THE ROLE OF DMRT1 IN POSTNATAL TESTIS DIFFERENTIATION

Abstract

DMRT1 (doublesex and maleabnormal-3 related transcription factor-1) is a transcription regulator that is expressed in Sertoli cells and germ cells of the testis, where it directs mammalian postnatal testis differentiation. It is currently the only mammalian gene within the sex determination and sex differentiation genetic pathways that is evolutionary conserved and shares a cysteine-rich DNA binding motif (DM-domain) with doublesex in the fruit fly and maleabnormal-3 in the roundworm. The work presented in this dissertation revealed numerous novel roles of DMRT1, including Sertoli cell-specific rescue, structural function in the testis, cell-specific pathways and targets, and a probable regulation by NANOS2 in germ cells; thus providing important insight into how DMRT1 regulates testis development. Recombinogenic engineering was invaluable in the generation of a novel Sertoli cell-rescue (Dmrt1-/-;tg) mouse model, which is a DMRT1-deficient germ cell model that was produced by breeding Dmrt1-null (Dmrt1-/-) mice with Wt1-Dmrt1 transgenic mice. The Wt1-Dmrt1 transgenic mice express a rat Dmrt1 cDNA in gonadal supporting cells by directing it from the Wilms' tumor locus carried on a yeast artificial chromosome transgene. Characterization of the Sertoli cell rescue mice revealed Sertoli cell-specific function of DMRT1 in the maintenance of seminiferous tubule morphology, autonomous and non-autonomous dose-dependent gene regulation, Sertoli cell differentiation, maintenance of testis size and sperm progressive motility, organization of testis-specific adherens junctions and maturation of Leydig cells and thus regulation of testosterone production. Furthermore, cell-specific direct and indirect pathways and targets regulated by DMRT1 in Sertoli cells and germ cells were identified using global gene expression profiles combined with ChIP-Seq. Analyses of the gene profiles identified potential functions for DMRT1 in vesicular transport, ubiquitination and RNA binding. Similarly, analysis of ChIP-Seq data identified 150 targets with the majority of DMRT1 binding sites located in introns, indicating that DMRT1 preferentially binds to distal regulatory regions. Finally, direct protein-protein interaction was shown between DMRT1 and NANOS2, an evolutionary conserved RNA-binding protein that plays a key role in male germ cell differentiation, by inhibiting entry into meiosis. NANOS2 was shown to act upstream, interact, partially co-localize and repress the in vitro transcriptional activity of DMRT1 in germ cells. Overall, these experiments uncovered several questions and new cell-specific roles of DMRT1 in testis differentiation. These findings and their implications are discussed further in this dissertation within the general scope of reproductive biology and the more defined scope of postnatal testis differentiation.