Auxin-degron system identifies immediate mechanisms of OCT4.

Lawrence E Bates,Mariana R.P Alves,José C.R Silva

doi:10.1016/j.stemcr.2021.05.016

Abstract

SummaryThe pluripotency factor OCT4 is essential for the maintenance of naive pluripotent stem cells in vitro and in vivo. However, the specific role of OCT4 in this process remains unknown. Here, we developed a rapid protein-level OCT4 depletion system that demonstrates that the immediate downstream response to loss of OCT4 is reduced expression of key pluripotency factors. Our data show a requirement for OCT4 for the efficient transcription of several key pluripotency factors and suggest that expression of trophectoderm markers is a subsequent event. In addition, we find that NANOG is able to bind to the genome in the absence of OCT4, and this binding is in fact enhanced. Globally, however, the active enhancer-associated histone mark H3K27ac is depleted. Our work establishes that, while OCT4 is required for the maintenance of the naive transcription factor network, at a normal embryonic stem cell levels it antagonizes this network through inhibition of NANOG binding.

Highlights

Naive pluripotent stem cells are the embryonic founders of the cells present in the adult animal
The endogenous Oct4 mRNA appears to be unusually stable in mouse embryonic stem cells (ESCs) (Abranches et al, 2013)
We found that conventional tamoxifen-induced CreER-driven genetic ablation of Pou5f1 resulted in a gradual reduction, and required over a day to fully deplete Oct4 RNA and protein (Figures 1A and 1B)

Summary

Introduction

Naive pluripotent stem cells (nPSCs) are the embryonic founders of the cells present in the adult animal. The transcription factor OCT4, expressed from the gene Pou5f1, is necessary for the maintenance of naive pluripotency in vivo and in vitro In both cases, loss of OCT4 leads to the exit from naive pluripotency and cells taking on characteristics of the trophoblast lineage, including expression of the marker genes Cdx, Pl-1, and Eomes and adoption of trophectoderm-like morphology (Nichols et al, 1998; Niwa et al, 2000). It seems likely that differentiation upon loss of OCT4 is the result of misregulation of a number of genes rather than of any single critical target

Methods

Results

Discussion

Conclusion