title: Use of altered-specificity binding Oct-4 suggests an absence of pluripotent cell-specific cofactor usage.
abstract: Oct-4 is a POU domain transcription factor that is critical for maintaining pluripotency and for stem cell renewal. Previous studies suggest that transcription regulation by Oct-4 at particular enhancers requires the input of a postulated E1A-like cofactor that is specific to pluripotent cells. However, such studies have been limited to the use of enhancer elements that bind other POU-protein family members in addition to Oct-4, thus preventing a 'clean' assessment of any Oct-4:cofactor relationships. Other attempts to study Oct-4 functionality in a more 'stand-alone' situation target Oct-4 transactivation domains to DNA using heterologous binding domains, a methodology which is known to generate artificial data. To circumvent these issues, an altered-specificity binding Oct-4 (Oct-4RR) and accompanying binding site, which binds Oct-4RR only, were generated. This strategy has previously been shown to maintain Oct-1:cofactor interactions that are highly binding-site and protein/binding conformation specific. This system therefore allows a stand-alone study of Oct-4 function in pluripotent versus differentiated cells, without interference from endogenous POU factors and with minimal deviation from bound wild-type protein characteristics. Subsequently, it was demonstrated that Oct-4RR and the highly transactive regions of its N-terminus determined here, and its C-terminus, have the same transactivation profile in pluripotent and differentiated cells, thus providing strong evidence against the existence of such a pluripotent cell-specific Oct-4 cofactor.
authors.affiliation: King's College London, Department of Haematological and Molecular Medicine, King's College School of Medicine, The Rayne Institute, 123 Coldharbour Lane, London SE5 9NU, UK. Alexander.e.smith@kcl.ac.uk
authors.forename: Alexander E F
authors.initials: AE
authors.lastname: Smith
authors.forename: Kevin G
authors.initials: KG
authors.lastname: Ford
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002454:Cell Differentiation; D002460:Cell Line; D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008297:Male; D050814:Octamer Transcription Factor-3; D039904:Pluripotent Stem Cells / Q000378:metabolism; D017434:Protein Structure, Tertiary; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
chemical_list: D004268:DNA-Binding Proteins; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D014157:Transcription Factors; D004247:DNA