Nanog is a homeobox-containing transcriptional factor required for maintaining the pluripotent state of stem cells. We investigated the nuclear localization signal (NLS) motif required for human Nanog (hNanog) nuclear import. Mutation analysis revealed that a mutant containing only the homeodomain (HD) was exclusively localized to the nucleus, while other mutants containing either the N- or C-terminal region (NR or CR) had impaired nuclear localization. In addition, NR and CR were exclusively localized to the nucleus when they were fused to the HD, indicating that complete nuclear localization is only driven by functional NLS motif(s) within the HD. Furthermore, partial loss of HD led to the incomplete localization of hNanog, suggesting that the intact HD is required for hNanog nuclear import. A series of deletion and site-directed mutagenesis within the HD revealed that two basic NLS motifs are located at the N-terminus and C-terminus of the HD and that both motifs are required for complete hNanog nuclear localization.
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