title: Gene expression profiling reveals the heterogeneous transcriptional activity of Oct3/4 and its possible interaction with Gli2 in mouse embryonic stem cells.
abstract: 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.
authors.affiliation: Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Yanzhen
authors.initials: Y
authors.lastname: Li
authors.affiliation: The W.M. Keck Center for Comparative and Functional Genomics, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Jenny
authors.initials: J
authors.lastname: Drnevich
authors.affiliation: The W.M. Keck Center for Comparative and Functional Genomics, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Akraiko
authors.affiliation: The W.M. Keck Center for Comparative and Functional Genomics, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Band
authors.affiliation: Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Dong
authors.initials: D
authors.lastname: Li
authors.affiliation: Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Fei
authors.initials: F
authors.lastname: Wang
authors.affiliation: DNA Chip Research Inc., Yokohama, Kanagawa 230-0045, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Matoba
authors.affiliation: Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA; Department of Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, IN 46556, USA. Electronic address: Tetsuya.Tanaka.9@nd.edu.
authors.forename: Tetsuya S
authors.initials: TS
authors.lastname: Tanaka
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000235:genetics*; D002467:Cell Nucleus / Q000235:genetics; D049109:Cell Proliferation; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D011691:Puromycin / Q000494:pharmacology; D015203:Reproducibility of Results; D015398:Signal Transduction; D014158:Transcription, Genetic; D000075746:Zinc Finger Protein Gli2
chemical_list: C000618551:Gli2 protein, mouse; D051741:Kruppel-Like Transcription Factors; D050814:Octamer Transcription Factor-3; D000075746:Zinc Finger Protein Gli2; D011691:Puromycin
keywords: Embryonic stem cell; Gli; Hh; Microarray; Mouse; Oct3/4; Transcriptional heterogeneity; hedgehog; mESCs; mouse embryonic stem cells; semi-quantitative reverse transcriptase polymerase chain reaction; sqRT-PCR