Germ Cell Nuclear Factor (GCNF) Represses Oct4 Expression and Globally Modulates Gene Expression in Human Embryonic Stem (hES) Cells

Hongran Wang,Xiaohong Wang,Xueping Xu,Michael Kyba,Austin J Cooney

doi:10.1074/jbc.m115.694208

Abstract

Oct4 is considered a key transcription factor for pluripotent stem cell self-renewal. It binds to specific regions within target genes to regulate their expression and is downregulated upon induction of differentiation of pluripotent stem cells; however, the mechanisms that regulate the levels of human Oct4 expression remain poorly understood. Here we show that expression of human Oct4 is directly repressed by germ cell nuclear factor (GCNF), an orphan nuclear receptor, in hES cells. Knockdown of GCNF by siRNA resulted in maintenance of Oct4 expression during RA-induced hES cell differentiation. While overexpression of GCNF promoted repression of Oct4 expression in both undifferentiated and differentiated hES cells. The level of Oct4 repression was dependent on the level of GCNF expression in a dose-dependent manner. mRNA microarray analysis demonstrated that overexpression of GCNF globally regulates gene expression in undifferentiated and differentiated hES cells. Within the group of altered genes, GCNF down-regulated 36% of the genes, and up-regulated 64% in undifferentiated hES cells. In addition, GCNF also showed a regulatory gene pattern that is different from RA treatment during hES cell differentiation. These findings increase our understanding of the mechanisms that maintain hES cell pluripotency and regulate gene expression during the differentiation process.

Highlights

Oct4 is precisely regulated throughout the entire embryonic and fetal developmental processes
Results showed that Germ cell nuclear factor (GCNF) expression was induced by either Dox, or Dox plus retinoic acid (RA) treatment; Oct4 expression was repressed about 80% more compared with RA treatment alone; and RA enhanced the repression of Oct4 in undifferentiated human ES (hES) cells (Fig. 3C)
Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) experiments showed that Dox induced overexpression of GCNF in undifferentiated and differentiated G-hES cells (Fig. 3, E and F); the level of Oct4 decreased considerably more in cells treated with RA plus Dox than in cells treated with RA alone. (Fig. 3, E and G)

Summary

Introduction

Oct4 is precisely regulated throughout the entire embryonic and fetal developmental processes. Results showed that GCNF expression was induced by either Dox, or Dox plus RA treatment; Oct4 expression was repressed about 80% more compared with RA treatment alone; and RA enhanced the repression of Oct4 in undifferentiated hES cells (Fig. 3C).

Results

Conclusion