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 4, 6, S6, S7, S11 we analysed the direct functional consequences of ZFP143 loss on nascent transcriptome, stable mRNA pool and protein abundance. The majority of genes affected by ZFP143 loss are down-regulated, consistent with its role as a transcriptional activator. Gene set enrichment analysis revealed that genes involved in mitochondrial translation and oxidative phosphorylation components show significant down-regulation in nascent and stable transcriptomes. Furthermore, ZFP143-bound genes that are down-regulated in transcription at early time points are manifested in the proteome at later time points. Taken together, these results show that ZFP143 is a transcriptional regulator of nuclear-encoded mitochondrial genes and its depletion has an immediate effect on their transcription, mRNA and protein levels. 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.

如图1所示，本研究通过免疫印迹技术证实，ZFP143可以从建立的mESCs中去除。据此，我们得出结论，ZFP143在dTAG-V1处理后的2小时内即可从建立的mESCs中去除。在图4、6、S6、S7、S11中，我们分析了ZFP143缺失对初生转录组、稳定mRNA池和蛋白质丰度的直接功能影响。受ZFP143缺失影响的多数基因表达下调，这与它作为转录激活因子的作用一致。基因集富集分析揭示了参与线粒体翻译和氧化磷酸化成分的基因在初生和稳定转录组中表现出显著的下调。此外，在早期时间点转录下调的ZFP143结合基因，在后期时间点的蛋白质组中得以体现。综合这些结果，表明ZFP143是核编码线粒体基因的转录调控因子，其去除对它们的转录、mRNA和蛋白质水平具有即时影响。在图5、S8和S9中，我们旨在研究ZFP143长期缺失对mESCs的影响。首先，我们评估了ZFP143缺失的mESCs集落形态，量化细胞生长，分析了细胞周期，并进行了Annexin V凋亡检测。ZFP143缺失的mESCs集落失去了穹顶状结构，细胞数量明显减少。ZFP143缺失后，我们未观察到细胞周期阶段的转换。然而，我们观察到S期细胞的EdU信号强度降低，表明EdU掺入率较低。Annexin V阳性细胞的比例在后期时间点略有增加，但总体上保持较低。其次，我们观察了活细胞中的线粒体，并测量了线粒体膜电位。在对照组mESCs中，我们观察到细长的线粒体和高度连通的线粒体网络，而ZFP143缺失的细胞显示出更多的大而圆形的线粒体，这些线粒体之间相互作用较少。此外，ZFP143缺失导致TMRE强度逐渐降低，表明随着dTAG-V1处理时间的延长，存在更多极化的线粒体。在图6、S10和S11中，我们研究了ZFP143在发育和分化中的作用。我们在原肠胚模型中去除ZFP143，分析了有和无ZFP143存在时原肠胚的生长动力学。然后，我们测试了ZFP143缺失是否会影响原肠胚的线粒体膜电位。我们发现，ZFP143缺失的原肠胚无法伸长，在最新时间点产生了小的球形聚集体。我们还观察到ZFP143缺失的原肠胚中细胞质膜极化的细胞增加。