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.
Ying
Ming
M
Department of Genetics and Center for Developmental Biology, College of Life Science, Wuhan University, Wuhan 430072, China.
Chen
Bo
B
Tian
Yihao
Y
Hou
Yu
Y
Li
Qin
Q
Shang
Xuan
X
Sun
Jinhua
J
Cheng
Hanhua
H
Zhou
Rongjia
R
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D019943:Amino Acid Substitution; D000818:Animals; D029742:Caenorhabditis elegans Proteins / Q000235:genetics; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics; D029721:Drosophila Proteins / Q000235:genetics; D006801:Humans; D008297:Male; D051379:Mice; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D012708:Sertoli Cells / Q000166:cytology / Q000378:metabolism*; D012733:Sex Differentiation / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D016335:Zinc Fingers / Q000235:genetics; D028961:beta Karyopherins / Q000378:metabolism
D029742:Caenorhabditis elegans Proteins; C110547:DMRT1 protein; D004268:DNA-Binding Proteins; C070369:DSX protein, Drosophila; D029721:Drosophila Proteins; C424166:KPNB1 protein, human; C110548:Mab-3 protein, C elegans; D019913:Nuclear Localization Signals; D014157:Transcription Factors; D028961:beta Karyopherins