A tale of two fates: the role of histone modifications in establishing bipotentiality

Abstract

Sex determination and differentiation is an essential process all mammals must undergo to reproduce. This process in mammals is initiated through the development of a bipotential tissue, the urogenital ridge (UGR), which differentiates into two morphologically different tissues; testes in male and ovaries in females. This is a genetically controlled process with the presence or absence of the Sry gene determining the sexual fate of an embryo. Many genetic pathways have been implicated in the development of the bipotential UGR but the epigenetic regulatory mechanisms involved are relatively unknown. Previously, to obtain a global view of genes active or actively repressed in the mouse UGR at E11.5, chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq) had been performed for active (H3K4me3) and repressive (H3K27me3) histone modifications. Analysis showed overrepresentation of genes linked to the Wnt signaling pathway targeted for epigenetic regulation from both histone modification data sets (Yang and Wilson, unpublished). Thus, we aimed to investigate the role of epigenetic mechanisms to histone proteins in the UGR prior to differentiation into a testis or ovary, on a set of targets involved in the Wnt signaling pathway. Four enrichment areas were chosen for ChIP-seq validation, Wnt10, Fzd6, Wnt4 and Fzd8. Significant enrichment for Wnt10a in association with H3K4me3 modifications and Wnt10a and Fzd6 in association with H3K27me3 modifications was observed through ChIP-qPCR (p<0.05). Expression patterns of Fzd6 and Wnt4 show stark differences in the UGR and surrounding mesonephros. Fzd6 expression was detected in the Wolffian duct and mesonephric tubules but not in the UGR itself whereas Wnt4 was restricted to the UGR. Further analysis of H3K4me3 and H3K27me3 modifications showed overlapping patterns of the distribution of these modifications in the UGR and mesonephric tubules Confirmation of Wnt signaling factors and their association with histone modifications when combined with the spatial patterns of the histone modifications, implements a mechanism by which the Wnt signaling pathway is regulated in the bipotential gonad. Motif analysis of regulatory gene regions enriched with the H3K27me3 histone modification (Yang and Wilson, unpublished) suggested that many of these genes are regulated by known pioneer factors, EBF1 and HNF6. RT-PCR confirmed the presence of EBF1 in the bipotential gonad at E11.5, however expression of HNF6 was not detected. Bioinformatic analysis using DAVID uncovered enrichment of genes involved in the MAPK signaling pathway along with processes relating to development and morphogenesis. The proposed model for the role of histone modifications in the regulation of gonad development is that they play an important role in maintaining the bipotential state of the gonad. Through the binding of EBF1, the gonad is kept in a poised transcriptional state, awaiting sex-specific signals to activate or repress pathways including the Wnt and MAPK signaling pathways