Fbx15 is a novel target of Oct3/4 but is dispensable for embryonic stem cell self-renewal and mouse development

Mol Cell Biol. 2003 Apr;23(8):2699-708. doi: 10.1128/mcb.23.8.2699-2708.2003.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3 Cells
  • Animals
  • Cell Division
  • Cell Line
  • DNA-Binding Proteins / deficiency
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Embryonic and Fetal Development / genetics
  • Embryonic and Fetal Development / physiology
  • Enhancer Elements, Genetic
  • F-Box Proteins
  • Gene Expression Regulation, Developmental
  • Gene Targeting
  • Ligases / genetics*
  • Ligases / metabolism
  • Male
  • Mice
  • Mice, Knockout
  • Mutagenesis, Site-Directed
  • Octamer Transcription Factor-3
  • Pluripotent Stem Cells / cytology*
  • Pluripotent Stem Cells / metabolism*
  • Transcription Factors / deficiency
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*
  • Ubiquitin / metabolism

Substances

  • DNA-Binding Proteins
  • F-Box Proteins
  • Fbx15 protein, mouse
  • Octamer Transcription Factor-3
  • Pou5f1 protein, mouse
  • Transcription Factors
  • Ubiquitin
  • Ligases