ZNF143 is a transcriptional regulator of nuclear-encoded mitochondrial genes that acts independently of looping and CTCF

In Figure 1, we performed immunoblotting to confirm that ZFP143 could be depleted from the established mESCs. We conclude that ZFP143 could be depleted from the established mESCs within 2 hours of dTAG-V1 treatment. In Figures 5, S8 and S9 we aimed to investigate the effect of prolonged loss of ZFP143 on mESCs. We first assessed the morphology of mESCs colonies, quantified cell growth, analysed the cell cycle and performed an Annexin V apoptosis assay. The mESCs colonies of cells depleted of ZFP143 lose their dome-like structure and show a clear reduction in cell number. We did not observe a shift in cell cycle phase following ZFP143 depletion. However, we observed a decrease in EdU signal intensity for the S phase cells, suggesting lower EdU incorporation rates. The percentage of Annexin V-positive cells was slightly increased at later time points, but remained consistently low overall. Secondly, we visualised mitochondria in live cells and measured the mitochondrial membrane potential. In control mESCs, we observed elongated mitochondria and mitochondrial networks with high connectivity, whereas ZFP143-depleted cells show increased amounts of larger, circular mitochondria that have fewer interactions with each other. In addition, ZFP143 depletion resulted in a gradual loss of TMRE intensity, indicating that more depolarised mitochondria were present with longer dTAG-V1 treatment time. In Figures 6, S10 and S11 we investigated the role of ZFP143 in development and differentiation. We depleted ZFP143 in gastruloids, a model of early development, and analysed the growth dynamics of gastruloids in the presence and absence of ZFP143. We then tested whether the loss of ZFP143 had an effect on the mitochondrial membrane potential in gastruloids. We found that ZFP143-depleted gastruloids failed to elongate and produced a small spherical aggregate at the latest time points. We also observed an increase in cells with depolarised mitochondria in ZFP143-depleted gastruloids.