NELFA is a novel regulator of the 2C-like state

Mouse embryonic stem cells (mESCs) are not homogenous; a rare subpopulation of cells can sporadically transit into a early-embryonic-like state and exhibits a gene expression program resembling that of the 2-cell (2C) embryo. We identified the maternal factor NELFA as a novel driver of the 2C-like gene expression through multiple mechanisms. We further demonstrate that the entry of mESCs into the 2C-like state is accompanied by epigenetic and metabolic reprogramming, and that perturbation of the metabolic state via a small molecule can promote this cell fate reversion in a NELFA-dependent manner, obviating the need for any genetic manipulation. Our findings thus place NELFA as one of the earliest regulators of 2C gene expression, extending contemporary knowledge of how totipotency may be regulated. To compare the gene expression differences between NELFAhigh and NELFAlow mESCs, we generated both a NELFA-StrepHA-P2A-EGFP reporter and a Dox-inducible NELFA- StrepHA-P2A-EGFP mESC stable line. The NELFA-reporter mESCs was treated with 4mM 2-DG for 4 days before harvesting total RNA for unbiased transcriptomic analysis with RNA-Seq, while Dox-inducible NELFA was induced with 0.4 µg/ml of Dox for 16 hours before FACS enrichment for both GFP positive and negative populations for RNA-seq, with an additional ERCC spike-in included in library preparation for normalization purposes in the downstream analysis for those two experiments. Both NELFA reporter and Dox-inducible mESCs were cultured in standard ESC media containing serum, LIF and 2i; briefly, knockout DMEM was supplemented with 15% FCS, L-Glutamine, non-essential amino acids, penicillin/streptomycin, 2-mercaptoethanol and supplemented with LIF, 1 µM PD0325901 and 3 µM CHIR99021.