ABSTRACT
The pluripotency of embryonic stem (ES) cells is thought to be maintained by a few key transcription factors, including Oct3/4 and Sox2. The function of Oct3/4 in ES cells has been extensively characterized, but that of Sox2 has yet to be determined. Sox2 can act synergistically with Oct3/4 in vitro to activate Oct-Sox enhancers, which regulate the expression of pluripotent stem cell-specific genes, including Nanog, Oct3/4 and Sox2 itself. These findings suggest that Sox2 is required by ES cells for its Oct-Sox enhancer activity. Using inducible Sox2-null mouse ES cells, we show that Sox2 is dispensable for the activation of these Oct-Sox enhancers. In contrast, we demonstrate that Sox2 is necessary for regulating multiple transcription factors that affect Oct3/4 expression and that the forced expression of Oct3/4 rescues the pluripotency of Sox2-null ES cells. These results indicate that the essential function of Sox2 is to stabilize ES cells in a pluripotent state by maintaining the requisite level of Oct3/4 expression.
Subject(s)
Cell Differentiation/physiology , DNA-Binding Proteins/metabolism , Embryonic Stem Cells/metabolism , Octamer Transcription Factor-3/metabolism , Organic Cation Transport Proteins/metabolism , Pluripotent Stem Cells/metabolism , Trans-Activators/metabolism , Animals , Cell Line , Cells, Cultured , DNA-Binding Proteins/genetics , Embryonic Development/physiology , Enhancer Elements, Genetic/genetics , Gene Expression Regulation, Developmental/genetics , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Nanog Homeobox Protein , Octamer Transcription Factor-3/genetics , Organic Cation Transport Proteins/genetics , SOXB1 Transcription Factors , Trans-Activators/genetics , Transcription Factors/genetics , Transcriptional Activation/genetics , Up-Regulation/geneticsABSTRACT
Although the POU transcription factor Oct3/4 is pivotal in maintaining self renewal of embryonic stem (ES) cells, little is known of its molecular mechanisms. We previously reported that the N-terminal transactivation domain of Oct3/4 is required for activation of Lefty1 expression (H. Niwa, S. Masui, I. Chambers, A. G. Smith, and J. Miyazaki, Mol. Cell. Biol. 22:1526-1536, 2002). Here we test whether Lefty1 is a direct target of Oct3/4. We identified an ES cell-specific enhancer upstream of the Lefty1 promoter that contains binding sites for Oct3/4 and Sox2. Unlike other known Oct3/4-Sox2-dependent enhancers, however, this enhancer element could not be activated by Oct3/4 and Sox2 in differentiated cells. By functional screening of ES-specific transcription factors, we found that Krüppel-like factor 4 (Klf4) cooperates with Oct3/4 and Sox2 to activate Lefty1 expression, and that Klf4 acts as a mediating factor that specifically binds to the proximal element of the Lefty1 promoter. DNA microarray analysis revealed that a subset of putative Oct3/4 target genes may be regulated in the same manner. Our findings shed light on a novel function of Oct3/4 in ES cells.
Subject(s)
HMGB Proteins/metabolism , Kruppel-Like Transcription Factors/metabolism , Membrane Proteins/genetics , Organic Cation Transport Proteins/metabolism , Promoter Regions, Genetic/genetics , Sex-Determining Region Y Protein/metabolism , Stem Cells/metabolism , Transforming Growth Factor beta/genetics , Animals , Base Sequence , Binding Sites , Cell Differentiation , Conserved Sequence , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Gene Expression/genetics , Genes, Reporter/genetics , HMGB Proteins/genetics , Humans , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Left-Right Determination Factors , Membrane Proteins/metabolism , Mice , Organic Cation Transport Proteins/genetics , Protein Binding , Sex-Determining Region Y Protein/genetics , Stem Cells/cytology , Transforming Growth Factor beta/metabolismABSTRACT
BACKGROUND: Rex1/Zfp42 has been extensively used as a marker for the undifferentiated state of pluripotent stem cells. However, its function in pluripotent stem cells including embryonic stem (ES) cells remained unclear although its involvement in visceral endoderm differentiation in F9 embryonal carcinoma (EC) cells was reported. RESULTS: We showed the function of Rex1 in mouse ES cells as well as in embryos using the conventional gene targeting strategy. Our results clearly indicated that Rex1 function is dispensable for both the maintenance of pluripotency in ES cells and the development of embryos. However, Rex1-/- ES cells showed the defect to induce a subset of the marker genes of visceral endoderm, when differentiated as embryoid body, as found in EC cells. CONCLUSION: Rex1 should be regarded just as a marker of pluripotency without functional significance like the activity of alkaline phosphatase.
Subject(s)
Cell Differentiation/genetics , Embryonic Stem Cells/cytology , Gene Expression Regulation, Developmental , Pluripotent Stem Cells/cytology , Transcription Factors/genetics , Animals , Biomarkers , Blotting, Northern , Cell Lineage , Cells, Cultured , Chimera , DNA Primers , Embryo, Mammalian/cytology , Endoderm/cytology , Female , Gene Expression Profiling , Genotype , Immunoblotting , Mice , Mice, Inbred C57BL , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction , Pregnancy , TransfectionABSTRACT
The growing use of mouse embryonic stem (ES) cells in research emphasizes their importance in studies of molecular mechanisms that maintain pluripotency and direct cellular differentiation. Although systems for regulatable transgene expression are essential for fine analysis of cellular processes at the molecular level, a strategy for the establishment of multiple ES cell lines carrying any of these systems has not yet been described. Here, we report our development of the ROSA-TET system, an effective system for the establishment of multiple ES cell lines carrying a tetracycline (Tc)-regulatable transgene at the Gt (ROSA)26asSor (ROSA26) locus. This system contains a knock-in step of a construct carrying both loxP and its mutant sequences into the ROSA26 locus, followed by a subsequent exchange step that introduces a cDNA to be Tc-regulated to the locus using the recombinase-mediated cassette exchange reaction. Both steps are demonstrated to give desired clones with high efficiency, suggesting that this system can be introduced readily into any ES cell lines, leading to the simultaneous establishment of multiple cell lines carrying different Tc-regulated cDNAs. We believe that use of this system will strongly accelerate molecular biological research using ES cells.
Subject(s)
Cell Line , Embryo, Mammalian/cytology , Mice , Stem Cells/cytology , Transcriptional Activation , Animals , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , GATA6 Transcription Factor , Mice/embryology , Proteins/genetics , RNA, Untranslated , Recombinases/genetics , Recombinases/metabolism , Stem Cells/metabolism , Tetracycline/pharmacology , Transcription Factors/genetics , Transcription Factors/metabolism , TransgenesABSTRACT
Trophectoderm (TE), the first differentiated cell lineage of mammalian embryogenesis, forms the placenta, a structure unique to mammalian development. The differentiation of TE is a hallmark event in early mammalian development, but molecular mechanisms underlying this first differentiation event remain obscure. Embryonic stem (ES) cells can be induced to differentiate into the TE lineage by forced repression of the POU-family transcription factor, Oct3/4. We show here that this event can be mimicked by overexpression of Caudal-related homeobox 2 (Cdx2), which is sufficient to generate proper trophoblast stem (TS) cells. Cdx2 is dispensable for trophectoderm differentiation induced by Oct3/4 repression but essential for TS cell self-renewal. In preimplantation embryos, Cdx2 is initially coexpressed with Oct3/4 and they form a complex for the reciprocal repression of their target genes in ES cells. This suggests that reciprocal inhibition between lineage-specific transcription factors might be involved in the first differentiation event of mammalian development.