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Programming asynchronous replication in stem cells

Nature Structural & Molecular Biology volume 24, pages 1132–1138 (2017)Cite this article

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Abstract

Many regions of the genome replicate asynchronously and are expressed monoallelically. It is thought that asynchronous replication may be involved in choosing one allele over the other, but little is known about how these patterns are established during development. We show that, unlike somatic cells, which replicate in a clonal manner, embryonic and adult stem cells are programmed to undergo switching, such that daughter cells with an early-replicating paternal allele are derived from mother cells that have a late-replicating paternal allele. Furthermore, using ground-state embryonic stem (ES) cells, we demonstrate that in the initial transition to asynchronous replication, it is always the paternal allele that is chosen to replicate early, suggesting that primary allelic choice is directed by preset gametic DNA markers. Taken together, these studies help define a basic general strategy for establishing allelic discrimination and generating allelic diversity throughout the organism.

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Figure 1: Allelic plasticity in mouse stem cells.
Figure 2: Asynchronous gene replication in daughter cells.
Figure 3: Replication time switching (late to early).
Figure 4: Replication time switching (early-to-late).
Figure 5: Initiation of asynchronous replication.

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Acknowledgements

We thank N. Grover for statistical analyses, J. Hannah (Weizmann Institute, Rehovot, Israel) for providing the BiPS cells and M. Berger for help with FACS sorting. This work was supported by research grants from the Israel Academy of Sciences (grant 419/10 to H.C., grant 734/13 to Y.B.), the Israel Cancer Research Foundation (grant 210910 to H.C., grant 211410 to Y.B.), The Binational Science Foundation (grant 2100289 to Y.B.), The Emanuel Rubin Chair in Medical Sciences (Y.B.), the European Research Council (grant 268614 to H.C.), Rosetrees Foundation (H.C.), the Israel Centers of Excellence Program (41/11 to H.C., grant 1796/12 to Y.B.) and Lew Sanders (H.C.).

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Author notes

  1. Hagit Masika and Marganit Farago: These authors contributed equally to this work.

  2. Yehudit Bergman and Howard Cedar: These authors jointly directed this work.

Authors and Affiliations

  1. Department of Developmental Biology and Cancer Research, Hebrew University Medical School, Jerusalem, Israel

    Hagit Masika, Marganit Farago, Merav Hecht, Reba Condiotti, Kirill Makedonski, Yosef Buganim, Yehudit Bergman & Howard Cedar

  2. The Institute of Dental Sciences, Faculty of Dental Medicine, Hebrew University–Hadassah, Jerusalem, Israel

    Tal Burstyn-Cohen

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Contributions

H.M. and M.F. designed and conducted the experiments, interpreted the results and assisted in manuscript preparation. M.H. validated the replication timing data by ReTiSH analysis. R.C. generated the mammospheres, K.M. and Y. Buganim generated ES lines 11 and 12 and assisted in generating the EpiSCs, T.B.-C. generated the neurospheres and assisted in designing methods for the other spheroids. H.C. and Y. Bergman directed the study and wrote the manuscript.

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Correspondence to Yehudit Bergman.

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Integrated supplementary information

Supplementary Figure 1 Generation of EpiSC and NPCs.

(a) Single-cell clones of EpiSCs were generated from ES (LN3). Briefly, ES (LN3) cells were grown on gelatin coated dishes in N2B27 medium containing LIF and 2i and replated after several days on plates that were incubated with MEF medium overnight. Cells were grown with N2B27 media, 1% KSR and supplemented with 12 ng/ml of βFGF and 20 ng/ml activin for two weeks. Cultures were split every three days over a period of 2 weeks and subjected to limiting dilution to obtain single cell clones that were then picked and fixed for FISH analysis. (b) An example of EpiSC colony morphological view from day 0 (ESCs) until two weeks after induction. (c) To verify the nature of these EpiSCs clones, they were analyzed by real-time PCR analysis, using specific primers, for genes specifically expressed in ES cells (Nanog, Oct4, klf4, klf5 and Rex1) versus genes that are specifically expressed in EpiSCs (Lin28b, Fgf5, Dnmt3b, Sox17, Wnt3, Gata4 and Gata6) (Table S1). (d) Single NPC cell clones were generated from ES (LN3) cells in a step-wise manner. Briefly, ES cells were grown on gelatin coated dishes in N2B27 medium containing LIF and 2i for 7 days and then replated on bacterial Petri-dish plates to induce neurosphere formation using N2B27 medium supplemented with 10 ng/ml EGF and 10 ng/ml βFGF. After 3 days the floating cells from the Petri-dish were plated again on gelatin-coated dishes using the same medium and supplements. Individual colonies of NPCs were picked under the microscope and subcloned using limiting dilution. Single cell colonies were dissociated and fixed for FISH analysis. (e) An NPC colony morphological view from day 0 (ES (LN3)) until NPCs were attached to the coated dishes and picked as single colonies. (f) To verify the nature of these NPC clones, they were subjected to real time PCR analysis. Specific primers were used to detect genes that are specifically expressed in ES cells (Nanog, Oct4, klf4 and Rex1) versus genes that are specifically expressed in NPC cells (Pax6, Olig2, Nestin and Vimentin) (Table S1). (g) The asynchronous replication timing pattern of the Igκ and the olfactory receptor (OlfR) loci on chromosome 6 is presented. ES cells (LN3), mamospheres, neurospheres, NPCs, EpiSCs and pre-B cells clones were labeled with BrdU and assayed for FISH analysis using the mentioned probes. A probe for the synchronously early replicating control gene, A2m (early-E) was also detected. In each case the percentage of single/single (SS), single/double (SD) and double/double (DD) signals in interphase nuclei was counted.

Supplementary Figure 2 Asynchronous Igκ replication.

ES cells were labeled with BrdU for 2 h and then harvested and separated into G1, S1-S5, G2 fractions by fluorescence activated sorting, BrdU DNA then isolated by immunoprecipitation and subjected to semi-quantitative PCR analysis with specific primers for the Igk locus, as an example of an asynchronously replicating gene. The replication time patterns of early (α -globin) (left arrow, S2) and late (Amylase) (right arrow, S5) controls are also shown. The graph represents a quantitative analysis of gel panels shown below.

Supplementary Figure 3 Fixed replication timing at synchronous replicating genes.

Illustration for the late-early (a) and early-early (b) ReTiSH assay (as described in Figs. 3 and 4) of synchronous genes: the light grey line represents the paternal allele of the synchronous genes; Adyc8 (Adenylatecyclase 8, on chromosome 11), a late replicating gene (a), and the early replicating synchronous gene A2m (Alpha-2-macroglobulin, on chromosome 6) (b). The black line represents the maternal allele of these synchronous replicating genes (a, b). Both genes show a fixed replicating pattern, with either early (A2m) or late (Adyc8) replication during every cell division. Single/Single dots (SS) are expected for Adyc8 (late replicating) in the late-early BrdU labeling protocol (green dots), and again Single/Single dots (SS) are expected for A2m (early replicating) in the early-early assay (green dots). (c) Late-early and early-early ReTiSH experiments using ES (LN3) cells. DAPI-stained nuclei were hybridized using the indicated probes. (d) The number of nuclei with two hybridization dots was counted (A2m, n=109; Adyc8, n=114). No Single or Single/Double nuclei were observed and this serves as a control in comparison to the switching seen for asynchronously replicating regions.

Supplementary Figure 4 Fixed replication timing at imprinted loci.

(a) Illustration for the late-early ReTiSH assay of the imprinted genes; H19 and Tfpi2. The black line represents the paternal chromosome and the grey line represents the maternal chromosome. Both genes showed a fixed replication pattern during every cell division. (b, c) Late-early ReTiSH experiments on imprinted genes in ES (LN3) cells. The number of nuclei with two hybridization dots was counted (H19, n=143; Tfpi2, n=108).

Supplementary Figure 5 Role of DNA methylation in replication time choosing.

(a) WT and TKO ES cells were grown in medium containing LIF and then 3 μM CHIR and 1 μM PD (2i) were added. After several passages the cell inhibitors were removed and cells were grown for several more passages. The cells were labeled with BrdU and then prepared and assayed for replication timing using probes for Nanog and Igκ. Replication time was determined by the percentage of cells carrying a single/double (SD) pattern. In each case 100-300 cells were counted. The graphs depict the approximate replication time of each allele (arrows). The red arrow shows the timing of the early allele (percent single/single (SS) nuclei), and the blue arrow shows the timing of the late allele (percent SS+SD nuclei). (b) TKO ES cells were grown as in (a). The degree of asynchrony was determined by the percent of cells carrying a single/double (SD) pattern. In each case 100-300 cells were counted. The percent SD in LIF as opposed to 2i was found to be statistically significant (P=0.01), while there was no difference in cells before and after removal of 2i (P>0.9), as determined by the exact Kruskal-Wallis Test.

(c) WT and TKO ES cells were grown as in (a). All samples were analyzed by real-time PCR analysis using specific primers for pluripotent genes; Nanog, Oct4, Lin28b, Dppa3, Utf1, Tfcp2I1, Sox2 and Rex1, (Marks, H. et al. Cell 149, 590–604, 2012). All normalized to the Ubc, Hprt and Ppia housekeeping control genes. Relative expression levels are shown, values are means and error bars indicate standard deviation.

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Masika, H., Farago, M., Hecht, M. et al. Programming asynchronous replication in stem cells. Nat Struct Mol Biol 24, 1132–1138 (2017). https://doi.org/10.1038/nsmb.3503

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