Identification Of Nanog Gene Cis-regulatory Element In Undifferentiated Human Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BM-MSCs) can self-renew and have the pluripotency to differentiate into many kinds of cells. Because of their features such as easy isolation, high proliferation and other advantages, they can be extensively used for many applications, including tissue engineering, cell transplantation and gene therapy. So far, little is known about the mechanisms for control of BM-MSC growth, and it is necessary to understand how BM-MSC growth and differentiation is regulated.This study tried to identify the cis-regulatory element from Nanog gene and determine if the key transcription factor Oct4 and Sox2 can regulate Nanog gene expression. Dual luciferase reporter system showed that Nanog gene fragment (from -380bp to +24bp) could be activated in undifferentiated BM-MSCs and could activate luciferase gene expression, but this Nanog gene fragment could not be activated in osteoblasts differentiated from BM-MSCs. In addition, after transfection of green fluorescent protein (GFP) into cells, this Nanog gene fragment could activate GFP gene expression in undifferentiated BM-MSCs. These results demonstrated that this Nanog gene fragment contained cis-regulatory element, which could be activated in undifferentiated BM-MSCs.RT-PCR analysis showed that undifferentiated BM-MSCs expressed Oct4 gene as well as Sox2 gene at lower level, but, osteoblasts differentiated from BM-MSCs did not express both Oct4 and Sox2 genes. The Nanog gene fragment (from -380bp to +24bp) contains nucleotide sequences, which can bind the transcription factors Oct4 and Sox2 in embryonic stem cells, and gel mobility shift assay revealed that transcription factors Oct4 and Sox2 could not bind to this nucleotide sequence in undifferentiated BM-MSCs. These results demonstrated that although Oct4 and Sox2 genes were expressed in undifferentiated BM-MSCs, transcription factors Oct4 and Sox2 could not regulate Nanog gene expression in these cells. Previous studies showed that transcrition factors Oct4 and Sox2 are important in the regulation of Nanog gene expression in embryonic stem cells. In contrast, this study showed that Oct4 and Sox2 could not regulate Nanog gene expression in undifferentiated BM-MSCs and some other unknown transcription factors may regulate Nanog gene expression in these cells. These results can provide important values in understanding the mechanisms for regulation of Nanog gene expression, controlling the growth and differentiation of BM-MSCs and applying BM-MSCs to tissue engineering.