Expression of NANOG and NANOGP8 in a variety of undifferentiated and differentiated human cells.
Expression of NANOG and NANOGP8 in a variety of undifferentiated and differentiated human cells.
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DOI：
10.1387/ijdb.103192sa
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发表时间：
2010
期刊：
影响因子：
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通讯作者：
Dominko T
中科院分区：
文献类型：
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作者：
Ambady S;Malcuit C;Kashpur O;Kole D;Holmes WF;Hedblom E;Page RL;Dominko T
转录因子NANOG对于维持胚胎干细胞的多能性至关重要。我们以前曾报道过NANOG在成人成纤维细胞中的表达;在这里，我们提出了一个更彻底的调查NANOG在一组分化和未分化的人类细胞中的表达。我们利用RT-PCR、qRT-PCR、克隆和测序、序列比对、限制性酶切消化、免疫细胞化学、蛋白质印迹和EMSA来研究NANOG在各种体细胞、转化细胞和干细胞表型中的表达。RT-PCR和qRT-PCR分析揭示了NANOG转录物在所有检查的细胞类型中的存在，尽管其数量级低于人胚胎干细胞。通过对几种细胞类型中表达的转录物进行单核苷酸多态性分析的进一步研究检测到NANOG假基因NANOGP 8，其是仅有的两个具有编码与胚胎NANOG（eNANOG）相似大小的蛋白质的潜力的NANOG假基因之一。我们的分析表明，尽管在几乎所有检查的细胞中检测到NANOG蛋白，但eNANOG和/或NANOGP 8转录物的表达以及蛋白质的亚细胞定位是细胞类型特异性的。另外，仅表达NANOGP 8的平滑肌细胞显示NANOG蛋白的核定位，表明NANOGP 8是可能作为转录因子起作用的蛋白质编码基因。最后，发现表达eNANOG和/或NANOGP 8的所有细胞类型都能够在体外结合NANOG共有序列。我们的结论是，eNANOG并不完全表达在未分化的细胞，这两个eNANOG和NANOGP 8可以作为转录因子在细胞类型特异性的方式。
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.