title: In silico tandem affinity purification refines an Oct4 interaction list.
abstract: BACKGROUND
  Octamer-binding transcription factor 4 (Oct4) is a master regulator of early mammalian development. Its expression begins from the oocyte stage, becomes restricted to the inner cell mass of the blastocyst and eventually remains only in primordial germ cells. Unearthing the interactions of Oct4 would provide insight into how this transcription factor is central to cell fate and stem cell pluripotency.
  
  
  METHODS
  In the present study, affinity-tagged endogenous Oct4 cell lines were established via homologous recombination gene targeting in embryonic stem (ES) cells to express tagged Oct4. This allows tagged Oct4 to be expressed without altering the total Oct4 levels from their physiological levels.
  
  
  RESULTS
  Modified ES cells remained pluripotent. However, when modified ES cells were tested for their functionality, cells with a large tag failed to produce viable homozygous mice. Use of a smaller tag resulted in mice with normal development, viability and fertility. This indicated that the choice of tags can affect the performance of Oct4. Also, different tags produce a different repertoire of Oct4 interactors.
  
  
  CONCLUSIONS
  Using a total of four different tags, we found 33 potential Oct4 interactors, of which 30 are novel. In addition to transcriptional regulation, the molecular function associated with these Oct4-associated proteins includes various other catalytic activities, suggesting that, aside from chromosome remodeling and transcriptional regulation, Oct4 function extends more widely to other essential cellular mechanisms. Our findings show that multiple purification approaches are needed to uncover a comprehensive Oct4 protein interaction network.
authors.affiliation: Stem Cell and Developmental Biology Group, Genome Institute of Singapore, 60 Biopolis Street, 138672, Singapore.
authors.forename: Clara Yujing
authors.initials: CY
authors.lastname: Cheong
authors.forename: Patricia Miang
authors.initials: PM
authors.lastname: Lon Ng
authors.forename: Rhonda
authors.initials: R
authors.lastname: Ponnampalam
authors.forename: Heng-Hang
authors.initials: HH
authors.lastname: Tsai
authors.forename: Guillaume
authors.initials: G
authors.lastname: Bourque
authors.forename: Thomas
authors.initials: T
authors.lastname: Lufkin
mesh_terms: D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D053595:Embryonic Stem Cells / Q000378:metabolism; D005804:Genes, Lethal; D013058:Mass Spectrometry; D051379:Mice; D057507:Mice, 129 Strain; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D050814:Octamer Transcription Factor-3 / Q000302:isolation & purification* / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism; D019076:Transgenes
chemical_list: D000345:Affinity Labels; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors