Regulation of the mitochondrial proteome during naïve-to-primed human embryonic stem cell transition

Using human embryonic stem cells (hESC) in regenerative medicine or in
disease modeling requires a complete understanding of their biology. Two
states of hESC, recapitulating distinct developmental stages, have been
stabilized in vitro: a naïve pre-implantation stage and a post-implantation
primed stage. Among the numerous changes observed during this
transition, one of the most dramatic is mitochondria remodeling. Naive
hESCs have poorly developed mitochondria, but rely on oxidative
phosphorylation (OXPHOS) for ATP production. Interestingly, primed
hESCs switch to an exclusively glycolytic state even though their
mitochondria exhibit a more mature morphology. To this date, proteomic
analysis of these two stages have failed to identify the regulatory
mechanisms of this transition in the mitochondria. The first major aim of this
project is the characterization of the mitochondrial proteome during the
naïve to primed transition via purification of mitochondrial fraction followed
by mass spectrometry. In addition, since post-translational modifications
have been shown to regulate the activity of mitochondrial proteins,
especially through acetylation, we plan to use these fractions to investigate
the mitochondrial acetylome. We will also investigate the abundance and
activity of complex IV proteins, which have been previously shown to be the
most downregulated during the transition. Finally, we aim to combine the
results of our proteomic analysis with several public RNAseq datasets and
use a bioinformatics approach to identify putative regulators of differentially
expressed mitochondrial genes. The candidates will be functionally
validated using CRISPR-Cas9 gene editing. Taken together, these results
will define the profile of mitochondria in the context of embryo implantation
and shed light on the role of this organelle during the earliest stages of
development.