Metabolic and Epigenetic Regulation in Development and in Embryonic Diapause

Abstract

Embryonic diapause is a state of dormancy that interrupts the normally tight connection between developmental stage and time. Regulation of this dormant state, however, is poorly understood. To better understand the processes underlying diapause in mammals, we characterized the transcriptional and metabolite profiles of mouse pre-implantation, post-implantation and diapause embryos. We identified a unique cellular regulation signature placing diapause at a distinct developmental state with highly activated lipolysis, glycolysis and metabolic pathways regulated by AMPK. Significant enrichment of AMP further indicated activation of the cellular starvation-sensor, AMPK. We show that starvation in pre-implantation ICM derived mouse embryonic stem cells induce a reversible dormant state, transcriptionally mimicking the in vivo hormonally controlled diapause stage. During starvation, a splice variant of an upstream kinase of AMPK, Liver kinase b1 (Lkb1), induces a reversible, mTOR dependent glycolytic, H4K16Ac negative, diapause-like quiescence state in vitro through AMPK. We furthermore show that, paradoxically, forced expression of a non- diapause Lkb1 splice variant results in a constitutive diapause-like state due to a phospho-AMPK dependent increase in glucose transporters and decrease in mTOR activation. Our analysis reveals increased lipolysis in diapause wherein triacylglycerol (TAG) and diacylglycerol (DAG) levels are highly reduced while their products, free fatty acids and phosphatidylcholine (PC) are enriched to support cell survival in diapause. Lipolysis is increased due to mTORC2 repression as both starvation-induced diapause-like state and knockout of Rictor show upregulation of lipolysis in mESC. Furthermore, glutamine transporters, SLC38A1/2 are highly enriched and essential for a H4K16Ac negative, diapause state. These data suggest that mTORC1/2 inhibition, regulated by amino acid levels is causal for diapause metabolism and epigenetic state. In addition to studying embryonic diapause, we also investigated how pre-implantation (naïve) human embryonic stem cells transition to the post-implantation (primed) stage. These pluripotent stem cells are refractory to regenerative aging and have the capacity to remain in a pluripotent stage either by culture conditions in vitro or in vivo by entering a diapause state. We have previously identified metabolic differences that regulate the ESC epigenetic state. Here we define a novel epigenetic regulator during implantation stage, SUV420H2. A previous screen from this laboratory for early ESC regulators revealed SUV4020H2 as a critical component in naïve to primed transition. Using the CRISPR/Cas9 system, we generated a SUV420H2 knockout naïve hESC line to study its role in the pre- and post-implantation embryonic stages. We show that SUV420H2 mutants do not enter in vitro diapause, but instead continue dividing. By immunoblotting, we also show mutant cells have higher levels of H4K16ac as well as increased rates of proliferation. These data suggest that mechanistically, H4K20me3 repressive marks in key target genes are a pre-requisite for the diapause stage. Furthermore, our functional metabolic assays show that SUV420H2 mutant cells have an increased levels of fatty acid β-oxidation, mitochondrial respiration and glycolysis compared to wildtype, suggesting SUV420H2 as a potential metabolic inhibitor. We conclude that increased OXPHOS and glycolytic metabolism might be due to blocking the inhibitory effect of SUV420H2 on PPAR-γ. The data reveal the mechanism for SUV420H2 requirement during naïve to primed embryonic transition. The epigenetic repression by H4K20me3 marks is a pre-requisite for the potential diapause and metabolic reprogramming that takes place during naïve to primed transition. Finally, we studied the role of mitochondria in quiescence. Both normal and cancer stem cells can arrest cell division, avoid apoptosis, and then regenerate following acute genotoxic insult. This protective, reversible proliferative arrest is still poorly understood. We have shown that mitochondrial activity is reduced in mouse diapause embryos. We asked if mitophagy was critical for cells to enter and exit diapause state. Here, we show that mTOR-regulated mitophagy is required for mTOR-inhibition induced quiescence in human induced pluripotent stem cells (hiPSCs). Depletion of mitophagy by mutating PINK, an essential protein for mitophagy, eliminates entry into quiescence, whereas wildtype cells can enter a reversible quiescent state. Mitochondrial number significantly decreases as cells enter a diapause-like state. Our data suggest that mitochondrial number coordinates reversible quiescence. We further identify that the mechanism of quiescence in human induced pluripotent stem cells (iPSCs) relies on mitophagy to deplete the mitochondrial pool of CycE and limit cell cycle progression. This alternative method of G1/S regulation may present new opportunities for therapeutic purposes.