Abstract
IntroductionOctamer-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.MethodsIn 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.ResultsModified 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.ConclusionsUsing 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.
Highlights
Octamer-binding transcription factor 4 (Oct4) is a master regulator of early mammalian development
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
Summary
Octamer-binding transcription factor 4 (Oct4) is a master regulator of early mammalian development. Oct4-null mouse embryos become restricted to a trophectoderm lineage at the blastocyst stage, leading to periimplantation lethality [5] Such cell fate restriction is observable in mouse embryonic stem (ES) cells when Oct levels decrease to less than 50% of the normal diploid expression. Previous studies strove to keep changes in Oct levels within perceived limits for ES cell maintenance, it is unknown whether this minor increase in dosage would affect embryonic development. This is a very real concern, since modulating Oct levels is an intrinsic mechanism used by the embryo to control cell fate [6]
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