DAX1 regulatory networks unveil conserved and potentially new functions

Abstract

DAX1 is an orphan nuclear receptor with actions in mammalian sex determination, regulation of steroidogenesis, embryonic development and neural differentiation. Conserved patterns of DAX1 gene expression from mammals to fish have been taken to suggest conserved function. In the present study, the European sea bass, Dicentrarchus labrax, DAX1 promoter was isolated and its conserved features compared to other fish and mammalian DAX1 promoters in order to derive common regulators and functional gene networks. Fish and mammalian DAX1 promoters share common sets of transcription factor frameworks which were also present in the promoter region of another 127 genes. Pathway analysis clustered these into candidate gene networks associated with the fish and mammalian DAX1. The networks identified are concordant with described functions for DAX1 in embryogenesis, regulation of transcription, endocrine development and steroid production. Novel candidate gene network partners were also identified, which implicate DAX1 in ion homeostasis and transport, lipid transport and skeletal development. Experimental evidence is provided supporting roles for DAX1 in steroid signalling and osmoregulation in fish. These results highlight the usefulness of the in silico comparative approach to analyse gene regulation for hypothesis generation. Conserved promoter architecture can be used also to predict potentially new gene functions. The approach reported can be applied to genes from model and non-model species.

Keywords: 18S; 18S ribosomal RNA; ACTB; ACTH; ANOVA; Arβ; CAH; CAM; CILP; CYP; Ca(2+)-calmodulin; DAX1; DBD; DMSO; DNA-binding domain; Dosage-sensitive sex reversal in Adrenal Hypoplasia Congenita critical region on X chromosome, gene 1; EF1α; ERα=ESR1; Erβ=ESR2; FSH; GO; Gene Ontology; Gene networks; GnRH; HPGA; HSD; IGF1; KCN; LH; MIS; MO; MS222; Müllerian inhibiting substance; N-(6-aminohexyl)-5- chloro-1-naphtalensulfonamide; Nr0B1; ORF; Osmoregulation; PKC; PRL; Pax; Promoter architecture; SCN; SF1; SHP=Nr0B2; SLC; StAR; Steroidogenesis; Steroidogenic acute regulatory protein; TFBS; TSS; Transcription binding sites; Transcription factor binding site; Tricaine methanesulfonate; W7; adrenocorticotrophic hormone; analysis of variance; androgen receptor beta; beta actin; cAMP; cartilage intermediate layer protein; congenital adrenal hypoplasia; cyclic adenosine monosphosphate; cytochrome P-450; dimethyl sulphoxide; elongation factor 1 alpha; follicle stimulating hormone; gonadotrophin releasing hormone; hydroxysteroid dehydrogenase; hypothalamic-pituitary-gonadal-adrenal axis; insulin-like growth factor I; luteinizing hormone; morpholino; oestrogen receptor alpha or 1; oestrogen receptor beta or 2; open reading frame; orphan nuclear receptor 0 B; paired box; potassium channel; prolactin; protein kinase C; qPCR; reverse-transcription quantitative polymerase chain reaction; runt-related transcription factor 2; runx 2; small heterodimer protein; sodium channel; solute carrier; steroidogenic factor 1; transcription start site.