Cis-Regulatory Control of Mammalian Sex Determination

Abstract

Sex determination is the process by which an initial bipotential gonad adopts either a testicular or ovarian cell fate. The inability to properly complete this process leads to a group of developmental disorders classified as disorders of sex development (DSD). To date, dozens of genes were shown to play roles in mammalian sex determination, and mutations in these genes can cause DSD in humans or gonadal sex reversal/dysfunction in mice. However, exome sequencing currently provides genetic diagnosis for only less than half of DSD patients. This points towards a major role for the non-coding genome during sex determination. In this review, we highlight recent advances in our understanding of non-coding, cis-acting gene regulatory elements and discuss how they may control transcriptional programmes that underpin sex determination in the context of the 3-dimensional folding of chromatin. As a paradigm, we focus on the Sox9 gene, a prominent pro-male factor and one of the most extensively studied genes in gonadal cell fate determination.

Keywords: 3D genome organisation; DSD; Enhancers; Ovary; Regulatory elements; Sex determination; Sox9; TAD; Testis.

Fig. 1

Organisation of the genome in 3 dimensions. a DNA is organised in the nucleus into chromosomal territories with each chromosome located in a specific location in a non-random fashion. b DNA within the nucleus is generally subdivided into 2 types of compartments: an active, Type A compartment, mostly localised at the centre of the nucleus, and an inactive, Type B compartment, mostly localised at the cell periphery termed the nuclear lamina/envelope. c Chromosomes are divided into megabase-scale units called Topologically Associating Domains (TADs). TADs are characterised by high levels of intra-TAD interactions and are separated by TAD boundaries over which interactions are relatively depleted. These boundaries are enriched for CTCF binding sites. TADs usually contain several genes and their regulatory elements and can be identified using Hi-C techniques. d Within TADs, regulatory elements can be brought near their target genes via chromosomal loop formation. e DNA is wrapped around histones to form nucleosomes. Enhancers and gene promoters are characterized by an accessible/open DNA signature due to binding of transcription factors at their target sites. Histone tails can undergo post-translational modifications including acetylation, phosphorylation, and methylation; this can attract or repel specific chromosomal proteins, thereby directly affecting genome function.

Fig. 2

Cascade of events leading to mammalian sex determination. The gonads originate from a common precursor called the bipotential gonad. Several genes including wt1, Sf1, Gata4, and Lhx9 are expressed in early gonads and have been implicated in controlling their function. In mammals, males contain XY sex chromosomes, and the expression of the Sry gene, located on the Y chromosome, initiates testis development at E11.5. SRY acts with SF1 to upregulate the expression of Sox9. Several regulatory loops exist that maintain high levels of Sox9 expression throughout life in Sertoli cells. SOX9 is thought to activate Sox8 expression and other testis genes while repressing pro-female genes such as ß-catenin. Females contain XX sex chromosomes and thus lack the Sry gene. In the absence of SRY, the activity of RSPO1 along with WNT4 leads to stabilization of ß-catenin which, together with FOXL2, is believed to initiate the expression of ovarian genes while repressing pro-male genes such as Sox9.

Fig. 3

The regulatory landscape of the Sox9 gene. In the mouse, Sox9 is located on chromosome 11, with the Kcnj2 gene located 1.7 Mb upstream of Sox9. Five gonad-specific enhancers in the Sox9 locus were identified termed Enh8, Enh13, Enh14, TES, and Enh32. Enh13 was shown to have a critical role in the regulation of Sox9 at the sex determination stage. The other enhancers may function as shadow enhancers with redundant functions. We speculate that Enh13, Enh14, TES, and Enh32 are bound by SRY, SF1, SOX9, and maybe other pro-male TFs and are brought into proximity of the Sox9 promoter via DNA looping, leading to upregulation of Sox9 and hence Sertoli cell fate. In the female, we hypothesise that these enhancers do not interact with the Sox9 promoter; instead Enh8, bound by FOXL2 and other pro-female TFs, is in close proximity to the Sox9 promoter, which represses Sox9 expression and promotes granulosa cell fate acquisition.