Publication
Two fundamental properties of embryonic stem cells (ESCs) are their ability to self-renew and differentiate into all somatic cell types. Maintenance of their identity faces major challenges when transitioning through mitosis, as most DNA-binding proteins are evicted from the DNA, enhancer-promoter connections are lost, and transcription is considerably reduced. It is unclear how ESCs maintain their cell identity and faithfully re-initiate pluripotent gene expression during mitotic exit. Previous studies on synchronized mitotic cells suggested that enhancer loops are reformed and gene expression is re-initiated during anaphase-telophase. Moreover, a specific ordering of transcription factor (TF) binding was proposed to take place after mitotic exit, but thus far, it has not been studied without the application of synthetic drugs. This thesis aims at studying the sequence of events leading to the global restoration of the chromatin landscape during mitotic exit in mouse ESCs using a cell cycle synchronization-free approach. We engineered a novel reporter cell line combining the oscillatory fluorescence of two cell cycle reporters to allow for precise separation of different temporal windows during mitotic exit. This cell line allows sorting mouse ESCs into an anaphase-telophase-enriched population and four temporal bins of early G1 separated by less than 30 minutes. We then used quantitative ChIP-seq to determine the sequential binding changes of OCT4, SOX2, and NANOG (OSN), ATAC-seq to quantify chromatin accessibility changes, and nuclear RNA-seq to quantify transcriptional reactivation in these temporal windows. We found that most genes involved in basic cellular processes and pluripotency were already highly expressed very early after mitotic exit, while genes involved in cell fate commitment were less expressed and likely regulated through transcriptional and post-transcriptional mechanisms. Moreover, we demonstrate that OSN does not bind to mitotic chromatin, re-binding is initiated in anaphase-telophase and completely restored at most regulatory regions within 40 minutes after cytokinesis. However, OSN exhibit distinct quantitative binding changes that differ in their dependency on chromatin accessibility. Although NANOG can bind to inaccessible chromatin at loci poorly enriched for OS, we found that OS directs NANOG binding genome-wide during mitotic exit. On the other hand, NANOG stabilizes OS binding. Interestingly, during the transition from naïve pluripotent stem cells to a more advanced developmental stage, the interaction between OCT4 and SN becomes more decisive to maintain the pluripotent regulatory landscape. Overall, our study suggests that OSN dynamically regulates genome reactivation during mitotic exit, and it emphasizes the importance of using unperturbed cells to study the sequential molecular changes occurring on chromatin during mitotic exit.