In the population of naïve murine embryonic stem cells (mESCs), primarily derived from the inner cell mass (ICM) of the mouse blastocyst, a subpopulation of cells spontaneously oscillates in and out a totipotent-like state. These cells, reminiscing of the 2-cell stage embryo, are called 2-cell-like cells (2CLCs). Previous results in the laboratory showed that upstream inhibition of the heme biosynthesis increased the expression of 2-cell-like markers in mESCs. This phenotype was not recovered by hemin supplementation but by diethyl-butylmalonate (BM), inhibiting succinate transport out of the mitochondria. Since metabolites have been previously reported to induce 2CL reprogramming in mESCs, we hypothesized that succinate could be involved in this phenomenon. We demonstrated here that atpenin A5 (AA5), an inhibitor of the succinate dehydrogenase (SDH), is able to provoke a rise in succinyllysine levels in the cells and to upregulate 2C-specific markers at transcript and protein levels, also reflected in the 2CLCs (ZSCAN4+ and MERVL+) population increase. Searching for the molecular mechanisms underlying the 2C-like reprogramming associated with succinate build-up, we first investigated its role in lysine residue succinylation. Using SIRT7i, an inhibitor of the nuclear dessucynilase sirtuin 7, we observed that while this inhibitor intensifies pansuccinyllysine (Pan-SuccK) nuclear levels in naïve cells, it does not increase the 2C-like reprogramming. Another role of succinate being the inhibition of 2-oxoglutarate-dependent dioxygenases (2OXs), this prompted us to use octyl-𝛼-ketoglutarate (OAKG) that prevented AA5 effects. Three relevant 2OGXs were investigated here: the hypoxia-related prolyl hydroxylase (PHD), the JMJC-containing domain histone demethylase (HDM), and 5-methylcytosine (5mC) demethylase of the ten eleven (TET) family. No implication of HIF-1𝛼 in 2C-like reprogramming was shown. The putative roles of HDMs and TETs were explored by the means of two specific inhibitors, JIB and C35, respectively. Immunofluorescence analyses showed that H3K4me3 and H3K27me3 were enriched upon AA5 treatment and also seemingly when treated with a combination of JIB and C35. These results were supported by the increase of the percentage of ZSCAN4+ cells in the naïve total population in AA5, JIB, C35, or JIB+C35-treated cells, although not significant. Unfortunately, even if a trend is observed for a reduced nuclear 5hmC signal in AA5-treated cells, no definite conclusion can be drawn on the levels of methylated DNA. Together, this enables us to conclude that the 2C-like reprogramming of naïve mESCs following succinate accumulation is characterised by a reduced demethylase activity on histones and DNA, supporting a metabolic regulation of the epigenetic landscape of the pluripotent state of mESCs.
- stem cells
- embryonic stem cells
Study of the role of succinate in naïve murine embryonic stem cells two-cell-like reprogramming
Feller, L. (Author). Jan 2022
Student thesis: Master types › Master in biochemistry and molecular and cell biology Research focus