CELL CYCLE 2016, VOL. 15, NO. 18, 2464–2475 http://dx.doi.org/10.1080/15384101.2016.1203492 REPORT Spatio-temporal re-organization of replication foci accompanies replication domain consolidation during human pluripotent stem cell lineage specification Korey A. Wilsona, Andrew G. Elefantyb,c,d, Edouard G. Stanleyb,c,d, and David M. Gilberta aDepartment of Biological Science, Florida State University, Tallahassee, FL, USA; bMurdoch Childrens Research Institute, Parkville, Australia; cDepartment of Pediatrics, University of Melbourne, Parkville, Victoria, Australia; dDepartment of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, Australia ABSTRACT ARTICLE HISTORY Lineage specification of both mouse and human pluripotent stem cells (PSCs) is accompanied by spatial Received 1 April 2016 consolidation of chromosome domains and temporal consolidation of their replication timing. Replication Revised 8 June 2016 timing and chromatin organization are both established during G1 phase at the timing decision point (TDP). Accepted 13 June 2016 Here, we have developed live cell imaging tools to track spatio-temporal replication domain consolidation KEYWORDS during differentiation. First, we demonstrate that the fluorescence ubiquitination cell cycle indicator (Fucci) differentiation; Fucci; G1/S; system is incapable of demarcating G1/S or G2/M cell cycle transitions. Instead, we employ a combination of PCNA; replication foci; fluorescent PCNA to monitor S phase progression, cytokinesis to demarcate mitosis, and fluorescent replication domains; stem nucleotides to label early and late replication foci and track their 3D organization into sub-nuclear cells 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. Introduction nucleus and nucleoli, and other heterochromatic regions.9 Indi- Eukaryotic DNA replication follows a defined spatial-temporal vidual replication foci labeled with nucleotide analogs and fol- sequence that is achieved by the nearly synchronous firing of lowed over numerous generations do not diminish in size or clusters of origins along 400–800 kb replication domains intensity, indicating that the DNA replicated within individual (RDs). Genome-wide studies have shown that this “replication foci remains stably associated through many cell cycles.10-13 timing program” is cell type specific and highly conserved Thus, replication foci are stable chromosome units that are between related species.1-3 RDs that replicate early correlate likely the equivalent of RDs identified by genomics methods, with transcriptional activity, suggesting that replication timing although this has not been directly demonstrated. reflects other chromosome functions.4-6 During development 3D maps of chromatin interactions (Hi-C) and lamina-asso- many RDs undergo changes in replication timing that are ciating domain (LAD) mapping have revealed a strong correla- accompanied by changes in subnuclear position and transcrip- tion between sub-nuclear position, chromatin interaction tional competence.2 In each cell cycle, replication timing is re- compartments and replication timing, providing molecular con- established coincident with the anchorage of chromosome firmation of this spatio-temporal organization of chromatin.1,14 domains at a discrete time during G1 termed the timing deci- In addition, Hi-C mapping has uncovered structures within sion point (TDP),7,8 demonstrating an intimate relationship chromatin compartments known as topologically associated between sub-nuclear position and replication timing. domains (TADs).15 We have recently shown that TADs share Cytogenetically, DNA replication can be observed to occur chromosomal boundaries with RDs, suggesting that TADs, RDs at discrete punctate sites, called replication foci, the spatial dis- and replication foci are reflections of the same developmentally tribution of which changes characteristically during the course stable, large-scale chromatin structures.16 Altogether, these of S phase.9 During early S phase replication foci are enriched observations suggest a model (“replication domain model”)in in the interior of the nucleus, whereas replication foci appear- which RDs and TADs are equivalent units of chromosome struc- ing in late S phase are enriched along the periphery of the tures, and the 3D folding of TAD/RDs creates compartments in CONTACT David M. Gilbert [email protected] Department of Biological Science, 319 Stadium Drive, Florida State University, Tallahassee, FL 32306-4295, USA. Color versions of one or more of the figures in this article can be found online at www.tandfonline.com/kccy. Supplemental data for this article can be accessed on the publisher’s website. © 2016 Taylor & Francis CELL CYCLE 2465 which TAD/RDs in close proximity replicate at similar times and faithfully report cell-cycle transitions in hPSCs by directly com- can be visualized as punctate replication foci. paring the expression of Fucci reporters to the presence or Both human and mouse embryonic stem cells (hPSCs and absence of a synthetic nucleotide analog, EdU, as well as the mESCs) exhibit a unique RD organization in which a higher detergent-resistant chromatin association of the mini-chromo- percentage of adjacent RDs replicate discordantly.5,17 Upon dif- some maintenance (MCM) helicase subunit Mcm5 (Fig. 1C). ferentiation RDs rapidly consolidate, both temporally to form MCMs are loaded onto chromatin during telophase, tightly larger coordinately replicated constant timing regions (CTRs), bound to chromatin throughout G1 phase, and removed from and spatially to form larger blocks of dense chromatin.5,17 This active replication forks throughout the course of S phase,29 consolidation was recently confirmed by Hi-C mapping,18 but while G2 phase cells completely lack detergent-resistant Mcm. its biological significance remains unknown. We have previ- H9 Fucci expressing cells were briefly pulse labeled with EdU ously demonstrated that replication timing is established dur- and expression of Fucci reporters was photographed by live cell ing G1 coincident with the anchorage of chromosome fluorescent microscopy. Thereafter, soluble Fucci proteins were positions inside of the nucleus, and the formation of TADs and extracted by a brief triton wash and the same cells were stained sub-nuclear compartments.7,19 However, hPSCs and mESCs for EdU and MCM and re-imaged. Results revealed that »10% are known to have an abbreviated G1 phase, which is length- of KO2C cells had initiated DNA replication (EdUC) with ened upon differentiation.20-23 Within this context, it is con- early S phase patterns (Fig. 1D). Thus, degradation of the KO2 ceivable that pluripotent cells initiate replication prior to the reporter occurs distinctly after entry into S phase. complete re-establishment of sub-nuclear compartments, while To confirm this result we transfected Fucci expressing the lengthening of G1 phase during differentiation could pro- cells with a fluorescent tagged replication fork protein, vide time for adjacent RDs to assemble into more consolidated PCNA, which forms prominent replication foci upon entry compartments.24 Global reorganization of chromosomal into S phase, and conducted live-cell imaging experiments. domains, consolidation and accumulation of heterochromatin Our results reveal that PCNA foci appear approximately 1 hr may contribute to the stable silencing of genes that are no lon- before the accumulation of the Az1-tagged APC-degron for ger required or are detrimental to lineage specification.25 geminin (Fig. 2A and B) and the targeted destruction of the Although the existence of spatio-temporal patterns of repli- SCF-degron derived from Cdt1 (Fig. 2C and D), confirming cation foci has been documented in mESCs,26 the dynamic that, in hPSCs, entry into S phase precedes the transition in organization of these patterns during differentiation has not Fucci reporters. Interestingly, these results are consistent been addressed. In this study we develop a live cell imaging sys- with an earlier report that geminin does not accumulate until tem to track the reorganization of RDs in single PSCs as they several hours after the onset of S phase in Chinese Hamster undergo cell-fate transitions. We find significant changes in the fibroblasts,30 suggesting that geminin is not necessary to pre- spatio-temporal patterns of replication foci during differentia- vent re-replication during early S phase. Together, our tion consistent with RD consolidation. However, these changes results demonstrate that Fucci is not able to identify the occurred without any detectable lengthening of G1 phase. We G1/S transition in hPSCs. Since Fucci is also unable to iden- employ our live cell imaging system to show that anchoring of tify the S/G2 or G2/M transitions,