Gene expression profiling reveals the heterogeneous transcriptional activity of Oct3/4 and its possible interaction with Gli2 in mouse embryonic stem cells.
We examined the transcriptional activity of Oct3/4 (Pou5f1) in mouse embryonic stem cells (mESCs) maintained under standard culture conditions to gain a better understanding of self-renewal in mESCs. First, we built an expression vector in which the Oct3/4 promoter drives the monocistronic transcription of Venus and a puromycin-resistant gene via the foot-and-mouth disease virus self-cleaving peptide T2A. Then, a genetically-engineered mESC line with the stable integration of this vector was isolated and cultured in the presence or absence of puromycin. The cultures were subsequently subjected to Illumina expression microarray analysis. We identified approximately 4600 probes with statistically significant differential expression. The genes involved in nucleic acid synthesis were overrepresented in the probe set associated with mESCs maintained in the presence of puromycin. In contrast, the genes involved in cell differentiation were overrepresented in the probe set associated with mESCs maintained in the absence of puromycin. Therefore, it is suggested with these data that the transcriptional activity of Oct3/4 fluctuates in mESCs and that Oct3/4 plays an essential role in sustaining the basal transcriptional activities required for cell duplication in populations with equal differentiation potential. Heterogeneity in the transcriptional activity of Oct3/4 was dynamic. Interestingly, we found that genes involved in the hedgehog signaling pathway showed unique expression profiles in mESCs and validated this observation by RT-PCR analysis. The expression of Gli2, Ptch1 and Smo was consistently detected in other types of pluripotent stem cells examined in this study. Furthermore, the Gli2 protein was heterogeneously detected in mESC nuclei by immunofluorescence microscopy and this result correlated with the detection of the Oct3/4 protein. Finally, forced activation of Gli2 in mESCs increased their proliferation rate. Collectively, it is suggested with these results that Gli2 may play a novel role in the self-renewal of pluripotent stem cells.
['Li|Yanzhen|Y|', 'Drnevich|Jenny|J|', 'Akraiko|Tatiana|T|', 'Band|Mark|M|', 'Li|Dong|D|', 'Wang|Fei|F|', 'Matoba|Ryo|R|', 'Tanaka|Tetsuya S|TS|']
D000818:Animals D002454:Cell Differentiation D002467:Cell Nucleus D049109:Cell Proliferation D002478:Cells, Cultured D053595:Embryonic Stem Cells D020869:Gene Expression Profiling D018507:Gene Expression Regulation, Developmental D051741:Kruppel-Like Transcription Factors D051379:Mice D050814:Octamer Transcription Factor-3 D020411:Oligonucleotide Array Sequence Analysis D039904:Pluripotent Stem Cells D011691:Puromycin D015203:Reproducibility of Results D015398:Signal Transduction D014158:Transcription, Genetic D000075746:Zinc Finger Protein Gli2
Oct3/4 Mouse mouse embryonic stem cells Embryonic stem cell mESCs Microarray hedgehog Gli Hh Transcriptional heterogeneity semi-quantitative reverse transcriptase polymerase chain reaction sqRT-PCR