Nuclear import of human sexual regulator DMRT1 is mediated by importin-beta

Human DMRT1 (Doublesex-Mab3-Related Transcription factor 1) encodes a male-specific transcriptional regulator with a conserved zinc-finger-like DNA-binding domain, so called DM domain, which is similar to male sexual regulatory genes doublesex of Drosophila and mab-3 of Caenorhabditis elegans. As a key transcription factor critical to sex determination and differentiation, however, human DMRT1 nuclear import mechanism remains unknown. We have identified a functional nuclear localization signal (NLS) located between the two intertwined zinc-binding sites of the DM domain. Site-directed mutagenesis indicates that K92 and R93 within the DM domain are critical for DMRT1 nuclear localization. Analysis of deletion mutants shows that importin-beta1 binds directly to DMRT1 via the DM domain, mediating its nuclear import. Co-immunoprecipitation analysis confirms the interaction of mouse Dmrt1 in Sertoli cells with importin-beta1 in vivo. In addition, in vitro docking or nuclear transport assay in digitonin-permeabilized cells shows that DMRT1 is docked at the nuclear pore complex (NPC) or accumulated in the nucleus when importin-beta1, but not importin-alpha1 added. Furthermore, transduction of anti-importin-beta1 antibody into live Sertoli cells effectively inhibits DMRT1 nuclear import. These results suggest that zinc finger domain of DMRT1 functions as a nuclear localization signal and DMRT1 is transported into the nucleus in an importinbeta1-mediated manner. Thus, effective nuclear import of DMRT1 and its interaction with importin-beta1 insure the nuclear retention of the DMRT1 and further exertion of its influence on downstream targets in the cascade of sexual development.