Abstract 18784: Identification of Long Non Coding RNAs Associated to Mesendoderm Differentiation and Cardiovascular Commitment of Early Precursors Generated by Naïve Mouse Embryonic Stem Cells
Introduction: Nitric oxide (NO) negatively regulates the zinc-finger E-box binding homeobox 1 and 2 (Zeb1, 2) factors via upregulation of miR-200 family members. Although Zeb1, 2 are well known inducers of epithelial-to-mesenchymal transition, an important step to heart formation, their role in mesendoderm differentiation is still poorly understood. In this study, we provide evidence that, in mouse embryonic stem cells (mES), the Zeb factors are downregulated by NO with important consequences on the expression of crucial mesendodermal genes and on a subset of NO-dependent long non-coding RNAs (lncRNAs).
Methods and Results: mES, classically cultured in the presence of serum and LIF (L), synthesize NO only late during differentiation. Instead, mES kept in the presence of three inhibitors (3i), namely L, GSK3i and ERKi realize the so called “naïve state”, express rapidly the endothelial nitric oxide synthase (eNOS) and generate detectable NO levels very early after release from stemness. In fact, as early as 2 hours of release from stemness, a NO-positive mES subpopulation (ESNO+) has been identified. Sorted ESNO+ cells expressed high levels of mesendodermal markers and rapidly behaved as cardiovascular precursors. Mechanistically, the endogenous NO production determined a rapid S-nitrosylation of HDAC2 and the reduction of its association with the transcription repression factor Zeb1. This condition significantly destabilized the Zeb1/chromatin interaction with its genomic targets. The effect of NO was further explored by an integrated full RNA-seq and genome wide ChIP-seq approach aimed at the identification of relevant Zeb1 targets. The results showed the induction of a number of Zeb1-dependent lineage specific mesendodermal markers and the concurrent down modulation of neuroectodermal markers. Among them, we identified 17 lncRNAs unknown to be NO-dependent or transcriptionally targeted by Zeb1. Although some lncRNAs, i.e. H19, have been previously associated to mesendodermal differentiation, our finding provides unprecedented mechanistic information about their transcriptional regulation in mES.
Conclusions: The eNOS-HDAC2-Zeb1 pathway is important for gene expression and lncRNA regulation in early mesendoderm precursors.
Author Disclosures: C. Cencioni: None. F. Spallotta: None. M. Savoia: None. C. Kuenne: None. S. Guenther: None. A. Re: None. S. Wingert: None. M. Rehage: None. D. Sürün: None. J. Heid: None. F. Schnütgen: None. H. von Melchner: None. M.A. Rieger: None. F. Martelli: None. A. Farsetti: None. T. Braun: None. A.M. Zeiher: None. C. Gaetano: None.
- © 2016 by American Heart Association, Inc.