In silico tandem affinity purification refines an Oct4 interaction list

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.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.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.Octamer-binding transcription factor 4 (Oct4) [1], also termed Oct3 or Pou5f1 [2], is an early developmental stage transcription factor. Oct4 expression begins in the oocyte from maternal sources and is continued by zygotic expression after the four-cell stage. Thereafter it becomes restricted to the inner cell mass, the epiblast and eventually the germ cells [3]. During this time, Oct4 expression serves to regulate pluripotency and cell fate development [4]. Oct4-null mouse embryos become restricted to a trophectoderm lineage at the blastocyst stage, leading to peri-implantation lethality [5]. Such cell fate restriction is also observable in mouse embryonic stem (ES) cells when Oct4 levels decrease to less than 50% of the normal diploid expression.