title: Fbx15 is a novel target of Oct3/4 but is dispensable for embryonic stem cell self-renewal and mouse development.
abstract: Embryonic stem (ES) cells are immortal and pluripotent cells derived from early mammalian embryos. Transcription factor Oct3/4 is essential for self-renewal of ES cells and early mouse development. However, only a few Oct3/4 target genes have been identified. In this study, we found that F-box-containing protein Fbx15 was expressed predominantly in mouse undifferentiated ES cells. Inactivation of Oct3/4 in ES cells led to rapid extinction of Fbx15 expression. Reporter gene analyses demonstrated that this ES cell-specific expression required an 18-bp enhancer element located approximately 500 nucleotides upstream from the transcription initiation site. The enhancer contained an octamer-like motif and an adjacent Sox-binding motif. Deletion or point mutation of either motif abolished the enhancer activity. The 18-bp fragment became active in NIH 3T3 cells when Oct3/4 and Sox2 were coexpressed. A gel mobility shift assay demonstrated cooperative binding of Oct3/4 and Sox2 to the enhancer sequence. In mice having a beta-galactosidase gene knocked into the Fbx15 locus, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining was detected in ES cells, early embryos (two-cell to blastocyst stages), and testis tissue. Despite such specific expression of Fbx15, homozygous mutant mice showed no gross developmental defects and were fertile. Fbx15-null ES cells were normal in morphology, proliferation, and differentiation. These data demonstrate that Fbx15 is a novel target of Oct3/4 but is dispensable for ES cell self-renewal, development, and fertility.
authors.affiliation: Laboratory of Animal Molecular Technology, Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan.
authors.forename: Yoshimi
authors.initials: Y
authors.lastname: Tokuzawa
authors.forename: Eiko
authors.initials: E
authors.lastname: Kaiho
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Maruyama
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Takahashi
authors.forename: Kaoru
authors.initials: K
authors.lastname: Mitsui
authors.forename: Mitsuyo
authors.initials: M
authors.lastname: Maeda
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Niwa
authors.forename: Shinya
authors.initials: S
authors.lastname: Yamanaka
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002455:Cell Division; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D005314:Embryonic and Fetal Development / Q000235:genetics / Q000502:physiology; D004742:Enhancer Elements, Genetic; D044783:F-Box Proteins; D018507:Gene Expression Regulation, Developmental; D018390:Gene Targeting; D008025:Ligases / Q000235:genetics* / Q000378:metabolism; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D016297:Mutagenesis, Site-Directed; D050814:Octamer Transcription Factor-3; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism*; D014157:Transcription Factors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D044783:F-Box Proteins; C518314:Fbx15 protein, mouse; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D014157:Transcription Factors; D025801:Ubiquitin; D008025:Ligases