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

Human DMRT 1 (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 DMRT 1 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-beta 1 binds directly to DMRT 1 via the DM domain, mediating its nuclear import. Co-immunoprecipitation analysis confirms the interaction of mouse Dmrt 1 in Sertoli cells with importin-beta 1 in vivo. In addition, in vitro docking or nuclear transport assay in digitonin-permeabilized cells shows that DMRT 1 is docked at the nuclear pore complex (NPC) or accumulated in the nucleus when importin-beta 1, but not importin-alpha 1 added. Furthermore, transduction of anti-importin-alpha 1 antibody into live Sertoli cells effectively inhibits DMRT 1 nuclear import. These results suggest that zinc finger domain of DMRT 1 functions as a nuclear localization signal and DMRT 1 is transported into the nucleus in an importin beta 1-mediated manner. Thus, effective nuclear import of DMRT 1 and its interaction with importin-beta 1 insure the nuclear retention of the DMRT 1 and further exertion of its influence on downstream targets in the cascade of sexual development. (c) 2007 Elsevier B.V. All rights reserved.