Role of heme biosynthesis in the early stages of pluripotency

PhD thesis defended by Damien DETRAUX (Prof. Patsy RENARD) - 26/04/2021

Prof. Patsy RENARD, UNamur, Laboratory of Cellular and Molecular Biology - URBC

  • Prof. Patsy Renard (UNamur), Promoter – Secretary
  • Prof. Thierry Arnould (UNamur), Co-Promoter
  • Prof. Nicolas Gillet (UNamur), President
  • Prof. Julie Mathieu (University of Washington - Seattle)
  • Prof. Frederic Lluis Vinas (KUL)
  • Prof. Michel Jadot (UNamur)

Using human embryonic stem cells (hESC) in regenerative medicine or in disease modeling requires a complete understanding of these cells. Developmentally speaking, two distinct states of ESC have been stabilized, a naïve pre-implantation stage and a post-implantation primed stage.  Among the numerous changes observed during this transition, the mitochondria show a transition to maturity on a morphologic point of view but also a strong reduction in its metabolic oxidative phosphorylation activity, suggesting another role for the organelle. Performing a deep analysis of two recently published CRISPR-Cas9 KO functional screens (Li et al., 2018; Mathieu et al., 2019), we identified the heme synthesis, a partly mitochondrial pathway, as critical for the naïve-to primed transition in vitro. We show here an impairment of the exit of the naïve state upon blockade of the pathway by reducing the heme synthesis rate with succinylacetone (SA), a chemical inhibitor of the ALAD, the second enzyme of the pathway. Interestingly, ESC pushed to exit the naïve stage with SA fail to activate the two crucial signaling pathways for the transition: the MAPK and TGFβ. In parallel, heme synthesis inhibition promoted the acquisition of features found in the 2-cell embryo, as well as in a subpopulation of ESCs. We showed that this effect was heme-independent and due to the accumulation of succinate in the mitochondria, leaking to the other cellular compartments, since blocking the mitochondrial exit of succinate was able to prevent the acquisition of the 2C-like features. We propose that extra-mitochondrial succinate induces the succinylation of proteins, including regulators of the 2C-like-cells (2CLCs) such as nucleolin and TRIM28. Further experiments are required to pinpoint the exact mechanisms controlling both the exit of the naïve state and the acquisition of 2CLCs features. Overall, this study aims to unveil the mechanisms underlying the maintenance of pluripotent cells in early development.