upregulated (A) or downregulated (B) genes following SOX4 and SOX8 overexpressions in TM4 Sertoli cells show the number of common (overlapping circles) and unique transcripts (non-overlapping circles). Ranked differential expression results for SOX4 (C) or SOX8 (D) were compared to pathway databases and filtered for junctions and adhesion functions using the GSEA 3.0 software and leading-edge analysis.
previous results showing a 1.4 fold induction of this gene in Sox8/ mouse testis [32]. Oppositely, Cldn3 expression was increased by 2.0 folds following SOX8 overexpression in TM4 cell, confirming previously reported implication of SOX8 in transcriptional regulation of Cldn3 in mouse testis [33]. Although the overexpression of SOX4 and SOX8 do not lead to major changes in gene expression related to cell-to-cell interactions, our transcriptomic study demonstrates a significant enrichment of downregulated genes for this biological process. Interestingly, SOX8 overexpression resulted in increased expression of Gata4 by 6.1 folds in TM4 Sertoli cells. Depletion of the transcription factor GATA4 in Sertoli cells was associated with altered expression of genes involved in tight and adherent junctions’ formation, including Cldn12, and extracellular matrix reorganization [34]. Thus, the regulation of genes responsible for cellto-cell interactions by SOX8 may be mediated, in part, by an
alteration of Gata4 expression. Our current study indicates that SOX4 and SOX8 overexpressions downregulate Gja1 expression. Importantly, such inhibitory effect may be reversed by cooperation between SOX transcription factors and cJUN [35]. Hence, protein-protein interactions and post-translational modifications may alter the activity of SOX factors, leading to a transcriptional regulation of target genes different from that observed during overexpression. 4.3. SOX8 may be implicated in Sertoli cells differentiation and development Our study provides new insights on SOX8-dependent transcriptional regulation participating in Sertoli cell differentiation and development. Indeed, Sox8 overexpression in TM4 Sertoli cells upregulated genes involved in Sertoli cell differentiation and
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Fig. 3. Quantitative real-time PCR for genes modulated by SOX4 or SOX8 over-expressions in TM4 Sertoli cells. Following transfections of TM4 Sertoli cells using a control, SOX4 or SOX8 expression plasmid, cells were selected with blasticidin (15 mg/ml for 48 h), followed by RNA extraction, reverse transcription and quantitative real-time PCR assays. Results are normalized using Rpl19 reference gene expression and presented relative to control (CTL) expression. Statistics were performed using nonparametric Mann-Whitney tests. Black bars correspond to control and grey bars to SOX4 or SOX8 overexpressed conditions.
development such as Atrx, Sox9, Akap9 and Safb2. Among the other genes being modulated and having an influence on the proliferation and differentiation of Sertoli cells, Smad5 transcript variant 1 was downregulated whereas the transcript variant 2 was upregulated by SOX4. Although weaker than in spermatogonia, Smad5 transcripts have been detected in mouse Sertoli cells from birth to adulthood [36] and may regulate cell proliferation [37]. Although Axl transcript variant 1 is downregulated by SOX4, the transcript variant 3 is rather upregulated by SOX8. This receptor tyrosine kinase is essential for male fertility and is expressed in Sertoli cells during postnatal development [38]. Normally, Fst is expressed in somatic cells of XX gonad and acts downstream of WNT4 during ovary development [39]. SOX8 and SOX4 overexpressions decreases the expression of Fst transcript variant 1 by 97.1% and 53.7% respectively, whereas the variant 3 was increased by 13.5 folds and 14.0% in TM4 Sertoli cells. The expression of Fst was reported to be increased in XY Sox8/ mice [40]. Moreover, Amh expression was increased by 3.1 folds in response to SOX8 overexpression in TM4 Sertoli cells, which supports previous findings in Sox8/ mice where Amh was decreased [40]. SOX8 is known to synergize with SF1 to activate Amh expression [41]. Overall, our results confirm that SOX4 and SOX8 influence gene expression related to regulation of blood-testis barrier assembly and are important mediators of Sertoli cell development and
differentiation. Further studies will be needed to characterize DNA recruitments of SOX4 and SOX8 and their regulatory mechanisms to modulate target genes expressions in Sertoli cells. Disclosure The authors declare that there is no conflict of interest that would prejudice their impartiality. Acknowledgements Current work was funded by the New Brunswick Innovation Foundation (#CV2017 to L.J.M.) and the Natural Sciences and Engineering Research Council (NSERC) of Canada (#386557 to L.J.M). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.03.096. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.03.096.
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