Identifying novel genes from integrated SOX9 knockout RNAseq/SOX9 ChIPseq datasets — ASN Events

Identifying novel genes from integrated SOX9 knockout RNAseq/SOX9 ChIPseq datasets (#165)

Aleisha Symon 1 , Rowena Lavery 1 , Massilva Rahmoun 2 , Maisie Hands 1 , Dimuthu Alankarage 1 , Janelle Ryan 1 , Stefan Bagheri-Fam 1 , Francis Poulat 2 , Vincent Harley 1
  1. Hudson Institute of Medical Research, Clayton, VIC, Australia
  2. Institute of Human Genetics, University of Montpellier, Montpellier, France

SOX9 is a key transcription factor and Sertoli cell fate determinant responsible the differentiation of the gonad into a testis rather than an ovary during embryonic development. Human SOX9 mutations cause Disorders of Sex Development (DSD) in XX males (SOX9 duplications) and XY females (SOX9 mutations/deletions) however most such patients do not receive a definitive genetic diagnosis.  We hypothesise that SOX9 target genes are candidate DSD genes.

To identify Sox9 target genes, we undertook RNAseq analysis on mouse Sox9 knock-out gonads from embryonic day E13.5, when Sox9 is abated in an intact Sertoli cell environment (‘Sox9-responsive’ dataset). We also performed SOX9 ChIPseq on wildtype E13.5 mouse testes and E90 bovine testes (‘Sox9-bound’ dataset). 240 genes were downregulated in the Sox9 knockout testes, thus activated by Sox9.4293 SOX9 ChIPseq peaks were common to the mouse and bovine testis. As Sox9 is involved in the cell fate decisions of many cell types, it is likely that it has a signature of binding partners to enable regulation of genes specifically in Sertoli cells. Analysis of SOX9 bound regions revealed enrichment of Dmrt1 and Gata4 binding motifs, indicating a Sertoli cell signature for Sox9 binding.

Overlapping the RNAseq and conserved ChIPseq datasets identified 119 genes whose gonadal chromatin is bound by Sox9, and whose gene expression is upregulated by SOX9. Here, we describe the validation of two candidate SOX9 target genes by two different approaches.

The first approach is to analyse the embryonic gonads of knockout mice available to determine whether SOX9 mediates testis development via the candidate gene. One such gene is Nedd9, a gene expressed in the developing testis but not ovary. Analysis of Nedd9 knockout embryos revealed abnormal testis morphology, including a mis-located coelomic blood vessel, a male-specific feature of gonadal vasculature. The second approach is to antagonise proteins with available drugs in a hanging-drop culture model where E11.5 XY gonads will develop into testes with cords encompassing Sertoli cells after 3 days. In the presence of an inhibitor of TRPC3 XY gonads showed disrupted cord formation.

These approaches are proving useful for revealing unsuspected roles for Sox9 such as blood vessel and testis cord formation. DSD patients with variants in these genes are being sought.

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