Sox9 gene regulation and the loss of the XY/XX sex-determining mechanism in the mole vole Ellobius lutescens
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- Bagheri-Fam, S., Sreenivasan, R., Bernard, P. et al. Chromosome Res (2012) 20: 191. doi:10.1007/s10577-011-9269-5
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In most mammals, the Y chromosomal Sry gene initiates testis formation within the bipotential gonad, resulting in male development. SRY is a transcription factor and together with SF1 it directly up-regulates the expression of the pivotal sex-determining gene Sox9 via a 1.3-kb cis-regulatory element (TESCO) which contains an evolutionarily conserved region (ECR) of 180 bp. Remarkably, several rodent species appear to determine sex in the absence of Sry and a Y chromosome, including the mole voles Ellobius lutescens and Ellobius tancrei, whereas Ellobius fuscocapillus of the same genus retained Sry. The sex-determining mechanisms in the Sry-negative species remain elusive. We have cloned and sequenced 1.1 kb of E. lutescens TESCO which shares 75% sequence identity with mouse TESCO indicating that testicular Sox9 expression in E. lutescens might still be regulated via TESCO. We have also cloned and sequenced the ECRs of E. tancrei and E. fuscocapillus. While the three Ellobius ECRs are highly similar (94–97% sequence identity), they all display a 14-bp deletion (Δ14) removing a highly conserved SOX/TCF site. Introducing Δ14 into mouse TESCO increased both basal activity and SF1-mediated activation of TESCO in HEK293T cells. We propose a model whereby Δ14 may have triggered up-regulation of Sox9 in XX gonads leading to destabilization of the XY/XX sex-determining mechanism in Ellobius. E. lutescens/E. tancrei and E. fuscocapillus could have independently stabilized their sex determination mechanisms by Sry-independent and Sry-dependent approaches, respectively.
Chromobox protein homolog 2
Dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome, gene 1
Dulbecco's modified Eagle's medium
Doublesex and mab-3-related transcription factor 1
Evolutionarily conserved region
Forkhead box L2
Human embryonic kidney carcinoma
High mobility group
Lymphoid enhancer factor-1
Polymerase chain reaction
Polled intersex syndrome regulated transcript 1
Plasmid Renilla luciferase-thymidine kinase
Plasmid University of California
Steroidogenic factor 1
SOX = SRY-related HMG-Box
Sex-determining region of the Y chromosome
Testis-specific enhancer of Sox9
Testis-specific enhancer of Sox9 core
Sex determination in mammals is chromosomally controlled with males and females carrying the XY and XX sex chromosomes, respectively. In most mammals, the Y chromosomal Sry gene triggers the fate of the bipotential gonad to develop into a testis rather than into an ovary resulting in male development (Sinclair et al. 1990; Koopman et al. 1991). The Sry gene evolved before the divergence of marsupials and placental mammals around 144–168 million years ago, and thus is absent in monotremes such as in platypus (Wallis et al. 2007, 2008). The SRY protein is the founder member of the SOX (SOX = SRY-related high mobility group (HMG)-Box) family of transcription factors which share at least 50% homology within their DNA-binding and bending HMG domain (Bowles et al. 2000; Schepers et al. 2002). Despite its pivotal role in mammalian male sex determination, a handful of rodent species have been identified who apparently determine sex in the absence of Sry and a Y chromosome. These include the mole voles Ellobius lutescens and Ellobius tancrei (Matthey 1958; Just et al. 1995; Vogel et al. 1998) and the spiny rats Tokudaia osimensis and Tokudaia tokunoshimensis (Soullier et al. 1998). The loss of the Y chromosome in these two genera can be considered as independent events because Ellobius and Tokudaia belong to different subfamilies, to Arvecolinae and Murinae, respectively. Their sex-determining mechanisms, i.e. which gene acts as the sex-determining switch, remain elusive. However, it has been demonstrated recently that males of both Sry-negative Tokudaia species have additional copies of Cbx2, a gene acting upstream of Sry (Katoh-Fukui et al. 1998), suggesting that CBX2 might be involved in male sex determination in Tokudaia (Kuroiwa et al. 2011). In E. lutescens, several genes have been excluded as the sex-determining switch genes namely Dax1, Sf1, Foxl2/Pisrt1, Sox9, Sox3 and Dmrt1 (reviewed in Just et al. 2007).
The genus Ellobius contains at least five species, namely Ellobius fuscocapillus, E. lutescens, the sister species E. tancrei and Ellobius talpinus, and Ellobius alaicus (Just et al. 2007; Romanenko et al. 2007). The only species of this genus with an XY/XX karyotype and for which the presence of the Sry gene could be demonstrated is E. fuscocapillus (Just et al. 1995). E. lutescens has an odd number of chromosomes, with both sexes having the karyotype 2n = 17,X (Matthey 1958). The sister species E. tancrei and E. talpinus have an even number of chromosomes ranging from 2n = 32,XX to 2n = 54,XX and a constant number of 2n = 54, respectively, in both sexes (Kolomiets et al. 1991; Romanenko et al. 2007). Since the Sry gene is present in E. fuscocapillus, it can be assumed that the common ancestor of these Ellobius species also possessed Sry, but that Sry was lost shortly after or during the speciation of Ellobius.
Evolution of new species can be driven by various genetic mechanisms, including gene mutations and gene duplications resulting in novel protein functions, gene loss and mutations in cis-regulatory elements altering gene regulation. A recent breakthrough in the field of mammalian sex determination was the finding that in the mouse, SRY up-regulates the Sertoli cell expressed gene Sox9 through direct binding to a 1.3-kb cis-regulatory element, termed TESCO (testis-specific enhancer of Sox9 core), which is located 13 kb upstream of Sox9 (Sekido and Lovell-Badge 2008). Like Sry, Sox9 is both required and sufficient for male sex determination, for example, XX transgenic mice over-expressing Sox9 develop as infertile males (Foster et al. 1994; Wagner et al. 1994; Vidal et al. 2001; Chaboissier et al. 2004; Barrionuevo et al. 2006). It is therefore possible that Sox9 is the only important target for SRY in the developing testis. The discovery of the testis-specific enhancer of Sox9 now opens the possibility to screen Sry-negative mammals for sequence variations in TESCO. This analysis might reveal clues as to which transcription factors bind to TESCO in those species and might shed light on the fundamentals of SRY–TESCO interaction. The genus Ellobius is a bona fide model for such analyses since closely related species exist with Sry (E. fuscocapillus) and without Sry (E. lutescens).
Materials and methods
Cloning of Ellobius TESCO sequences
DNA sequence analysis
Four pGEM®T Easy clones containing the 2.2-kb E. lutescens fragment and two pGEM®T Easy clones containing the 200-bp E. fuscocapillus fragment were sequenced with standard vector primers Sp6 and T7, using an Applied Biosystems 3130xl Genetic Analyzer fitted with an 80-cm array to generate read lengths of approximately 1,000 bases. The four pGEM®T Easy clones containing the 2.2-kb E. lutescens fragment were also sequenced with the internal primers NestF: 5′-AGCAAGGCAGGACTCAGACA-3′ and NestR: 5′-ATCCGGTCCAGCATTCACCT-3′. The ECRs (200 bp PCR fragment) from five E. lutescens, two E. tancrei and three E. talpinus individuals were amplified and sequenced using primers hSox9TE2F and eSox9TE3R.
Identification of transcription factor binding sites
To identify putative transcription factor binding sites within the ECRs of E. lutescens, E. tancrei and E. fuscocapillus, we used the online program MatInspector (www.genomatix.de) with core and matrix similarity set to 1.00 and optimized, respectively. The MatInspector library at the time of analysis was the Matrix Family Library version 8.4 (June 2011).
The TESCO-E1b-Luc reporter was constructed by cloning the mouse 1.3-kb testis-specific enhancer of Sox9 (TESCO) (Sekido and Lovell-Badge 2008) by PCR into the E1b-luciferase reporter (Bernard et al. 2008). The TESCO-Δ14-E1b-Luc construct was generated by site-directed mutagenesis (Stratagene Kit) of the TESCO-E1b-Luc construct using primers SOXDELMF: 5′-CACAAAATAACAATGCCTTCTGGCTAAGAAAGAGAAGACTCC-3′ and SOXDELMR: 5′-GGAGTCTTCTCTTTCTTAGCCAGAAGGCATTGTTATTTTGTG-3′ according to the manufacturer's instructions.
In vitro luciferase assays
Human embryonic kidney carcinoma (HEK293T) cells were cultured at 37°C with 5% CO2 in Dulbecco's modified Eagle's medium (DMEM), high glucose, GlutaMAX media (Invitrogen) containing 10% foetal bovine serum, 1% sodium pyruvate and 1% penicillin-streptomycin. For in vitro luciferase assays, 30,000 cells were seeded into each well of a 96-well tissue culture plate 24 h prior to transfection. Cells in each well were co-transfected with the reporter construct TESCO-Δ14-E1b-Luc (10 ng), TESCO-E1b-Luc (10 ng) or the empty vector E1b-Luc (8 ng), together with 40 ng of each of the expression constructs pCDNA3-SF1, pCDNA3-SRY or pCDNA3-SOX9. pRL-TK-Renilla (Promega; 1 ng) was included as an internal control. Total DNA amount was made up to 100 ng per well using the plasmids pCDNA3 and pUC. The cells were transfected with FuGENE6 Transfection Reagent (Roche) following the manufacturer's instructions. Cell lysis was performed 46 h after transfection, and firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase activity (Luc) was normalised against that of Renilla luciferase (Ren). Five independent assays were performed, each in triplicate. Paired t tests were performed for statistical analysis.
Cloning of the ECRs of E. lutescens, E. tancrei, E. talpinus and E. fuscocapillus
Identification of a 14-bp deletion (Δ14) within the ECR of Ellobius
DNA sequence identities (percent) calculated between the different mammalian ECRs
Notably, all Ellobius species carry a large deletion within the ECR (14 bp when compared to mouse/rat (Δ14) and 15 bp when compared to other vertebrates), which removes most of the evolutionarily conserved module ECRiii including its SOX/TCF site, as well as additional 3′-flanking sequences (Fig. 2, highlighted in cyan). In the remainder of module ECRiii, E. lutescens shows a unique T to C change. The 14-bp deletion in Ellobius is an intriguing finding since no deletions in module ECRiii are found in the ECR sequences of 41 vertebrate species (including 37 mammalian species) obtained from the nucleotide collection (nr/nt), high throughput genomic sequences (htgs) and whole-genome shotgun reads (wgs) databases at the National Centre of Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov). Moreover, only two of the 37 mammalian species show sequence variation in ECRiii, namely the Chinese hamster (CTTTCAG to CTTTGGA) and the bottlenose dolphin (CTTTCAG to CTTTTAG). Another noteworthy sequence variation present in all Ellobius species is a C to A change in module ECRv.
Only E. fuscocapillus shows an A to G change in module ECRii, predicted to disrupt the SOX site R5 (Fig. 2, highlighted in cyan) which is important for SRY and SOX9-mediated activation of TESCO in the mouse (Sekido and Lovell-Badge 2008). In all Ellobius species, no sequence variation was found in modules ECRi (SOX/TCF site) and ECRiv (GATA site; Fig. 2).
Sequence variation in the Ellobius ECRs alter the prediction of potential transcription factor binding sites
Δ14 increases basal activity and SF1-mediated activation of mouse TESCO in HEK293T cells
Through a PCR-based approach using degenerate primers, we were able to clone 1.1 kb of E. lutescens TESCO (including the entire ECR) which shares 75% sequence identity with mouse TESCO. This shows that TESCO is present in this species with high sequence conservation despite the loss of Sry. We did not expect a complete loss of TESCO in E. lutescens since TESCO is controlled by additional important transcription factors including SF1, SOX9 (SOX9 auto-regulation) and FOXL2 (SOX9 repression) (Sekido and Lovell-Badge 2008; Uhlenhaut et al. 2009). Moreover, TESCO sequences are conserved in non-mammalian species including chicken, lizard and frog (Bagheri-Fam et al. 2010) which all lack Sry indicating that TESCO might be important for testicular Sox9 expression independent of the sex-determining switch mechanism. It is thus possible that Sox9 expression in E. lutescens is still regulated via TESCO by factors such as SOX9, SF1 and FOXL2 and potentially also by the new sex-determining switch gene which remains to be identified.
We could also clone the ECR of E. tancrei (which also lacks Sry) and of the Sry-positive species E. fuscocapillus spanning all evolutionarily conserved modules (ECRi-ECRv) which allowed us to compare the Ellobius ECR sequences with each other and to other vertebrate ECRs. All three Ellobius species carry a 14-bp deletion (Δ14) removing module ECRiii and additional 3′-flanking sequences. As a result, a putative SOX/TCF site is removed in all three Ellobius species, and new transcription factor binding sites are created, an ETS binding site in E. lutescens and a Heat protein binding site in E. fuscocapillus. The only sequence difference between the Sry-positive species E. fuscocapillus and the two Sry-negative Ellobius species within the highly conserved ECR modules is the A to G change in module ECRiii predicted to abolish SRY binding to the bona fide target site R5 (Sekido and Lovell-Badge 2008).
There are various possible new sex-determining mechanisms to replace Sry in Ellobius. For example, in the Sry-negative Tokudaia species, additional copies of Cbx2 might trigger male sex determination (Kuroiwa et al. 2011). A potential link between a mutation in the testis-specific enhancer of Sox9 and the evolution of a Sry-independent sex-determining mechanism is an intriguing hypothesis which remains to be tested.
This work was supported by the National Health and Medical Research Council (NHMRC, Australia) Program Grants 334314 and 546517 to V.R.H., by Project Grant 1004992 to S.B. and by the Victorian Government's Operational Infrastructure Support Program (OIS). V.R.H. is the recipient of the NHMRC (Australia) Research Fellowship 441102; Prince Henry's Institute audit number 11-24.