Polycomb Ezh1 maintains muscle stem cells quiescence through non-canonical regulation of the Notch signaling

Organismal homeostasis and regeneration are predicated on committed stem cells which, in tissues and organs with low turnover, reside for long periods in a reversible cell cycle arrest, defined as quiescence. Inability to exit or premature escape from quiescence, as occurring in pathological conditions and aging, is detrimental as it results in either lack of stem cell mobilization or pool depletion with consequent defective tissue homeostatis and regeneration. It is therefore unsurprising that quiescence is safeguarded by multilayered regulatory mechanisms. Here, we report that Polycomb Ezh1 confers quiescence to muscle stem cells (MuSCs) through a non-canonical function. In the absence of Ezh1, MuSCs spontaneously exit quiescence and, following repeated injuries, the stem cell pool is depleted resulting in failure to sustain appropriate muscle regeneration. Rather than regulating repressive Polycomb-dependent histone H3K27 methylation, Ezh1 actively maintains the Notch signaling pathway in MuSCs. Accordingly, selective genetic reconstitution of the Notch signaling corrects stem cell number and re-establishes quiescence of Ezh1-/- MuSCs. To establish the role of polycomb Ezh1 in MuSCs biology, we performed RNASeq, ATACSeq, and H3K27me3 ChIPseq in FACS-purified MuSCs from the hindlimbs of wild-type (WT) or Ezh1-/- mice in homeostatic conditions. To further evaluate potential differences between WT and Ezh1-/- mice, tibialis anterior (TA) muscles of WT and Ezh1-/- mice were injured with notexin (NTX), and an additional NTX injection was performed twenty-eight days after the first injury. We conducted scRNAseq experiments in uninjured and regenerating MuSCs (collected three days after the second muscle injury).