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, we conclude it is not use- domains is accomplished within the abbreviated G1, putting to ful to measure cell cycle phase lengths. rest the hypothesis that hPSCs’ unique chromosome domain structure is a consequence of replication initiating prior to the An improved imaging system for live cell imaging studies complete reorganization of RDs during G1. Finally, we of replication in hPSCs exploited this system to study the cell cycle regulation of cell fate choice, confirming with single cell analyses that hPSCs PCNA has been used to image replication foci and track their preferentially respond to induction factors during G1. spatio-temporal changes during S phase in living cells.31 We reasoned that the use of fluorescently tagged PCNA, coupled with visible changes in cell morphology during mitosis, would Results be sufficient to track all the transitions in the phases of the cell cycle in hPSCs, in addition to tracking the spatio-temporal The Fucci system does not demarcate the G1 to S phase changes in replication foci. We transfected H9 hPSCs tran- transition siently with RFP-PCNA and subjected the cells to long-term, We aimed to examine dynamics of spatio-temporal organiza- live-cell imaging. To track multiple cells simultaneously, and tion of replication foci in real time, which required a means to to reduce phototoxicity, long term imaging was performed at identify S-phase cells. The Fucci cell cycle indicator system27 low magnification using an Olympus VivaView incubator reports cell-cycle phases using fluorescent proteins, Kusabira- microscope. When conducting live cell imaging experiments Orange 2 (KO2) and Azami-Green-1 (Az1), fused to Skp, we selected cells with moderate to low levels of RFP expression Cullin, F-box containing complex (SCF) and anaphase-promot- to avoid potential toxic effects of PCNA over-expression. In ing complex (APC) targeted degrons derived from Cdt1 and addition, we confirmed that expression of PCNA at these Geminin, respectively. Cell cycle phases are then reported based observed levels did not impair the cell cycle by comparing on the accumulation vs. targeted destruction of these 2 cell- the doubling times of hPSCs transfected with RFP-PCNA, to cycle-regulated proteins. The Fucci system is illustrated sche- those expressing RFP-H2B or untagged RFP (Fig. S1). Cell matically in Figure 1A, and has recently been adapted for cycle lengths in all cases were similar and consistent with previ- hPSCs (Fig. 1B).28 We assessed the ability of this system to ously published methods21,23,32 reporting an average doubling 2466 K. A. WILSON ET AL.

Figure 1. Single-cell evaluation of Fucci in hPSCs. (A) Diagram of Fucci reporters’ anticipated expression patterns during the cell cycle. (B) Diagram of adapted Fucci reporters driven by the PSC-expressed CAG promoter and linked with selectable markers through an internal ribosome entry site (IRES). (C) Fluorescent microscopy images directly comparing Fucci reporters (pre-extract, top panels) to cell cycle specific markers MCM5 and EdU (post-extract, lower panels) within the same cells. Fucci expressing hPSCs were pulse labeled with EdU prior to imaging. One KO2C cell is EdUC indicating this cell initiated replication before degradation of KO2. (D) Table com- paring Fucci expressing hPSCs (Fucci expression reported on rows, DN D double negative, DP D double positive) to cell cycle position based on the presence/absence of EdU and extraction-resistant MCM5 (columns). time of »18 hrs, indicating that the ectopic expression of RFP- monitoring PCNA foci relative to cell divisions. Furthermore, PCNA does not impact overall cell cycle length. As the panels even at low magnification, we were able to distinguish the in Figure 3A show, we were able to identify cell cycle phases by changes in spatio-temporal replication foci patterns during S CELL CYCLE 2467

Figure 2. The Fucci system does not accurately designate the G1 to S phase transition. (A) Panels taken from a live-cell-imaging video of Fucci expressing hPSCs tran- siently transfected with RFP-PCNA. Top panels correspond to KO2 & RFP-PCNA, middle panels correspond to Az1. PCNA foci appear prior to the accumulation of Az. (B) Quantification of Time (in hours) after mitosis that PCNA foci and Az1 are detected in live cell imaging videos. PCNA foci appear »1 hr prior to the detection of Az1. (C) Panels from a live-cell-imaging video of Fucci expressing hPSCs transiently transfected with GFP-PCNA. Top panels correspond to KO2, middle panels are GFP-PCNA & Az1. PCNA foci appear prior to the disappearance of KO2-Cdt1. (D) Quantification of Time (in hours) after mitosis that PCNA foci are detected and KO2-Cdt1 signal disap- pears in live-cell imaging videos. PCNA foci appear »1 hr prior to the disappearance of KO2-Cdt1. phase, which are consistent with previous reports and our The appearance and disappearance of replication foci rela- higher-resolution images (Fig. 3A and Fig. S2).9,26,31 In the first tive to cell division by definition demarcates the G1/S & S/G2 replication foci pattern, Pattern I, sites of replication are dis- boundaries, respectively. Thus, by transiently transfecting cells tributed throughout the interior, euchromatic, nucleoplasm, with our RFP-PCNA construct and performing live-cell imag- with little proximity to the nucleolus or nuclear periphery. ing we were able to determine the exact lengths of G1, S, and After »5hrs, Pattern II emerges in which sites of replication G2 phases in individual hPSCs. We show that H9 hPSCs have become apparent around the periphery of nucleus and nucle- an abbreviated G1 of »2 hrs, S phase »9 hrs, and G2 phase is oli, and the number of foci in the interior of the nucleus is »5 hrs (Fig. 3B). We extended this analysis to include multiple reduced. In Pattern III, replication foci are further depleted in hESC cell lines as well as an induced pluripotent stem cell the interior while regions of replication around the periphery (iPSC) line and show that this unique profile is highly con- of the nucleus and nucleoli become more prominent. By Pat- served among hPSCs (Fig. 3C). These results are corroborated tern IV, sites of replication become larger in size and fewer in by fluorescence activated cell sorting (FACS) analysis of H9 number and are distributed within the nuclear interior. Finally, hPSCs stained for DNA content, which confirm a smaller per- at the end of S phase, the last pattern of replication emerges, centage of cells are present in G1 than S or G2 phase (Data not Pattern 5, in which there are still fewer and larger sites of repli- shown). Importantly, our results are the first direct measure- cation visible. ments of G1 length in human pluripotent stem cells. 2468 K. A. WILSON ET AL.

Figure 3. An improved imaging system for live cell imaging studies of replication in hPSCs. (A) Panels from a live-cell-imaging video of RFP-PCNA expressing hPSCs with deduced cell cycle position indicated. Mitosis can easily be detected by drastic morphological changes to nuclei. G1 phase cells lack PCNA foci but PCNA foci are readily detectable upon entry into S phase. The 5 distinct spatial temporal foci patterns observed in hPSCs are indicated in the panels. G2 phase cells can be detected either by the disappearance of replication foci, or based on subsequent entry into mitosis. (B) Individual cell cycle phase lengths recorded from live-cell imaging videos of RFP- PCNA expressing H9 hPSCs. (C) Table of the cell cycle parameters for cell lines examined. Numbers indicate hours for each phase.

Sox17 promoter drives mCherry expression.34 Several lines of Spatio-temporal re-organization of replication foci evidence establish that under these conditions Sox17 is a reli- accompanies RD consolidation able marker for definitive endoderm differentiation.35-37 In Both human and mESCs share a unique chromatin organiza- our hands we detect »70% mCherryC cells following 48 hrs tion characterized by more discordantly replicating RDs,5,17 of stimulation (Fig. 4B). and less clustered TADs18 that consolidate upon differentia- Next, before investigating the association of G1 length tion. Since spatio-temporal patterns of DNA replication are a and RD reorganization in single cells, we confirmed the con- reflection of the 3D organization of chromatin, we reasoned solidation of domains in our reporter cell line under our that domain consolidation during differentiation might be differentiation conditions by genome-wide replication timing detectable as changes in the amount of the genome organized analysis (Fig. 4C). Briefly, populations of unstimulated cells within the various spatio-temporal replication patterns. For and cells stimulated for 48 hrs (DE) were pulse labeled with instance, since pattern 1 is characterized by a more dispersed 50-bromo-20-deoxyuridine (BrdU), retroactively synchronized distribution of replication foci, domain consolidation would into early and late S phase factions by flow cytometry, then be predicted to result in a lower fraction of the genome orga- BrdU incorporated DNA from early and late S phase frac- nized within replication pattern 1 in differentiated cells com- tions was immunoprecipitated and sequenced. Sequences pared to PSCs. We chose the definitive endoderm (DE) were mapped back to the genome in 6kb bins and plotted as differentiation system as our model because of the rapid and a ratio of early to late replicating DNA. We detected changes robust differentiation scheme available.33 hPSCs stimulated by in genome-wide replication timing that are consistent with high concentrations of Activin A reach DE in approximately differentiation to DE5 (Fig. S3) and confirmed consolidation 48 hrs, after passing through a mesendoderm intermediate. of RDs in our reporter cell line. Figure 4D shows an exem- The differentiation protocol is outlined in Figure 4A. To mon- plary region of chromosome 8 in which smaller domains that itor for differentiation in single cells we employed an endo- replicate at different times in PSCs merge to form larger derm reporter cell line, H9 Sox17-mCherry, in which the coordinately replicated domains upon differentiation. CELL CYCLE 2469

Figure 4. Sox17-mCherry reporter line exhibits consolidation of RDs upon differentiation. (A) Table of differentiation scheme and diagram showing established cell type markers. (B) Fluorescent microscopy images of H9 Sox17-mCherry cells undifferentiated (top panels) and after 48 hrs of differentiation (lower panels) taken at 20x magni- fication. (C) Schematic of genome-wide replication timing (Repli-seq) protocol. (D) Replication timing profiles of chromosome 8 for ESCs and DE. Regions of consolidation are outlined by boxes. To test the hypothesis that domain consolidation can be genomics methods coincides with a spatial consolidation of visualized as a reduction in the duration of replication pat- replication foci observed cytogenetically by dynamically tern 1, we differentiated H9 Sox17-mCherry cells harboring tracking spatio-temporal patterns. a GFP-PCNA reporter and measured the percentage of S Both human and mouse PSCs share an abbreviated G1 that phase cells displaying each replication pattern. Results has been claimed to lengthen during differentiation.21-23,38 As revealed that, upon differentiation, there is a significant we discussed in the introduction, in principle, a reasonable decrease in the percent of S phase dedicated to replicating explanation for domain consolidation is that the longer G1 pattern 1 and a corresponding increase in the duration of phase in differentiated cells could permit more time for later patterns (Fig. 5). These results demonstrate that consol- domains to consolidate before S phase begins.24 However, pre- idation of domains during ESCs differentiation observed by vious studies measuring G1 phase length of ESCs employed 2470 K. A. WILSON ET AL.

* G1 length 33 60

22 Tme (hrs) Time (hrs) Time 11

40 00

ESC Sox17+ Percent of S phase

20

0 Pattern 1 Pattern 2 Pattern 3 Pattern 4 Pattern 5

Figure 5. Spatio-temporal re-organization of replication foci accompanies differentiation without G1 phase lengthening. The % of S phase spent replicating each pattern for GFP-PCNA expressing H9 Sox17-mCherry cells using live-cell-imaging videos (Exemplary patterns shown in Fig. 2A). Undifferentiated hPSCs are in black, differentiated (mCherryC) cells are in red. G1 lengths of cells observed are shown in the inset of figure. Ãp value < 0.05. either the Fucci system21 to measure G1 length, which we show exhibited a characteristic mid-S pattern of replication foci that in Figures 1 and 2 does not detect the G1 to S phase transition, are relatively immobile throughout interphase. This pattern or prolonged drug-induced cell cycle arrest,22 which perturbs was easily distinguished from the random chromatin 3D orga- cell cycle structure. To investigate whether domain consolida- nization and rapid movement of replication foci seen immedi- tion accompanies a lengthened G1 phase, we measured G1 ately after nuclear membrane re-assembly.39 We therefore length in the same cells used to track replication foci patterns. tracked cells with labeled mid-S replication foci through mito- Results indicated that G1 length did not increase during the sis and into the next G1 phase using live-cell microscopy, and course of 48 hours of differentiation within these individual related the spatial organization and movement of those foci to cells that induced Sox17-RFP (Fig. 5, inset). Our results are not the appearance of PCNA foci in the same cells, indicative of necessarily inconsistent with G1 phase lengthening during dif- the onset of S phase. This experiment revealed that, despite ferentiation observed by others. It is possible that differentia- the abbreviated G1 phase, chromosome positions and tion for a longer period of time may lengthen G1. We also restricted motion of foci were completed prior to the appear- would not have scored cells that had exited the view field, and ance of PCNA foci (Fig. 6). Interestingly, when we repeated cells with longer G1 phases would have had more time to exit this experiment using Fucci expressing hPSCs, we found that the view field. Regardless, our results demonstrate that, changes the KO2-tagged Cdt1-degron was stabilized prior to the re- in the spatio-temporal replication patterns occur without an establishment of chromosomal positions (Fig. S4), indicating accompanying increase in G1 length. that double-negative Fucci cells can be employed to study cell cycle events that occur prior to the re-establishment of chro- mosome organization and replication timing at the TDP. hPSC RD re-organization following mitosis is complete prior to DNA replication Sox17 is preferentially induced from G1 phase The results presented above show domain consolidation occurs despite the maintenance of a short G1. Thus, either 3D Recent evidence indicates that hPSCs are more responsive to positioning does not need to be completed before S phase induction factors during G1.40-43 However, the intrinsic cell to begins, or RDs re-position within the abbreviated G1 phase cell variation in responsiveness has not been addressed. Our live time. In fibroblasts, re-positioning takes 2–3 hours after mito- cell imaging approach provided an opportunity to investigate sis, which is close to the length of an ESC G1 phase.7,19 To the relationship between cell cycle position and differentiation in distinguish these possibilities, we labeled RDs by pulse-label- single cells, in real time. We tracked the appearance of Sox17- ing RFP-PCNA expressing hPSCs with fluorescently labeled mCherry in cells stimulated to differentiate and traced each cell nucleotides. hPSCs in mid-S phase at the time of labeling back to identify the cell-cycle phase of each individual cell when CELL CYCLE 2471

Figure 6. hPSC RD re-organization following mitosis is complete prior to DNA replication. Panels taken from a live cell imaging video of RFP-PCNA expressing hPSCs with fluorescently labeled RDs. RDs are labeled by briefly pulse-labeling hPSCs with green fluorescent conjugated nucleotides, which are rapidly incorporated into replicating DNA. Using live-cell-imaging, a cell in Mid-S phase at the time of pulse-labeling was identified based on its Mid-S phase foci pattern (middle panels) and tracked through mitosis until the subsequent S phase, indicated by the appearance of RFP-PCNA foci (top panels). Similar results were obtained in 20 cells. differentiation factors were added (time D 0). Our results show Discussion that hPSCs in G1 phase at the time of induction induced Sox17 in a cell cycle earlier (Fig. 7A) and more rapidly (Fig. 7B), than We have developed a system to track RDs during the cell cycle cells in S or G2 phase. Cells in early S took the longest to induce in human PSCs in real time and we have investigated whether Sox17, suggesting they must come around a full cell cycle to the chromosome domain consolidation during human PSC differ- next G1 before they can elicit a response to induction factors. entiation is related to G1 lengthening. Our results represent the These results demonstrate the utility of our novel system to mon- first characterization of replication patterns in living cells dur- itor cell cycle phase transitions during cell fate commitment. ing differentiation. We show that the spatial distribution and

Figure 7. Sox17 is preferentially induced from G1 phase. (A) Diagram of cell cycle phase induction of Sox17-mCherry in hPSCs. Observations were recorded from live-cell- imaging videos of Sox17-mCherry, GFP-PCNA expressing hPSCs. mCherryC cells were traced back to time D 0, the stage of cell cycle that the cell was positioned at time of stimulation was deduced from GFP-PCNA as described in Figure 2. The cell cycle phase that the cell was stimulated is indicated by “X.” Point at which cell became mCherryC indicated by red bar. (B) Quantification of when cells were turned mCherryC (Y axis, time in hours) relative to phase of cell cycle from which cells were posi- tioned at time of induction (indicated on X axis). 2472 K. A. WILSON ET AL. temporal organization of DNA replication foci changes in a seen by genome-wide replication timing maps, and TADs seen manner that is consistent with the consolidation of RDs in Hi-C data, and clusters of adjacent replication foci/TADs/ detected by genome-wide RT5,17 and Hi-C analysis.18 Further- RDs replicate at very similar times, giving the appearance of more, we establish that domain consolidation can occur with- larger CTRs.49 out an accompanying increase in G1 phase length. This finding puts to rest a long-standing hypothesis that the more discor- Differentiation from G1 phase dant replication and compartmentalization of RDs observed in We have employed our live single cell imaging system to show PSCs is a consequence of replication origins firing prior to the that PSCs induce definitive endoderm marker Sox17 more rap- 24 complete spatial re-organization of RDs. idly when stimulated prior to the onset of DNA replication. This result is consistent with ensemble methods that have ’ fi Biological significance of domain consolidation reported that PSCs capacity to differentiate is signi cantly reduced as cells exit G1 phase.40-42 The prevailing view is that We conclude that G1 lengthening does not account for domain G1 represents a critical opportunity for cells to elicit a response consolidation; although, the significance of this unique domain to differentiation signals, and once cells pass this window of structure is still unclear. The conservation of more discordantly opportunity, cells are refractory to such factors until the next replicated and spatially separated RDs in both human and cell cycle. Moreover, recent evidence shows that undergoing mouse PSCs suggests consolidation serves some role in lineage DNA replication is a requirement for successful cellular reprog- 5,17 committment. Discordantly replicating RDs may reflect a ramming and gene repositioning.50-52 It is tempting to specu- specialized feature of PSC chromosome structure in which late that passage through S phase is required to remodel domains have flexibility to organize into different types of chromatin as it is repackaged at the replication fork. Indeed, more restricted spatial-temporal patterns during lineage specifi- different types of chromatin replicate at different times during cation. Spatio-temporal consolidation could provide a scaffold S phase, thus, a switch in replication timing could facilitate a to assemble more stable epigenetic states that define cell line- domain-wide remodeling of chromatin. Additionally, the repli- ages. While their exact role is still unclear, RDs reflect meaning- cation machinery has been shown to interact with chromatin ful aspects of chromosome biology and the study of RD remodelers53,54 and components of replication machinery have dynamics is likely to reveal an important molecular handle for been identified as important factors in gene positioning.51 Thus executing proper differentiation programs. Our results demon- it is possible that cells experiencing differentiation signals dur- strate that this consolidation is not a passive result of the time ing G1 elicit a response via changes to the replication-timing permitted for chromatin to re-organize but is a feature of the program. This model would explain correlations observed pluripotent cell interphase chromatin architecture. between RT, chromatin organization, and transcription. It also raises the interesting question as to the order in which these Replication foci and replication domains events change during a single cell type transition. Addressing this question will bring us closer to understanding how these The preponderance of evidence suggests that replication foci events are casually linked and provide a foundation for future correspond to the replication domains seen by genome-wide studies of mechanism. replication timing analyses, which are equivalent to TADs.16 Once labeled, replication foci remain visible as stable units for many generations.10,12,13 DNA fiber studies reveal that RDs Materials and methods fi replicate by the synchronized ring of clusters of origins across Cell culture hundreds of kilobases.44-47 Moreover, the estimated amount of DNA per replication focus46 is consistent with the sizes of RDs, Cell lines H7, H9, BG01, BG02, and iPSK3 were cultured under and replication foci appear quite uniform in size, similar to RDs, feeder-free conditions on Geltrex (Thermo Fisher, A14133) which range from 400–800 kb.1,17 The sizes of coordinately rep- coated dishes, and maintained in StemPro (Thermo Fisher, licating segments of chromosomes in any give cell type on the A100701) culture media per manufacture’s specifications. Cell other hand are far less uniform in size, ranging from 400 kb to passaging was achieved by brief treatment (»6–8 min) with several Mb, due to the coordinate replication of adjacent RDs. In Accutase (Thermo Fisher, A1110501). After detachment, cells fact, studies of PCNA in living cells have shown that, frequently, were gently collected, centrifuged for 5 min at 200 g, and re- upon the completion of replication at one focal site, replication plated on freshly coated Geltrex dishes. The H9 derived Sox17- initiates rapidly within an adjacent site, which would give rise to mCherry reporter cell line was provided by our collaborators a large segment of coordinated DNA replication (or a large Edouard Stanley and Andrew Elefanty and maintained in CTR) in the ensemble genome-wide data.48 Consolidation dur- DMEM F12 (Invitrogen, 11320–033), 20% Knockout Serum ing differentiation results in an increase in the number of adja- Replacement (Invitrogen, 10828–028), 2 mM Glutamax (Invi- cent RDs that are coordinately replicating and spatially trogen, 35050–061), 100uM Nonessential amino acids (Invitro- consolidated.5,17 Our results here show that, during differentia- gen, 11140–050), and 10ng/ml bFGF (Thermo Fisher, tion, replication foci spatially consolidate to reduce the amount PHG0261). of time cells spend replicating the more disperse pattern I foci, coincident with domain consolidation measured by other Definitive endoderm differentiation methods. Altogether, this gives rise to a model in which replica- Detailed differentiation conditions may be found at Schultz tion foci are the cytogenetic equivalent of replication domains et al., 2010. Briefly, cultures at »70% confluency were washed CELL CYCLE 2473

2x with cold PBS and stimulated to differentiation by the addi- Repli-seq produces indistinguishable results from array hybrid- tion of RPMI (Invitrogen), 50ng/ml Wnt (CellGS, GFM77), ization.16 RT datasets were normalized using limma package in 100ng/ml Activin A (CellGS, GFH6). 24hrs after stimulation R (R Core Team 2015) and rescaled to equivalent ranges by media was replaced with RPMI containing 0.2% FBS, and quintile normalization. 100 ng/ml Activin A. Tracking re-organization of RDs after mitosis Generation of cell cycle reporter lines To track the re-establishment of RDs following mitosis, cells The FUCCI reporter plasmids (described in Fig. 1B) were gen- were labeled with Alexa Fluor 488 dUTP (Thermo Fisher, erously provided by Amar Sigh (Steve Dalton’s lab, UGA). Plas- C11397). For this purpose, cells were seeded onto 35 mm glass mids were transfected into hPSCs using FugeneHD (Promega, bottom dishes (MatTek) and transfection was performed at E2311) following manufactures specifications. Four days fol- 70% confluency. For each dish, 3 ml Fugene6 (Promega, E2691) lowing transfection, Fucci expression hPSCs were selected was mixed with 1 ml dUTP analog (10 minutes; 0C) and 17 ml based on resistance to antibiotic drugs Neomycin and Puromy- PBS (further 5 minutes; 0C) before applying to cells cin. Drug-resistant colonies were pooled. RFP-PCNA and (10 minutes; 0C). Dishes were rinsed with PBS and growth GFP-PCNA plasmids were provided by Michael Davidson media was returned. Cells were imaged using either the Olym- (FSU). Plasmids were transfected into hPSCs using FugeneHD pus VivaView, or Andor Revolution Spinning disk equipped following manufactures guidelines. with 60x oil objective and Andor Clara camera for high resolu- tion image capture. Immunofluorescence To compare Fucci with established cell cycle markers EdU and MCM5, H9 Fucci expressing hPSCs were pulse labeled with Abbreviations EdU (10 uM) for 20 min and subsequently imaged using an APC Anaphase Promoting Complex image restoration microscope system (DeltaVision; Applied Az1 Azami-Green 1 Precision) attached to a fluorescence microscope (Olympus, BrdU 50-bromo-20-deoxyuridine IX-71) equipped with a Plan Apo1.40 NA oil objective lens CTR Constant timing region (Olympus). Stage positions where images were captured were DE Definitive Endoderm recorded. Soluble Fucci reporters were then extracted by 0.5% Fucci Fluorescence ubiquitination cell cycle indicator Trition X-100 diluted in ice-cold CSK buffer supplemented GFP Green Fluorescent Protein with protease inhibitors (1:50, Cocktail III, VWR, 80053–852). hESC Human embryonic stem cell Following extraction, cells were fixed by incubating cells in 4% KO2 Kusabira Orange 2 paraformaldehyde for 15min on ice. Cells were washed 3x with LADs Latin associated domain cold PBS and incubated in blocking buffer containing: 10% MCM Mini-chromosome maintenance normal goat serum, 0.2% BSA, diluted in cold PBS for 1hr. mESC Mouse embryonic stem cell DNA-incorporated EdU was then labeled by Click-it Alexa PSCs Pluripotent stem cells Fluor 488 (Thermo Fisher, C10337). MCM5 was labeled by PCNA Proliferating Cell Nuclear Antigen incubating cells for 1hr with MCM5 primary antibody (1:100, RFP Red Fluorescent Protein Santa Cruz) diluted in blocking buffer. Cells were washed 3x RD Replication domain with cold PBS and incubated for 1hr in blocking buffer contain- SCF Skp, Cullin, F-box containing complex ing secondary antibody Alexa Fluor 594. Cells were washed and TAD Topologically associated domain stained with DAPI (2 ug/ml). Stage positions were re-visited TDP Timing decision point using Olympus Deltavision software and cells were re-imaged.

Long-term, live-cell imaging of cell cycle and RDs For imaging experiments in which cell cycle parameters and Disclosure of potential conflicts of interest replication foci patterns were recorded, cells were plated onto No potential conflicts of interest were disclosed. Geltrex-coated, glass-bottom dishes (MatTek). Live cell imag- ing was performed with a fluorescent incubator microscope (Olympus VivaView FL) equipped with a 40x objective and a Acknowledgements highly sensitive EM-CCD camera (Hamamatsu, ImagEM). We thank H. Bass and V. Dileep for helpful discussions. Time-lapse images were collected every 12–20 min for 48 hrs at minimal exposure times to avoid phototoxicity. Image analysis was performed using the Olympus VivaView software package, Funding Metamorph. S-phase replication foci patterns were visually This work was funded by NIH grant GM085354 to DMG. identified.

Genome-wide replication timing analysis Notes on contributors Genome-wide RT profiles were constructed as described previ- 17 Manuscript writing: KAW; Conception and design: KAW and DMG; ously. Array hybridization was replaced by next generation Reporter cell line construction: AGE and EGS; data analysis and interpre- sequencing (NGS) (Repli-seq). We have previously shown tation: KAW and DMG; Manuscript editing: KAW, AGE, EGS, DMG 2474 K. A. WILSON ET AL.

References [16] Pope BD, Ryba T, Dileep V, Yue F, Wu W, Denas O, Vera DL, Wang Y, Hansen RS, Canfield TK, et al. Topologically associat- [1] Ryba T, Hiratani I, Lu J, Itoh M, Kulik M, Zhang J, Schulz TC, Rob- ingdomainsarestableunitsofreplication-timing regulation. ins AJ, Dalton S, Gilbert DM. Evolutionarily conserved replication Nature 2014; 515:402-5; PMID:25409831; http://dx.doi.org/ timing profiles predict long-range chromatin interactions and distin- 10.1038/nature13986 guish closely related cell types. Genome Res 2010; 20:761-70; [17] Hiratani I, Ryba T, Itoh M, Yokochi T, Schwaiger M, Chang C-W, PMID:20430782; http://dx.doi.org/10.1101/gr.099655.109 Lyou Y, Townes TM, Schubeler€ D, Gilbert DM. Global reorganiza- [2] Hiratani I, Ryba T, Itoh M, Rathjen J, Kulik M, Papp B, Fussner E, tion of replication domains during embryonic stem cell differentia- Bazett-Jones DP, Plath K, Dalton S, et al. Genome-wide dynamics of tion. PLoS Biol 2008; 6:e245; PMID:18842067; http://dx.doi.org/ replication timing revealed by in vitro models of mouse embryogene- 10.1371/journal.pbio.0060245 sis. Genome Res 2010; 20:155-69; PMID:19952138; http://dx.doi.org/ [18] Dixon JR, Jung I, Selvaraj S, Shen Y, Antosiewicz-Bourget JE, Lee AY, 10.1101/gr.099796.109 Ye Z, Kim A, Rajagopal N, Xie W, et al. Chromatin architecture reor- [3] Hansen RS, Thomas S, Sandstrom R, Canfield TK, Thurman RE, ganization during stem cell differentiation. Nature 2015; 518:331-6; Weaver M, Dorschner MO, Gartler SM, Stamatoyannopoulos JA. PMID:25693564; http://dx.doi.org/10.1038/nature14222 Sequencing newly replicated DNA reveals widespread plasticity in [19] Dileep V, Ay F, Sima J, Vera DL, Noble WS, Gilbert DM. Topologi- human replication timing. Proc Natl Acad Sci U S A 2010; 107:139- cally associating domains and their long-range contacts are estab- 44; PMID:19966280; http://dx.doi.org/10.1073/pnas.0912402107 lished during early G1 coincident with the establishment of the [4] Hiratani I, Takebayashi S, Lu J, Gilbert DM. Replication timing and replication-timing program. Genome Res 2015; 25:1104-13; transcriptional control: beyond cause and effect–part II. Curr Opin PMID:25995270; http://dx.doi.org/10.1101/gr.183699.114 Genet Dev 2009; 19:142-9; PMID:19345088; http://dx.doi.org/ [20] Lange C, Calegari F. Cdks and cyclins link G1 length and differentia- 10.1016/j.gde.2009.02.002 tion of embryonic, neural and hematopoietic stem cells. Cell cycle [5] Rivera-Mulia JC, Buckley Q, Sasaki T, Zimmerman J, Didier RA, Georg Tex 2010; 9:1893-900; PMID:20436288; http://dx.doi.org/ Nazor K, Loring JF, Lian Z, Weissman S, Robins AJ, et al. Dynamic 10.4161/cc.9.10.11598 changes in replication timing and gene expression during lineage spec- [21] Calder A, Roth-Albin I, Bhatia S, Pilquil C, Lee JH, Bhatia M, Leva- ification of human pluripotent stem cells. Genome Res 2015; 25:1091- doux-Martin M, McNicol J, Russell J, Collins T, et al. Lengthened G1 103; PMID:26055160; http://dx.doi.org/10.1101/gr.187989.114 phase indicates differentiation status in human embryonic stem cells. [6] Lubelsky Y, Prinz JA, DeNapoli L, Li Y, Belsky JA, MacAlpine DM. Stem Cells Dev 2013; 22:279-95; PMID:22827698; http://dx.doi.org/ DNA replication and transcription programs respond to the same 10.1089/scd.2012.0168 chromatin cues. Genome Res 2014; 24:1102-14; PMID:24985913; [22] Becker KA, Stein JL, Lian JB, van Wijnen AJ, Stein GS. Human http://dx.doi.org/10.1101/gr.160010.113 embryonic stem cells are pre-mitotically committed to self-renewal [7] Dimitrova DS, Gilbert DM. The spatial position and replication tim- and acquire a lengthened G1 phase upon lineage programming. J ing of chromosomal domains are both established in early G1 phase. Cell Physiol 2010; 222:103-10; PMID:19774559; http://dx.doi.org/ Mol Cell 1999; 4:983-93; PMID:10635323; http://dx.doi.org/10.1016/ 10.1002/jcp.21925 S1097-2765(00)80227-0 [23] White J, Dalton S. Cell cycle control of embryonic stem cells. Stem [8] Pope BD, Hiratani I, Gilbert DM. Domain-wide regulation of DNA Cell Rev 2005; 1:131-8; PMID:17142847; http://dx.doi.org/10.1385/ replication timing during mammalian development. Chromosome SCR:1:2:131 Res 2010; 18:127-36; PMID:20013151; http://dx.doi.org/10.1007/ [24] Gilbert DM. Cell fate transitions and the replication timing decision s10577-009-9100-8 point. J Cell Biol 2010; 191:899-903; PMID:21115801; http://dx.doi. [9] O’Keefe RT, Henderson SC, Spector DL. Dynamic organization of org/10.1083/jcb.201007125 DNA replication in mammalian cell nuclei: spatially and temporally [25] Meshorer E, Misteli T. Chromatin in pluripotent embryonic stem defined replication of chromosome-specific alpha-satellite DNA cells and differentiation. Nat Rev Mol Cell Biol 2006; 7:540-6; sequences. J Cell Biol 1992; 116:1095-110; PMID:1740468; http://dx. PMID:16723974; http://dx.doi.org/10.1038/nrm1938 doi.org/10.1083/jcb.116.5.1095 [26] Panning MM, Gilbert DM. Spatio-temporal organization of DNA [10] Jackson DA, Pombo A. Replicon clusters are stable units of chromo- replication in murine embryonic stem, primary, and immortalized some structure: evidence that nuclear organization contributes to the cells. J Cell Biochem 2005; 95:74-82; PMID:15723284; http://dx.doi. efficient activation and propagation of S phase in human cells. J Cell org/10.1002/jcb.20395 Biol 1998; 140:1285-95; PMID:9508763; http://dx.doi.org/10.1083/ [27] Sakaue-Sawano A, Kurokawa H, Morimura T, Hanyu A, Hama H, jcb.140.6.1285 Osawa H, Kashiwagi S, Fukami K, Miyata T, Miyoshi H, et al. Visual- [11] Maya-Mendoza A, Olivares-Chauvet P, Shaw A, Jackson DA. S phase izing spatiotemporal dynamics of multicellular cell-cycle progression. progression in human cells is dictated by the genetic continuity of Cell 2008; 132:487-98; PMID:18267078; http://dx.doi.org/10.1016/j. DNA foci. PLoS Genet 2010; 6:e1000900; PMID:20386742; http://dx. cell.2007.12.033 doi.org/10.1371/journal.pgen.1000900 [28] Singh AM, Chappell J, Trost R, Lin L, Wang T, Tang J, Matlock BK, [12] Ma H, Samarabandu J, Devdhar RS, Acharya R, Cheng PC, Meng C, Weller KP, Wu H, Zhao S, et al. Cell-Cycle Control of Developmen- Berezney R. Spatial and temporal dynamics of DNA replication sites tally Regulated Transcription Factors Accounts for Heterogeneity in in mammalian cells. J Cell Biol 1998; 143:1415-25; PMID:9852140; Human Pluripotent Cells. Stem Cell Reports 2014; 2:398; http://dx.doi.org/10.1083/jcb.143.6.1415 PMID:24371808; http://dx.doi.org/10.1016/j.stemcr.2014.02.009 [13] Sparvoli E, Levi M, Rossi E. Replicon clusters may form structurally [29] Kuipers MA, Stasevich TJ, Sasaki T, Wilson KA, Hazelwood KL, stable complexes of chromatin and chromosomes. J Cell Sci 1994; McNally JG, Davidson MW, Gilbert DM. Highly stable loading of 107(Pt 1):3097-103; PMID:7699008 Mcm proteins onto chromatin in living cells requires replication to [14] Peric-Hupkes D, Meuleman W, Pagie L, Bruggeman SWM, Solo- unload. J Cell Biol 2011; 192:29-41; PMID:21220507; http://dx.doi. € vei I, Brugman W, Graf S, Flicek P, Kerkhoven RM, van Lohui- org/10.1083/jcb.201007111 zen M, et al. Molecular maps of the reorganization of genome- [30] Sasaki T, Li A, Gillespie PJ, Blow JJ, Gilbert DM. Evidence for a nuclear lamina interactions during differentiation. Mol Cell 2010; mammalian late-G1 phase inhibitor of replication licensing distinct 38:603-13; PMID:20513434; http://dx.doi.org/10.1016/j.molcel. from geminin or Cdk activity. Nucleus 2:455-64; PMID:21983086; 2010.03.016 http://dx.doi.org/10.4161/nucl.2.5.17859 [15] Dixon JR, Selvaraj S, Yue F, Kim A, Li Y, Shen Y, Hu M, Liu JS, Ren [31] Leonhardt H, Rahn HP, Weinzierl P, Sporbert A, Cremer T, Zink D, B. Topological domains in mammalian genomes identified by analy- Cardoso MC. Dynamics of DNA replication factories in living cells. J sis of chromatin interactions. Nature 2012; 485:376-80; Cell Biol 2000; 149:271-80; PMID:10769021; http://dx.doi.org/ PMID:22495300; http://dx.doi.org/10.1038/nature11082 10.1083/jcb.149.2.271 CELL CYCLE 2475

[32] Becker KA, Ghule PN, Therrien JA, Lian JB, Stein JL, van Wijnen AJ, [43] Singh AM, Chappell J, Trost R, Lin L, Wang T, Tang J, Wu H, Zhao Stein GS. Self-renewal of human embryonic stem cells is supported S, Jin P, Dalton S. Cell-cycle control of developmentally regulated by a shortened G1 cell cycle phase. J Cell Physiol 2006; 209:883-93; transcription factors accounts for heterogeneity in human pluripo- PMID:16972248; http://dx.doi.org/10.1002/jcp.20776 tent cells. Stem Cell Rep 2013; 1:532-44; PMID:24371808; http://dx. [33] Schulz TC, Young HY, Agulnick AD, Babin MJ, Baetge EE, Bang AG, doi.org/10.1016/j.stemcr.2013.10.009 Bhoumik A, Cepa I, Cesario RM, Haakmeester C, et al. A scalable [44] Edenberg HJ, Huberman JA. Eukaryotic chromosome replication. system for production of functional pancreatic progenitors from Annu Rev Genet 1975; 9:245-84; PMID:55095; http://dx.doi.org/ human embryonic stem cells. PLoS One 2012; 7:e37004; 10.1146/annurev.ge.09.120175.001333 PMID:22623968; http://dx.doi.org/10.1371/journal.pone.0037004 [45] Hand R. Eucaryotic DNA: organization of the genome for replica- [34] Loh KM, Ang LT, Zhang J, Kumar V, Ang J, Auyeong JQ, Lee KL, tion. Cell 1978; 15:317-25; PMID:719745; http://dx.doi.org/10.1016/ Choo SH, Lim CYY, Nichane M, et al. Efficient endoderm induction 0092-8674(78)90001-6 from human pluripotent stem cells by logically directing signals con- [46] Ma H, Siegel AJ, Berezney R. Association of chromosome territories trolling lineage bifurcations. Cell Stem Cell 2014; 14:237-52; with the nuclear matrix. J Cell Biol 1999; 146:531-42; PMID:24412311; http://dx.doi.org/10.1016/j.stem.2013.12.007 PMID:10444063; http://dx.doi.org/10.1083/jcb.146.3.531 [35] Qu X-B, Pan J, Zhang C, Huang S-Y. Sox17 facilitates the differentia- [47] Berezney R, Dubey DD, Huberman JA. Heterogeneity of eukaryotic tion of mouse embryonic stem cells into primitive and definitive replicons, replicon clusters, and replication foci. Chromosoma 2000; endoderm in vitro. Dev Growth Differ 2008; 50:585-93; 108:471-84; PMID:10794569; http://dx.doi.org/10.1007/ PMID:19238729; http://dx.doi.org/10.1111/j.1440-169X.2008.01056.x s004120050399 [36] Wang P, Rodriguez RT, Wang J, Ghodasara A, Kim SK. Targeting [48] Sporbert A, Gahl A, Ankerhold R, Leonhardt H, Cardoso MC. DNA SOX17 in human embryonic stem cells creates unique strategies for polymerase clamp shows little turnover at established replication isolating and analyzing developing endoderm. Cell Stem Cell 2011; sites but sequential de novo assembly at adjacent origin clusters. Mol 8:335-46; PMID:21362573; http://dx.doi.org/10.1016/j.stem. Cell 2002; 10:1355-65; PMID:12504011; http://dx.doi.org/10.1016/ 2011.01.017 S1097-2765(02)00729-3 [37] Kanai-Azuma M, Kanai Y, Gad JM, Tajima Y, Taya C, Kurohmaru [49] Dileep V, Rivera-Mulia JC, Sima J, Gilbert DM. Large-scale chroma- M, Sanai Y, Yonekawa H, Yazaki K, Tam PPL, et al. Depletion of tin structure-function relationships during the cell cycle and develop- definitive gut endoderm in Sox17-null mutant mice. Development ment: insights from replication timing. Cold Spring Harb Symp 2002; 129:2367-79; PMID:11973269 Quant Biol 2015; PMID:26590169 [38] Borghese L, Dolezalova D, Opitz T, Haupt S, Leinhaas A, Steinfarz B, [50] Tsubouchi T, Soza-Ried J, Brown K, Piccolo FM, Cantone I, Landeira Koch P, Edenhofer F, Hampl A, Brustle€ O. Inhibition of notch sig- D, Bagci H, Hochegger H, Merkenschlager M, Fisher AG. DNA syn- naling in human embryonic stem cell-derived neural stem cells thesis is required for reprogramming mediated by stem cell fusion. delays G1/S phase transition and accelerates neuronal differentiation Cell 2013; 152:873-83; PMID:23415233; http://dx.doi.org/10.1016/j. in vitro and in vivo. Stem Cells 2010; 28:955-64; PMID:20235098; cell.2013.01.012 http://dx.doi.org/10.1002/stem.408 [51] Shachar S, Voss TC, Pegoraro G, Sciascia N, Misteli T. Identification [39] Manders EMM, Kimura H, Cook PR. Direct Imaging of DNA in Liv- of gene positioning factors using high-throughput imaging mapping. ing Cells Reveals the Dynamics of Chromosome Formation. J Cell Cell 2015; 162:911-23; PMID:26276637; http://dx.doi.org/10.1016/j. Biol 1999; 144:813-22; PMID:10085283; http://dx.doi.org/10.1083/ cell.2015.07.035 jcb.144.5.813 [52] Pop R, Shearstone JR, Shen Q, Liu Y, Hallstrom K, Koulnis M, Grib- [40] Sela Y, Molotski N, Golan S, ItskovitzÀEldor J, Soen Y. Human nau J, Socolovsky M. A key commitment step in erythropoiesis is embryonic stem cells exhibit increased propensity to differentiate synchronized with the cell cycle clock through mutual inhibition during the G1 phase prior to phosphorylation of pRB. Stem cells between PU.1 and S-phase progression. PLoS Biol 2010; 8:e1000484; Dayt Ohio 2012; 30:1097-108; http://dx.doi.org/10.1002/stem.1078 PMID:20877475; http://dx.doi.org/10.1371/journal.pbio.1000484 [41] Pauklin S, Vallier L. The cell-cycle state of stem cells determines cell [53] Zhang Z, Shibahara K, Stillman B. PCNA connects DNA replication fate propensity. Cell 2013; 155:135-47; PMID:24074866; http://dx. to epigenetic inheritance in yeast. Nature 2000; 408:221-5; doi.org/10.1016/j.cell.2013.08.031 PMID:11089978; http://dx.doi.org/10.1038/35048530 [42] Pauklin S, Madrigal P, Bertero A, Vallier L. Initiation of stem cell dif- [54] Shibahara K, Stillman B. Replication-dependent marking of DNA by ferentiation involves cell cycle-dependent regulation of developmen- PCNA facilitates CAF-1-coupled inheritance of chromatin. Cell tal genes by Cyclin D. Genes Dev 2016; 30:421-33; PMID:26883361; 1999; 96:575-85; PMID:10052459; http://dx.doi.org/10.1016/S0092- http://dx.doi.org/10.1101/gad.271452.115 8674(00)80661-3