Identification of a putative nuclear export signal motif in human NANOG homeobox domain.

NANOG is a homeobox-containing transcription factor that plays an important role in pluripotent stem cells and tumorigenic cells. To understand how nuclear localization of human NANOG is regulated, the NANOG sequence was examined and a leucine-rich nuclear export signal (NES) motif ((125)MQELSNILNL(134)) was found in the homeodomain (HD). To functionally validate the putative NES motif, deletion and site-directed mutants were fused to an EGFP expression vector and transfected into COS-7 cells, and the localization of the proteins was examined. While hNANOG HD exclusively localized to the nucleus, a mutant with both NLSs deleted and only the putative NES motif contained (hNANOG HD-ΔNLSs) was predominantly cytoplasmic, as observed by nucleo/cytoplasmic fractionation and Western blot analysis as well as confocal microscopy. Furthermore, site-directed mutagenesis of the putative NES motif in a partial hNANOG HD only containing either one of the two NLS motifs led to localization in the nucleus, suggesting that the NES motif may play a functional role in nuclear export. Furthermore, CRM1-specific nuclear export inhibitor LMB blocked the hNANOG potent NES-mediated export, suggesting that the leucine-rich motif may function in CRM1-mediated nuclear export of hNANOG. Collectively, a NES motif is present in the hNANOG HD and may be functionally involved in CRM1-mediated nuclear export pathway.

Regulation of OCT4 gene expression by liver receptor homolog-1 in human embryonic carcinoma cells.

We demonstrate the regulation of OCT4 gene expression mediated by liver receptor homolog-1 (LRH-1) in human embryonic carcinoma cells. LRH-1 and OCT4 are co-expressed in undifferentiated NCCIT cells and decreased during retinoic acid-induced differentiation. Dose-dependent overexpression of LRH-1 transactivated the OCT4 promoter activity and its dominant negative form with a deletion of activation function-2 motif reduced the activity even in the presence of LRH-1. The OCT4 promoter contains potent three LRH-1 binding sites; one within conserved region (CR) 1 and two within CR2. Mutagenesis of each binding site affected the decrease in OCT4 promoter activity and the 2nd binding site mutant most significantly reduced the transcriptional activity, compared to that of 1st and 3rd binding site mutants, respectively. Simultaneous disruption of 2nd and 3rd binding sites led to significant down-regulation of the activity even in the presence of 1st binding site-containing CR1. Moreover, mutation of each binding element within native or exogenous minimal promoter-driven CR1 or CR2 also decreased the promoter activity to some different extent, suggesting that three binding elements may be implicated in the induction of the full-activity of OCT4 promoter. In vivo binding assay revealed the 2nd and 3rd binding motifs within CR2 were more enriched than the 1st one within CR1. Taken together, our study indicates that LRH-1 acts as a transcriptional activator in the regulation of OCT4 gene expression through the cooperative interaction with three binding sites directly or/and indirectly.

Regulation of human growth and differentiation factor 3 gene expression by NANOG in human embryonic carcinoma NCCIT cells.

We investigated transactivation by NANOG in regulating growth and differentiation factor 3 (GDF3) expression in NCCIT cells. GDF3 expression was affected by shRNA-mediated downregulation and by exogenous overexpression of NANOG specifically, as well as by retinoic acid-mediated differentiation. GDF3 transcription was activated by NANOG, and the upstream region (-183 to -1) was sufficient to induce minimal transcriptional activity. Moreover, NANOG binds to the GDF3 minimal promoter in vivo and the transcriptional activity is mediated by NANOG transactivation domain. This study provides the first evidence that NANOG is a transcriptional activator of the expression of the oncogenic growth factor GDF3 in embryonic carcinoma cells.