Expression of NANOG and NANOGP8 in a variety of undifferentiated and differentiated human cells.

The transcription factor NANOG is essential for maintaining pluripotency in embryonic stem cells. We have previously reported the expression of NANOG in adult human fibroblasts; here we present a more thorough investigation into the expression of NANOG in a panel of both differentiated and undifferentiated human cells. We utilize RT-PCR, qRT-PCR, cloning and sequencing, sequence alignment, restriction digestion, immunocytochemistry, Western blotting, and EMSA to investigate expression of NANOG in a variety of somatic, transformed and stem cell phenotypes. RT-PCR and qRT-PCR analysis revealed the presence of NANOG transcripts in all the cell types examined, albeit at magnitudes lower than human embryonic stem cells. Further investigation by single nucleotide polymorphism analysis of expressed transcripts in several cell types detected a NANOG pseudogene, NANOGP8, one of only two NANOG pseudogenes with the potential of encoding a similar size protein to embryonic NANOG (eNANOG). Our analysis demonstrates that although the NANOG protein is detected in nearly all cells examined, expression of the eNANOG and/or NANOGP8 transcript as well as the sub-cellular localization of the protein is cell type-specific. Additionally, smooth muscle cells, which express exclusively NANOGP8, display nuclear localization of NANOG protein, indicating that NANOGP8 is a protein coding gene possibly functioning as a transcription factor. Lastly, all cell types expressing eNANOG and/or NANOGP8 were found to be capable of binding a NANOG consensus sequence in vitro. We conclude that eNANOG is not exclusively expressed in undifferentiated cells and that both eNANOG and NANOGP8 may function as transcription factors in a cell type-specific manner.