SOX1b as a novel candidate sex-determination gene
Sex chromosome turnovers are triggered by a different gene becoming the master sex determiner. A gene can become sex-determining if it has alleles that are beneficial to one sex but harmful to the other, if it is physically linked to such a gene with sexually-antagonistic variation, or if a new mutation changes the timing and/or tissue of its expression (Bachtrog et al., 2014; Graves, 2013). Some gene families repeatedly take on the role of a sex-determining gene in diverse independently evolved systems, and genes of the SOX family are often recruited (Graves, 2013). SOX genes govern major vertebrate developmental mechanisms, including testis development, central nervous system neurogenesis, oligodendrocyte development, chondrogenesis, and neural crest cell development, and are thus expressed in many tissues (Kiefer, 2007; Sarkar & Hochedlinger, 2013). Three genes in particular,SOX9 , SOX8 (Koopman, 2005) and SOX3 , a progenitor of the SRY gene in mammals (Graves, 2013; Takehana et al., 2014), have a role in male sex determination in unrelated species, their expression in the early gonad inducing testes development. In mammals, brief expression of SRY in a small number of undifferentiated gonadal cells stimulates differentiation of Sertoli cells and up-regulates SOX9 expression, triggering testis development (Koopman, 2005). SRY mediates SOX9 upregulation by binding to several SOX9 enhancers, some of which cause XX or XY sex reversal in humans when duplicated or deleted, respectively (Croft et al., 2018). In the Indian ricefish Oryzias dancena , a Y-chromosome-specific cis-regulatory element of SOX3 acts as a sex-determining locus, driving early gonadal SOX3 expression in XY individuals, which initiates testicular differentiation by upregulating expression of gsdf gene (Takehana et al., 2014). In medaka Oryzias latipes, SOX5 has a dual role in sex-determination: it acts as an evolutionarily conserved regulator of germ-cell number, and as a de novo regulator of expression of another transcription factor, dmrt1, during primary sex determination (Schartl et al., 2018). Several other SOX genes also play important roles in sex differentiation in fish (Hu, Wang, & Du, 2021).
Although SOX1 has not been reported to play a role in sex determination or sex differentiation, it belongs to the same B1 subfamily of transcription activators as SOX3 , and thus could have acquired a novel sex-determining function in Percichthyidae.SOX1a and SOX1b are fish-specific co-orthologs of tetrapodSOX1 , produced as a result of a teleost-specific genome duplication early in fish evolution, enabling diversification of gene function (Taylor, Braasch, Frickey, Meyer, & Van de Peer, 2003; Zhang et al., 2018). We hypothesize that in Macquarie perch, gonadal development is controlled by male-specific expression of SOX1bgene in a bipotential gonad during embryo development, modulated by the variation in a distal enhancer (the 146-bp sexing region). A somewhat similar mechanism of switching the master sex-determining gene exists inOryzias luzonensis , where a mutation in a cis-regulatory region of the sex-determining gene gsdf drives high expression in males during sex differentiation (Myosho et al., 2012). So far, male-specific regulation of SOX1b appears to be unique to Macquarie perch, and potentially to only some populations. In the golden perch genome, a locus with a female-specific allele is co-located with SOX1a,suggesting the potential involvement of this gene in sex determination or sex differentiation in that species. In other fishes, SOX1band SOX1a genes play roles in the early processes of neural development, defining the neural subdivisions and the workings of the central nervous system (Gao et al., 2015; Lekk et al., 2019; Okuda et al., 2006).
The 146-bp sexing region as a candidate distal enhancer of SOX1b
Non-coding DNA under evolutionary constraint and clustering of transcription-factor binding-site motifs are the two genomic features used to predict cis-regulatory elements, i.e. DNA sequences required to regulate tissue- and time-specific gene expression (Hardison & Taylor, 2012). Accordingly, the Macquarie perch 146-bp sexing region, occurring ~38 Kb upstream of SOX1b in a non-coding region of the genome, could act as distal enhancer of SOX1b : it shows evolutionary conservation across multiple orders of fish and multiple matches to known transcription factor binding motifs, with mutations on Y-chromosomes resulting in potential gains and losses of transcription factor binding sites (Appendix F). The loss on Y-haplotype of the binding site for SOX14 , a negative regulator of transcription, provides a plausible candidate mechanism of male-specific transcription of SOX1b , with additional potential changes in regulation suggested by gains and losses of binding sites for homeodomain transcription factors. Future functional experiments, such as those performed by Takehana et al. (2014) and/or investigation of 3D organization of chromatin (Shlyueva, Stampfel, & Stark, 2014; Zheng & Xie, 2019) will be required to clarify the role of SOX1b, SOX1a , and the putative SOX1b enhancer in gonad differentiation and the sex-determination pathway in Percichthyidae. Changes in gene regulation are a quick and efficient ways by which genes can acquire novel functions during sex chromosome turnover (Schartl et al., 2018).
Enhancers can be located a million bases away from their target genes (Hardison & Taylor, 2012), but studies of cis-regulatory regions ofSOX genes in other organisms generally report smaller distances. For example, in chicken, time-specific cranial and vagal/trunkSOX10 expression in embryo is regulated by two clusters of ~160 bp and ~267 bp located within a 3.5-Kb genomic fragment ~1-Kb downstream of SOX10(Betancur, Bronner-Fraser, & Sauka-Spengler, 2010). In zebrafish (Danio rerio ) a SOX10 cis-regulatory region is located within a 4.9-Kb region upstream of SOX10 (Carney et al., 2006). In Japanese flounder Paralichthys olivaceus , many putative transcription factor binding sites were identified within 2 Kb upstream of SOX1a and SOX1b , including those for factors regulating neurogenesis, pluripotent or stem-cell properties and diverse cellular processes (Gao et al., 2015).