A Zeb1 Hdac2 eNOS feedback circuitry identifies early cardiovascular precursors in naïve mouse embryonic stem cells (RNA-Seq)

Soon after release from stemness, a relevant portion of mouse embryonic stem cells (mES), kept in naïve state by MEK inhibitor, GSK3 inhibitor and LIF (2i/L), expresses endothelial nitric oxide synthase (eNOS) and endogenously synthesizes nitric oxide (NO). In this condition, an eNOS/NO-positive subpopulation (ESNO+) has been recognized by 4-amino-5-methylamino-2',7'-difluorescein (DAF) fluorescence. Sorted ESNO+ cells expressed high levels of mesendodermal markers and efficiently generated cardiovascular precursors compared to eNOS/NO-negative cells (ESNO-). Mechanistically, the endogenous NO production deter- mined a rapid S-nitrosylation of Hdac2, a post-translational modification that compromised Hdac2 association with the transcription repression factor Zeb1. This condition destabilized Zeb1/chromatin interaction with genomic loci encoding for mesendodermal genes. Remarkably, Zeb1 or Hdac2 targeting activated an unscheduled transcription of mesendodermal lineage-associated genes in naïve mES revealing the Hdac2-Zeb1 pathway important for stemness maintenance in 2i/L. In contrast, eNOS targeting significantly reduced the endogenous NO production preventing the activation of mesendodermal gene transcription. Overall design: ES cell in Mesendodermal and Spontaneous differentiation media