Epigenetic regulation of sex determination in the mouse

Abstract

Sex determination is a valuable model to study cell fate decisions. Prior to sex determination, the undifferentiated gonad forms as a bipotential organ, able to form either a testis or an ovary. Commitment to either the testis or ovary pathway is under the control of the gene Sry on the Y chromosome. Sry drives the initiation of the testis pathway, causing the supporting cell lineage to differentiate into Sertoli cells. A complex network of factors maintains supporting cell fate by activating one differentiation program while simultaneously repressing the alternative program. Along with the network of transcription factors required to maintain differentiation, increasing evidence suggests that sex-determination is epigenetically regulated. The goal of this work is to better characterize the role for epigenetics during sex determination, including histone modifications and their erasers as well as larger-order chromatin structure, and put these results in the broader context of how mechanisms of sex determination arose during evolution. Previous studies have shown that mammalian Sry is regulated epigenetically, notably through the removal of repressive histone modifications (Kuroki et al., 2013). In temperature-dependent sex determination systems, such as in the red-eared slider turtle, removal of the repressive histone modification H3K27me3 by the demethylase KDM6B is required downstream of temperature to activate the male-determining factor, Dmrt1. We hypothesized that a similar de-repression mechanism may be in place in mammals. Removal of the H3K27me3 mark may be critical for gene activation. While passive loss of this mark through proliferation could explain the loss of H3K27me3 in XY cells, this cannot explain loss of H3K27me3 in XX cells, which are in cell cycle arrest throughout this period of development. There are two characterized H3K27 demethylases in both mice and humans: Kdm6b and Utx (Kdm6a). These demethylases are part of the Jumonji domain-containing family of proteins and have been shown to specifically act on H3K27me2 and H3K27me3 marks (Swigut &Wysocka, 2007; Shpargel et al., 2012). A homologue of Utx, called Uty, exists on the Y chromosome, but a mutation has eliminated its demethylase activity. Both Kdm6b and Utx have been implicated as critical factors in early embryonic development through both demethylase-dependent and demethylase-independent mechanisms (Shpargel et al., 2012). Expression of Dmrt1 in the red-eared slider turtle is activated by KDM6B, whose expression is controlled by the temperature of incubation of the egg. Kdm6b is activated in red-eared slider turtles (T. scripta) at the male-promoting temperature and removes H3K27me3 from Dmrt1. In the absence of KDM6B, turtles exhibit male-to-female sex reversal, and the gonads will develop as ovaries even at the male-promoting temperature (Ge et al., 2018; Weber et al., 2020). This finding led us to hypothesize that proteins which remove the repressive H3K27me3 histone modification may be required for activation of the testis-determining pathway in mammals as well. Using mutant mouse models for Kdm6b, Utx, and Uty we demonstrate their roles in sex determination through immunofluorescent imaging, RNA expression analyses, and chromatin immunoprecipitation. Furthermore, we also report experiments to determine the role of UTX and UTY during mouse sex determination. Consistent with findings in turtles, here we report Kdm6b is necessary to establish the male pathway in mice through regulating the expression of Sry. UTX does not appear to have a critical role in the establishment or maintenance of the downstream pathway that controls supporting cell fate. UTY is a candidate male sex determining gene; however, the partial sex reversal phenotype is not fully penetrant. These results are significant, as they identify Kdm6b as a critical epigenetic regulator of the top of the sex-determining cascade in both mice and turtles, which determine sex by widely different mechanisms. The results are hypothesis generating for novel research on the evolution of upstream sex determining factors, such as Sry in mice and Dmrt1 in turtles, and how these factors are regulated.