Molecular Cloning And Functional Analysis Of Embryonic Stem Cell And Germ Cell Specific Protein ESGP And Roles Of Nanog Protein In F9 Embryonal Carcinoma Cells And Their Differentiated Endoderm Counterparts

In the present study, we investigated the structure, expression and function ofembryonic stem cell and germ cell specific protein ESGP in the self-renewal anddifferentiation of embryonic stem cells. Next we also investigated the roles of Nanogprotein in F9 embryonal carcinoma cells and their differentiated counterparts.(1). In previous study, we cloned several cDNA fragments of putativedownstream genes of Oct-4 using suppression-subtractive hybridization (SSH)method. We cloned the full-length cDNA of one of the novel genes by RACE andnamed it ESGP. It is 801 base pairs (bp) in whole length and contains a 255 bp openreading frame (ORF), which encodes an 84 amino acids small protein. Sequenceanalysis of nucleotide and protein showed that ESGP has no significant homology toany known genes. There is a signal peptide at the N-terminal domain of the ESGPaccording to the prediction of the Seqweb (GCG). The result of immunofluorescencealso suggested that ESGP might encode a small secretory protein. The expressionpattern of ESGP was consistent with the expression of Oct-4 during the mouseembryonic development. The expression of ESGP was first detected in fertilizedoocyte and sustained from 3.5-day postcoitum (dpc.) embryos to 17.5 dpc. embryos.In adult the expression could only be detected in testis and ovary tissues. In vitro, theESGP expression was restricted in pluripotent cell lines as same as Oct-4, such asembryonic stem cells, embryonic germ cells and embryonic carcinoma cells, but nottheir differentiated progenies. In spite of the specific expression pattern of ESGP,overexpression of ESGP was not indispensable for the effect of Oct-4 on theself-renewal of embryonic stem cells. And it had no obvious effects on the in vitro andin vivo differentiation of embryonic stem cells into three germ layers.(2). We also investigated the role of Nanog protein in F9 embryonal carcinomacells and their differentiated counterparts. Nanog is a newly found homeodomaintranscription factor, which plays an important role in the pluripotency maintenanceand endoderm differentiation inhibition of embryonic stem cells and early embryos.Murine F9 embryonal carcinoma cell is a good model system for the study ofendoderm lineage differentiation. We demonstrated for the first time thatoverexpression of the full length Nanog protein could return the F9 cells to the earlystatus of embryonic stem cells and repressed the primitive endoderm and parietalendoderm differentiation, but not the visceral endoderm differentiation. We also foundthat the two subdomains of Nanog, the N-terminal domain and C-terminal domain,both have indispensable roles in the endoderm inhibition function of Nanog. Thedeletion of either domain of Nanog could destroy this function. But the activity ofthese two subdomains was not identical in F9 cells. Overexpression of theC-terminal-domain-truncated Nanog spontaneously promoted the endodermdifferentiation of F9 cells. These data revealed that Nanog was required to sustain theproper undifferentiated status of F9 cells. The two subdomains of Nanog, theN-terminal domain and C-terminal domain, were both indispensable for the endodermsuppression function of Nanog, and the C-terminal domain of Nanog transduced themost effects in repressing primitive endoderm and parietal endoderm differentiation inF9 cells.