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ABSTRACT
Lineage specification of both mouse and human pluripotent stem cells (PSCs) is accompanied by spatial consolidation of chromosome domains and temporal consolidation of their replication timing. Replication timing and chromatin organization are both established during G1 phase at the timing decision point (TDP). Here, we have developed live cell imaging tools to track spatio-temporal replication domain consolidation during differentiation. First, we demonstrate that the fluorescence ubiquitination cell cycle indicator (Fucci) system is incapable of demarcating G1/S or G2/M cell cycle transitions. Instead, we employ a combination of fluorescent PCNA to monitor S phase progression, cytokinesis to demarcate mitosis, and fluorescent nucleotides to label early and late replication foci and track their 3D organization into sub-nuclear chromatin compartments throughout all cell cycle transitions. We find that, as human PSCs differentiate, the length of S phase devoted to replication of spatially clustered replication foci increases, coincident with global compartmentalization of domains into temporally clustered blocks of chromatin. Importantly, re-localization and anchorage of domains was completed prior to the onset of S phase, even in the context of an abbreviated PSC G1 phase. This approach can also be employed to investigate cell fate transitions in single PSCs, which could be seen to differentiate preferentially from G1 phase. Together, our results establish real-time, live-cell imaging methods for tracking cell cycle transitions during human PSC differentiation that can be applied to study chromosome domain consolidation and other aspects of lineage specification.
Abbreviations
| APC | = | Anaphase Promoting Complex |
| Az1 | = | Azami-Green 1 |
| BrdU | = | 5′-bromo-2′-deoxyuridine |
| CTR | = | Constant timing region |
| DE | = | Definitive Endoderm |
| Fucci | = | Fluorescence ubiquitination cell cycle indicator |
| GFP | = | Green Fluorescent Protein |
| hESC | = | Human embryonic stem cell |
| KO2 | = | Kusabira Orange 2 |
| LADs | = | Latin associated domain |
| MCM | = | Mini-chromosome maintenance |
| mESC | = | Mouse embryonic stem cell |
| PSCs | = | Pluripotent stem cells |
| PCNA | = | Proliferating Cell Nuclear Antigen |
| RFP | = | Red Fluorescent Protein |
| RD | = | Replication domain |
| SCF | = | Skp, Cullin, F-box containing complex |
| TAD | = | Topologically associated domain |
| TDP | = | Timing decision point |
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
Acknowledgements
We thank H. Bass and V. Dileep for helpful discussions.
Notes on contributors
Manuscript writing: KAW; Conception and design: KAW and DMG; Reporter cell line construction: AGE and EGS; data analysis and interpretation: KAW and DMG; Manuscript editing: KAW, AGE, EGS, DMG
Funding
This work was funded by NIH grant GM085354 to DMG.
