8 research outputs found
Core sex determining gene, SOX9, regulates novel target genes during testis development
No full textSOX9 is a transcriptional activator, expressed in the XY gonads, that plays an essential role during male sex development. In humans, dysregulation of SOX9 is accompanied by sex reversal (46, XY female, 46, XX male). In mice, early or late loss of SOX9 leads to sex reversal or male infertility, respectively. SOX9 functions as the key regulator of testis development and fertility, driving differentiation of Sertoli cells directly, and the development and maintenance of other testicular cell types by juxtacrine and paracrine signalling. However, the mechanisms by which SOX9 regulate downstream testicular functions remain poorly understood. The use of transcriptomic analysis has become a useful tool in the exploration of transcription factor function. Here, we have utilised human Sertoli-like NT2/D1 cells to transiently over-express SOX9 and performed microarray analysis of the RNA. In order to uncover genes that are positively regulated by SOX9, genes that increased in expression were pursued further. One such gene, Ets variant factor 5 (ETV5), is explored in chapter 3. We show that SOX9 binds to and activates the proximal ETV5 promoter via a conserved SOX site in vitro. Examination of Etv5 expression during mouse embryonic development indicates that Etv5 is strongly expressed by the XY gonads. ETV5 protein localises to Sertoli cell nuclei, as well as germ cells and interstitial cells of the embryonic testis. In two Sox9 KO XY mouse models, Etv5 expression is decreased before and after sex determination. Results of this chapter reveal SOX9 as a key regulator of ETV5 expression and therefore of ETV5 mediated functions in the testis. Chapter 4 describes the investigation of desert hedgehog (DHH) as a target gene of SOX9. DHH is an important signalling factor for testis organogenesis and maintenance of fertility. We show that DHH is a target of SOX9 transcriptional activity in human and mouse Sertoli cell lines. In the human Sertoli-like cell line, NT2/D1, transfection of a mutant SOX9 associated with a 46,XY DSD patient (SOX9-A158T) could not increase DHH expression. Chromatin immunoprecipitation revealed that SOX9 was bound to the endogenous DHH promoter at a conserved SOX consensus DNA binding site. Dhh expression was down-regulated in foetal XY gonads of two Sox9 knock-out mouse models before and after sex determination. Conversely, ectopic expression of Sox9 in XX mouse gonads resulted in Dhh up-regulation. Findings of this chapter suggest that SOX9 is a transcriptional regulator of Dhh expression in Sertoli cells of the developing testis and thereby controls DHH-mediated paracrine signalling required for the differentiation of multiple cell types in the testis. That SOX9 mediates its action via DHH is consistent with the overlapping reproductive phenotypes observed in 46,XY DSD patients with mutations in either gene. In conclusion, the work within this thesis details the regulation of novel target genes of SOX9 and adds to the current knowledge regarding molecular mechanisms of testis development. Ultimately, it is hoped that the identification of these target genes would contribute to the knowledge of human sex development and human disorders, facilitating easier diagnosis of DSD
Dataset of differentially expressed genes from SOX9 over-expressing NT2/D1 cells
No full textAbstractThe data presents the genes that are differentially up-regulated or down-regulated in response to SOX9 in a human Sertoli-like cell line, NT2/D1. The dataset includes genes that may be implicated in gonad development and are further explored in our associated article, “SOX9 Regulates Expression of the Male Fertility Gene Ets Variant Factor 5 (ETV5) during Mammalian Sex Development” (D. lankarage, R. Lavery, T. Svingen, S. Kelly, L.M. Ludbrook, S. Bagheri-Fam, et al., 2016) [1]. The necessity of SOX9 for male sex development is evident in instances where SOX9 is lost, as in 46, XY DSD where patients are sex reversed or in mouse knock-out models, where mice lacking Sox9 are sex reversed. Despite the crucial nature of this transcriptional activator, downstream target genes of SOX9 remain largely undiscovered. Here, we have utilized NT2/D1 cells to transiently over-express SOX9 and performed microarray analysis of the RNA. Microarray data are available in the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-3378
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Functional characterization of a novel PBX1 de novo missense variant identified in a patient with syndromic congenital heart disease.
No full textPre-B cell leukemia factor 1 (PBX1) is an essential developmental transcription factor, mutations in which have recently been associated with CAKUTHED syndrome, characterized by multiple congenital defects including congenital heart disease (CHD). During analysis of a whole-exome-sequenced cohort of heterogeneous CHD patients, we identified a de novo missense variant, PBX1:c.551G>C p.R184P, in a patient with tetralogy of Fallot with absent pulmonary valve and extra-cardiac phenotypes. Functional analysis of this variant by creating a CRISPR-Cas9 gene-edited mouse model revealed multiple congenital anomalies. Congenital heart defects (persistent truncus arteriosus and ventricular septal defect), hypoplastic lungs, hypoplastic/ectopic kidneys, aplastic adrenal glands and spleen, as well as atretic trachea and palate defects were observed in the homozygous mutant embryos at multiple stages of development. We also observed developmental anomalies in a proportion of heterozygous embryos, suggestive of a dominant mode of inheritance. Analysis of gene expression and protein levels revealed that although Pbx1 transcripts are higher in homozygotes, amounts of PBX1 protein are significantly decreased. Here, we have presented the first functional model of a missense PBX1 variant and provided strong evidence that p.R184P is disease-causal. Our findings also expand the phenotypic spectrum associated with pathogenic PBX1 variants in both humans and mice
Functional characterization of a novel PBX1 de novo missense variant identified in a patient with syndromic congenital heart disease.
No full textQuantitative trait and transcriptome analysis of genetic complexity underpinning cardiac interatrial septation in mice using an advanced intercross line
No full textUnlike single-gene mutations leading to Mendelian conditions, common human diseases are likely to be emergent phenomena arising from multilayer, multiscale, and highly interconnected interactions. Atrial and ventricular septal defects are the most common forms of cardiac congenital anomalies in humans. Atrial septal defects (ASD) show an open communication between the left and right atria postnatally, potentially resulting in serious hemodynamic consequences if untreated. A milder form of atrial septal defect, patent foramen ovale (PFO), exists in about one-quarter of the human population, strongly associated with ischaemic stroke and migraine. The anatomic liabilities and genetic and molecular basis of atrial septal defects remain unclear. Here, we advance our previous analysis of atrial septal variation through quantitative trait locus (QTL) mapping of an advanced intercross line (AIL) established between the inbred QSi5 and 129T2/SvEms mouse strains, that show extremes of septal phenotypes. Analysis resolved 37 unique septal QTL with high overlap between QTL for distinct septal traits and PFO as a binary trait. Whole genome sequencing of parental strains and filtering identified predicted functional variants, including in known human congenital heart disease genes. Transcriptome analysis of developing septa revealed downregulation of networks involving ribosome, nucleosome, mitochondrial, and extracellular matrix biosynthesis in the 129T2/SvEms strain, potentially reflecting an essential role for growth and cellular maturation in septal development. Analysis of variant architecture across different gene features, including enhancers and promoters, provided evidence for the involvement of non-coding as well as protein-coding variants. Our study provides the first high-resolution picture of genetic complexity and network liability underlying common congenital heart disease, with relevance to human ASD and PFO
Myhre syndrome is caused by dominant-negative dysregulation of SMAD4 and other co-factors
No full textMyhre syndrome is a connective tissue disorder characterized by congenital cardiovascular, craniofacial, respiratory, skeletal, and cutaneous anomalies as well as intellectual disability and progressive fibrosis. It is caused by germline variants in the transcriptional co-regulator SMAD4 that localize at two positions within the SMAD4 protein, I500 and R496, with I500 V/T/M variants more commonly identified in individuals with Myhre syndrome. Here we assess the functional impact of SMAD4-I500V variant, identified in two previously unpublished individuals with Myhre syndrome, and provide novel insights into the molecular mechanism of SMAD4-I500V dysfunction. We show that SMAD4-I500V can dimerize, but its transcriptional activity is severely compromised. Our data show that SMAD4-I500V acts dominant-negatively on SMAD4 and on receptor-regulated SMADs, affecting transcription of target genes. Furthermore, SMAD4-I500V impacts the transcription and function of crucial developmental transcription regulator, NKX2-5. Overall, our data reveal a dominant-negative model of disease for SMAD4-I500V where the function of SMAD4 encoded on the remaining allele, and of co-factors, are perturbed by the continued heterodimerization of the variant, leading to dysregulation of TGF and BMP signaling. Our findings not only provide novel insights into the mechanism of Myhre syndrome pathogenesis but also extend the current knowledge of how pathogenic variants in SMAD proteins cause disease
Functional genomics and gene-environment interaction highlight the complexity of congenital heart disease caused by Notch pathway variants
No full textCongenital heart disease (CHD) is the most common birth defect and brings with it significant mortality and morbidity. The application of exome and genome sequencing has greatly improved the rate of genetic diagnosis for CHD but the cause in the majority of cases remains uncertain. It is clear that genetics, as well as environmental influences, play roles in the aetiology of CHD. Here we address both these aspects of causation with respect to the Notch signalling pathway. In our CHD cohort, variants in core Notch pathway genes account for 20% of those that cause disease, a rate that did not increase with the inclusion of genes of the broader Notch pathway and its regulators. This is reinforced by case-control burden analysis where variants in Notch pathway genes are enriched in CHD patients. This enrichment is due to variation in NOTCH1. Functional analysis of some novel missense NOTCH1 and DLL4 variants in cultured cells demonstrate reduced signalling activity, allowing variant reclassification. Although loss-of-function variants in DLL4 are known to cause Adams-Oliver syndrome, this is the first report of a hypomorphic DLL4 allele as a cause of isolated CHD. Finally, we demonstrate a gene-environment interaction in mouse embryos between Notch1 heterozygosity and low oxygen- or anti-arrhythmic drug-induced gestational hypoxia, resulting in an increased incidence of heart defects. This implies that exposure to environmental insults such as hypoxia could explain variable expressivity and penetrance of observed CHD in families carrying Notch pathway variants
A metabolic signature for NADSYN1-dependent congenital NAD deficiency disorder
No full textNicotinamide adenine dinucleotide (NAD) is essential for embryonic development. To date, biallelic loss-of-function variants in 3 genes encoding nonredundant enzymes of the NAD de novo synthesis pathway — KYNU, HAAO, and NADSYN1 — have been identified in humans with congenital malformations defined as congenital NAD deficiency disorder (CNDD). Here, we identified 13 further individuals with biallelic NADSYN1 variants predicted to be damaging, and phenotypes ranging from multiple severe malformations to the complete absence of malformation. Enzymatic assessment of variant deleteriousness in vitro revealed protein domain–specific perturbation, complemented by protein structure modeling in silico. We reproduced NADSYN1-dependent CNDD in mice and assessed various maternal NAD precursor supplementation strategies to prevent adverse pregnancy outcomes. While for Nadsyn1+/– mothers, any B3 vitamer was suitable to raise NAD, preventing embryo loss and malformation, Nadsyn1–/– mothers required supplementation with amidated NAD precursors (nicotinamide or nicotinamide mononucleotide) bypassing their metabolic block. The circulatory NAD metabolome in mice and humans before and after NAD precursor supplementation revealed a consistent metabolic signature with utility for patient identification. Our data collectively improve clinical diagnostics of NADSYN1-dependent CNDD, provide guidance for the therapeutic prevention of CNDD, and suggest an ongoing need to maintain NAD levels via amidated NAD precursor supplementation after birth.</p
