Quiescence loosens epigenetic constraints in somatic cells and improves their reprogramming into totipotency

Reprogramming by nuclear transfer (NT) cloning forces cells to lose their lineage-specific epigenetic marks and re-acquire totipotency. This process often produces molecular anomalies that compromise clone development. We hypothesised that quiescence alters the epigenetic status of somatic NT donor cells and elevates their reprogrammability. To test this idea, we compared chromatin composition and cloning efficiency of serum-starved quiescent (G0) fibroblasts vs non-starved mitotically-selected (G1) controls. We show that G0 chromatin down-regulated Polycomb group proteins EED, SUZ12, PHC1 and RING2, as well as histone variant H2A.Z. Using quantitative confocal immunofluorescence microscopy and fluorometric ELISA, we further show that G0 induced DNA and histone hypomethylation, specifically at H3K4me3, H3K9me2/3 and H3K27me3, but not H3K9me1. Collectively, these changes resulted in a more relaxed G0 chromatin state. Following NT, G0 donors developed into blastocysts which retained H3K9me3 hypomethylation, both in the inner cell mass and trophectoderm. G0 blastocysts from different cell types and cell lines developed significantly better into adult offspring. In conclusion, quiescence induced long-term epigenetic changes, specifically hypotrimethylation, that provide a mechanistic explanation for increased somatic cell reprogrammability.