PhD Student, Monash University/ Murdoch Childrens research institute/Hudson Institute
Investigating how the genes involved in sex determination are activated at the right time and place
Disorders of Sex Development (DSDs) encompass a wide spectrum of conditions and often patients present with atypical gonads or genitalia, resulting in sex reversal or an intersex. The majority of DSD patients cannot be given an accurate diagnosis, which severely comprises their clinical management, leaving families unable to predict how their child will develop. While mutations in coding regions of genes known to be involved with making a testis or an ovary have been important in understanding some of the causes of DSD. Little attention has focussed on how the genes are turned on at the right time and place, or the regulatory regions of gonad genes. Recent reports by us and others of 46,XX testicular DSD patients (female to male sex reversal) with duplications and 46,XY gonadal dysgenesis patients (male to female sex reversal) carrying deletions upstream of SOX9, an important testis forming gene, suggest there is an important gonad specific regulatory region located in this area of the genome. Investigating the overlap between these patients has led to the discovery of novel testis specific regulatory switches, or enhancers, for SOX9. Using a comprehensive tiling luciferase assay and bioinformatic approaches, we have identified three novel enhancers upstream of SOX9. Enhancers that showed the strongest activity in vitro were used to generate transgenic mice. The enhancers showed expression in embryonic mouse gonads at the time of gonad differentiation. Indicating they are gonad specific. Analysis of these enhancers showed that in response to the transcription factors SF1/SRY and SF1/SOX9 they recapitulate the initiation, upregulation and maintenance of SOX9 expression exhibited during gonad differentiation. Our results strongly suggest that deletions or duplications (CNVs) of these enhancers may lead to DSD.
Abstract: Disorders of sex development (DSD), ranging in severity from mild genital abnormalities to complete sex reversal, represent a major concern for patients and their families. DSD are often due to disruption of the genetic programs that regulate gonad development. Although some genes have been identified in these developmental pathways, the causative mutations have not been identified in more than 50% 46,XY DSD cases. We used the Affymetrix Genome-Wide Human SNP Array 6.0 to analyse copy number variation in 23 individuals with unexplained 46,XY DSD due to gonadal dysgenesis (GD). Here we describe three discrete changes in copy number that are the likely cause of the GD. Firstly, we identified a large duplication on the X chromosome that included DAX1 (NR0B1). Secondly, we identified a rearrangement that appears to affect a novel gonad-specific regulatory region in a known testis gene, SOX9. Surprisingly this patient lacked any signs of campomelic dysplasia, suggesting that the deletion affected expression of SOX9 only in the gonad. Functional analysis of potential SRY binding sites within this deleted region identified five putative enhancers, suggesting that sequences additional to the known SRY-binding TES enhancer influence human testis-specific SOX9 expression. Thirdly, we identified a small deletion immediately downstream of GATA4, supporting a role for GATA4 in gonad development in humans. These CNV analyses give new insights into the pathways involved in human gonad development and dysfunction, and suggest that rearrangements of non-coding sequences disturbing gene regulation may account for significant proportion of DSD cases.
Pub.: 17 Mar '11, Pinned: 31 Aug '17
Abstract: Despite recent advances in our understanding, most cases of disorders of sex development (DSD) cannot be explained by mutations in known genes. In genome-wide screens of DSD patients, we and others detected duplications or deletions of potential regulatory regions of known or suspected DSD genes. It is therefore likely that a significant proportion of DSD cases may be explained by disrupted transcriptional regulation of gonad genes. Despite many recent technological advances, limited availability of relevant tissues - especially human embryonic material - can make the identification of long-range regulatory elements extremely difficult. In an attempt to overcome this limitation, we evaluated the usefulness of publicly available DNaseI hypersensitivity data from the Roadmap Epigenomics Project. For this feasibility study we used the 'gene desert' around the SOX9 gene and a genomic locus downstream of GATA4. Over 60% of our selected candidate regions had significant enhancer activity in luciferase assays. We show that this approach facilitates the detection of strong enhancer candidates worthy of further analysis.
Pub.: 15 Apr '16, Pinned: 31 Aug '17
Abstract: During mouse sex determination, transient expression of the Y-linked gene Sry up-regulates its direct target gene Sox9, via a 3.2 kb testis specific enhancer of Sox9 (TES), which includes a core 1.4 kb element, TESCO. SOX9 activity leads to differentiation of Sertoli cells, rather than granulosa cells from the bipotential supporting cell precursor lineage. Here, we present functional analysis of TES/TESCO, using CRISPR/Cas9 genome editing in mice. Deletion of TESCO or TES reduced Sox9 expression levels in XY fetal gonads to 60 or 45% respectively relative to wild type gonads, and reduced expression of the SOX9 target Amh. Although human patients heterozygous for null mutations in SOX9, which are assumed to have 50% of normal expression, often show XY female sex reversal, mice deleted for one copy of Sox9 do not. Consistent with this, we did not observe sex reversal in either TESCO-/- or TES-/- XY embryos or adult mice. However, embryos carrying both a conditional Sox9 null allele and the TES deletion developed ovotestes. Quantitative analysis of these revealed levels of 23% expression of Sox9 compared to wild type, and a significant increase in the expression of the granulosa cell marker Foxl2. This indicates that the threshold in mice where sex reversal begins to be seen is about half that of the ~50% levels predicted in humans. Our results demonstrate that TES/TESCO is a crucial enhancer regulating Sox9 expression in the gonad, but point to the existence of additional enhancers that act redundantly.
Pub.: 04 Jan '17, Pinned: 31 Aug '17