Research ArticleCELL CYCLE

Global increase in replication fork speed during a p57KIP2-regulated erythroid cell fate switch

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Science Advances  26 May 2017:
Vol. 3, no. 5, e1700298
DOI: 10.1126/sciadv.1700298
  • Fig. 1 S-phase shortening at the transition from self-renewal to differentiation in vivo.

    (A) Schematic depicting sequential flow cytometric fetal liver subsets S0 to S5 during erythroid differentiation (see flow cytometric profile in fig. S1A). Seventy percent of S0 and all of S1 cells have CFUe potential. The transition from S0 to S1 marks a switch from CFUe self-renewal to differentiation. The S0/S1 switch is S phase–dependent and takes place in the early S phase of the last CFUe generation (23). (B) Double-deoxynucleoside label approach. Pregnant female mice were injected with EdU at t = 0 and with BrdU following a time interval I. Fetal livers were harvested and analyzed shortly after the BrdU pulse. Cells in S phase of the cycle at the time of the EdU pulse incorporate EdU into their DNA and retain this label as they progress through the cycle (represented as green cells). Cells entering S phase continue to take up EdU during the interval I, until EdU is cleared from the blood (shown as dashed green circles). Similarly, cells that are in S phase during the BrdU pulse become labeled with BrdU (red). The “green only” cells (EdU+BrdU) represent cells that were in S phase during the first EdU pulse but have exited S phase during the interval I. This cell fraction f is proportional to the length of the interval I, as long as I is shorter than the gap phase (G2 + M + G1). By trial and error, we found that, for I to be shorter than the gap phase of S1 cells, it needs to be ≤2 hours. The linear relationship between I and f can be expressed in terms of the cell cycle length I/Tc = f, where f is the cells that exited S phase in interval I [measured as % (EdU+BrdU) cells], I is the interval between EdU and BrdU pulses, and Tc is the cell cycle length. This relationship gives the length of the cycle as Tc = I/f. The length of S phase, Ts, can then be calculated from fraction s, of all cycling cells that take up the BrdU label (%BrdU+), as Ts = s × Tc. In preliminary experiments, we used longer BrdU pulses. These showed that nearly all cells were labeled with BrdU, suggesting that essentially all cells are cycling. Therefore, we made no corrections for the fraction of cycling cells. (C) Representative experiment as described in (B). Pregnant female mice were pulsed with EdU, followed by a BrdU pulse after an interval of 2 hours. Upper two panels show fetal liver cells from mice that were pulsed only with EdU, or only with BrdU, but processed for labeling for both deoxynucleosides in the same way as the double-labeled mice. Lower panels show EdU and BrdU labeling in fetal liver subsets that were explanted after the second pulse, sorted by flow cytometry, and then each processed for EdU and BrdU incorporation. In this specific experiment, a third of all S1 and S2 cells exited S phase in a 2-hour interval, giving a cell cycle length of 2/0.33 = 6 hours; only 14% of S0 exited S phase in the same time interval, giving a cell cycle length of 2/0.14 = 14.3 hours. Because 64% of S1 and 44% of S0 cells are BrdU+, their S-phase lengths are 0.64 × 6 = 3.84 hours and 0.44 × 14.3 = 6.3 hours, respectively. (D) Summary of five independent experiments as described above. The linear relationship between I and the cells that exited S phase, f, allows calculation of a mean cycle and length. S1, empty symbols; S0, filled symbols. r2 = 0.95 for S1 and 0.85 for S0. Results in table are mean ± SE. Pulses were reversed (BrdU first, EdU second) in one experiment, without effect on results (included here). The y axis intercepts are not at 0 because it takes approximately 20 min for peak absorption of each deoxynucleoside (fetal livers are explanted 20 min after the second injection). (E) Durations of cell cycle and cell cycle phases. The length of each cell cycle phase was calculated by multiplying the fraction of cells in each cell cycle phase following the second pulse by the total cell cycle length [measured as in (D)].

  • Fig. 2 p57KIP2 regulates intra–S-phase DNA synthesis rate.

    (A) DNA content histograms of freshly explanted and sorted fetal liver cells enriched for either G1 or S phase, from either the S0 or S1 subsets. DNA content histograms of the sorted subsets are overlaid on the DNA content histograms of the parental S0 or S1 populations. DNA content was visualized with the DNA dye Hoechst 33342. Representative of five independent sorts. (B) p57KIP2 mRNA expression is highest in S phase of S0 cells. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed in samples sorted as in (A). Data are means ± SEM for five independent sorting experiments. Statistical significance was assessed with the Mann-Whitney test. (C) Western blotting on fetal liver samples sorted as in (A). Representative of two experiments, each with 30 fetal livers. (D) Intra–S-phase DNA synthesis rate following retroviral transduction of S0 cells with shRNA targeting p57KIP2 or with a nonsilencing control shRNA (NS). Cells were pulsed with BrdU for 30 min, 20 hours after transduction. (E) Summary of four independent p57KIP2 knockdown experiments as in (D). Intra–S-phase DNA synthesis is expressed relative to controls transduced with nonsilencing shRNA. Samples were pulsed with BrdU for 30 min, 20 to 60 hours after transduction. Statistical significance was assessed with a paired t test. (F) p57KIP2 exerts dose-dependent inhibition of DNA synthesis within S-phase cells. S0 cells were transduced with p57KIP2–internal ribosomal entry site (IRES)–hCD4 (ICD4) or with “empty” vector (MICD4). Eighteen hours after transduction, cells were pulsed with BrdU for 30 min. Transduced cells were divided into gates 1, 2, and 3 (top left panel), expressing increasing levels of hCD4. Corresponding cell cycle analyses for each gate are shown (top right panels), including BrdU MFI for the S-phase gates in italics. See also fig. S2. Lower panels: Similar analysis relating MICD4, p57-ICD4, or p57T329A-ICD4 expression (hCD4-MFI) with either the number of cells in S phase (lower left panel) or BrdU MFI (lower right panel). Representative of six independent experiments.

  • Fig. 3 Anemia and abnormal erythropoiesis in p57KIP2-deficient embryos.

    (A) p57KIP2+/−m and wild-type littermate embryos on embryonic day 13.5 (E13.5). The p57KIP2+/−m embryo is pale and has a smaller fetal liver. (B) p57KIP2-deficient embryos are anemic, as seen from their significantly reduced hematocrit (n = 64 embryos, E13.5). (C) Fewer fetal liver cells in p57KIP2-deficient embryos compared with wild-type littermates (n = 121 embryos). (D) Abnormal erythroid differentiation in p57KIP2-deficient embryos. A representative CD71/Ter119 plot showing fewer S3 erythroblasts in the p57KIP2−/− embryo compared with wild-type littermate. (E) Increased frequency of S0 cells in p57KIP2-deficient fetal livers, measured as in (D), from 7.9 ± 0.46% (mean ± SE, wild-type embryos, n = 36) to 10.6 ± 1% (p57KIP2−/−, n = 14) and 10.0 ± 0.38% (p57KIP2−/−m, n = 25). The absolute number of S0 cells is unchanged because the total number of fetal liver cells is reduced proportionally to 67% of wild-type for both p57KIP2−/−m and p57KIP2−/− embryos [see (C)]. (F) Reduced ratio of S3 to S0 erythroblast number, measured as in (D), for a total of 74 embryos. (G) Inverse correlation between apoptosis in the S0 or S1 subsets and S3 frequency in the fetal livers of p57KIP2-deficient embryos. No significant correlation was seen in the fetal livers of wild-type littermates. Fetal livers were freshly explanted and immediately stained for annexin V binding. n = 75 embryos.

  • Fig. 4 Prematurely short S-phase and replication-associated DNA damage in p57KIP2-deficient fetal liver.

    (A) Premature increase in intra–S-phase DNA synthesis rate, measured as BrdU MFI, in p57KIP2-deficient embryos. Representative cell cycle analysis of p57KIP2+/−m and wild-type littermate embryos. Embryos were pulsed in vivo with BrdU for 30 min before fetal livers were explanted. (B) Premature increase in intra–S-phase DNA synthesis rate in p57KIP2-deficient embryos. Data summary, analyzed as in (A); n = 29 (+/+), 12 (−/−), and 18 (+/−m). For each embryo, S-phase BrdU MFI in S0 is expressed as a percentage of S-phase BrdU MFI in S1 of the same fetal liver. (C) Increased γH2AX in S0 cells of p57KIP2-deficient embryos. Representative examples of freshly explanted fetal livers of p57KIP2-deficient embryos and wild-type littermates, labeled with an antibody against γH2AX. DNA content was measured using 7-amino-actinomycin D (7AAD). See also fig. S3. (D) Increased γH2AX in S0 cells of p57KIP2-deficient embryos. Summary of data obtained as in (C) for a total of n = 79 embryos. γH2AX measured in arbitrary fluorescence units. (E) Distribution of γH2AX labeling, DNA content, and BrdU incorporation in G1, S, or G2-M phases of the cell cycle and in γH2AX-positive cells, all in the S0 subset of a single p57KIP2−/− fetal liver. Embryos were pulsed in vivo with BrdU for 30 min, and fetal livers were harvested, fixed, and labeled for BrdU incorporation, DNA, and γH2AX. S0 cells were subdivided digitally into cell cycle phase gates based on their DNA content and BrdU incorporation. Cells positive for γH2AX were gated based only on γH2AX signal regardless of cell cycle phase. (F) p57KIP2-deficient γH2AX-positive S0 cells are slowed or arrested in S phase. Data summary for 17 p57KIP2−/+m fetal livers, analyzed as in (E). Each data point is BrdU MFI (bottom) or median DNA content (top) for all cells in a specific category (G1, S, G2-M, or γH2AX-positive) in a single fetal liver. BrdU MFI and median DNA content were normalized to their respective values in G1 cells of the S0 subset in each fetal liver.

  • Fig. 5 p57KIP2 is essential to CFUe self-renewal in vitro.

    (A) p57KIP2 mRNA levels during CFUe self-renewal in vitro and following the switch to differentiation. Wild-type S0 cells were cultured for 5 days in self-renewal medium (“Dex + Epo”). Cells were washed and then placed either in differentiation medium (“Epo”) or back in self-renewal medium (“Epo + Dex”). Dex withdrawal leads to rapid down-regulation of p57KIP2 and to concurrent rapid induction of erythroid genes, such as α-globin or β-globin. mRNA measured by qRT-PCR, normalized to β-actin, and expressed relative to t = 0. (B) Western blot and protein band quantification during CFUe self-renewal in vitro and following the switch to differentiation. Experiment as in (A); on day 0, CFUe progenitors were replaced either in self-renewal medium (Epo + Dex) or in differentiation medium (Epo). The p57KIP2 band was identified using control cells transduced with retroviral vector expressing p57KIP2, as in (E). The uppermost band on the p57KIP2 blot is an unrelated cross-reacting band seen in Epo + Dex cultures [see (E)]. Legend as in (A), except that blue diamonds and red circles represent α-globin. (C) Cell cycle status of CFUe during self-renewal in vitro (Epo + Dex) and 20 to 60 hours following Dex withdrawal (Epo). BrdU MFI in Epo is expressed relative to its value in matched control cells undergoing Dex-dependent self-renewal. Top: Representative example. Bottom: Summary of seven matched cultures from three independent experiments. Statistical significance, paired t test. (D) p57KIP2-deficient S0 CFUe cells fail to self-renew in vitro. S0 cells derived from individual wild-type or littermate p57KIP2-deficient fetal livers were cultured in medium containing Epo + Dex. Cell numbers are relative to t = 0, mean ± SE of four (for +/−m) or five (for −/−) embryos. See also fig. S4A. (E) Western blot of p57KIP2 protein on day 9 of Dex-dependent CFUe self-renewal in vitro. Control 3T3 cells transduced with either empty vector or vectors expressing each of two p57KIP2 isoforms are also shown. Fetal liver cells express only the shorter 335–amino acid isoform of p57KIP2. Low levels of the p57KIP2 protein are also detectable in p57KIP2+/−m cells following 9 days of culture in Epo + Dex (see also fig. S4, B and C). Note that the top band is an unrelated cross-reacting band. (F) Increased intra–S-phase DNA synthesis rate in p57KIP2-deficient CFUe undergoing Dex-dependent self-renewal in vitro for 6 days. BrdU MFI in the S-phase gate is expressed relative to the wild-type littermate value. Representative of three independent experiments. (G) Increased number of γH2AX-positive cells in p57KIP2−/− CFUe undergoing Dex-dependent self-renewal, relative to wild-type littermate culture. Representative of three independent experiments. (H) p57KIP2 rescues p57KIP2-deficient CFUe self-renewal. p57KIP2-deficient S0 cells were transduced with low-titer virus (viral supernatant at the indicated dilutions) encoding p57KIP2 or with empty vector. Cells transduced with p57KIP2 showed significant improvement in self-renewal. Wild-type S0 cells transduced in parallel showed a reduction in self-renewal rate. (I) The CDK inhibitor drug roscovitine rescues self-renewal of p57KIP2-deficient CFUe. p57KIP2−/− S0 cells and wild-type S0 cells from littermate embryos were harvested and cultured in self-renewal medium containing Epo + Dex in the presence or absence of roscovitine. See also fig. S6B.

  • Fig. 6 Global increase in replication fork speed at the transition from S0 to S1, regulated by p57KIP2.

    (A) Experimental design. Fetal livers were individually explanted and allowed to recover for 4 hours at 37°C. After genotyping, all embryos of the same genotype (either +/+ or +/−m) were pooled and pulsed with IdU for 10 min, followed by CldU. Cells were then placed at 4°C, sorted into S0 and S1 subsets by flow cytometry, and processed for DNA combing. (B) Portion of a DNA fiber illustrating the identification and measurement of replication structures. For clarity, the same fiber is shown twice, with (bottom) or without (top) the blue fluorescence channel. IdU tracks are labeled green and are used to measure fork speed. CldU tracks are labeled red or yellow because, in addition to CldU (red), they contain DNA incorporating residual IdU (green). The red/yellow tracks are used to obtain fork directionality. Note that for fiber sections, where there is equivalent staining for red, blue, and green (which may occur during the second pulse), the fiber appears white (the sum total of red, green, and blue in the RGB color format); this does not reflect saturation of signal. See also fig. S9 for an example of a full microscope field. The blue fluorescence allows assessment of the DNA fiber continuity between replication bubbles or forks. This therefore allows localization of origins (marked “o”) as either equidistant from two forks proceeding in opposite directions or in the center of a green (IdU) track bordered by two red (CldU) tracks. The former is an origin that fired before the IdU pulse, whereas the latter is an origin that fired during the IdU pulse. Identification of origins allows the measurement of inter-origin distances as shown. Fork speed was measured as the length of green (IdU) tracks (in kilobase) for forks that moved throughout the IdU pulse (immediately adjacent blue, green, and red tracks) and divided by the duration of the pulse (10 min). (C) Scatter plots showing inter-origin distances (top) and fork speeds (bottom) for a single experiment, in which littermates p57KIP2+/−m and wild-type embryos were analyzed. See fig. S7 for additional analysis including fiber length distributions. P values are for a two-tailed t test of unequal variance between the indicated samples. Inter-origin distance was not significantly different between any of the samples. (D) Examples of fork trajectories during the 10-min IdU pulse from the data set in (C). The yellow dashed line indicates the transition from IdU to CldU. Only the IdU track was used for measurement of fork speed. (E) Violin plots for the data shown in (C).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/5/e1700298/DC1

    fig. S1. The transition from self-renewal to differentiation at S0/S1 is associated with a transient increase in intra–S-phase DNA synthesis rate.

    fig. S2. p57KIP2 exerts dose-dependent inhibition of DNA synthesis within S-phase cells.

    fig. S3. Increased γH2AX in p57KIP2-deficient fetal liver.

    fig. S4. Analysis of p57KIP2-deficient CFUe undergoing self-renewal in vitro.

    fig. S5. Increased cell death in p57KIP2−/− S0 CFUe during self-renewal in vitro.

    fig. S6. CFUe self-renewal requires the CDK-binding and inhibition functions of p57KIP2.

    fig. S7. DNA combing analysis of freshly explanted fetal livers from wild-type and p57KIP2+/−m embryos associated with the experiment in Fig. 6.

    fig. S8. DNA combing experiments.

    fig. S9. DNA combing: Example of fluorescence image file used for scoring data.

    data file S1. Spreadsheet containing the data set for the DNA combing experiment in Fig. 6.

    Image files

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. The transition from self-renewal to differentiation at S0/S1 is associated
      with a transient increase in intra–S-phase DNA synthesis rate.
    • fig. S2. p57KIP2 exerts dose-dependent inhibition of DNA synthesis within S-phase
      cells.
    • fig. S3. Increased γH2AX in p57KIP2-deficient fetal liver.
    • fig. S4. Analysis of p57KIP2-deficient CFUe undergoing self-renewal in vitro.
    • fig. S5. Increased cell death in p57KIP2−/− S0 CFUe during self-renewal in vitro.
    • fig. S6. CFUe self-renewal requires the CDK-binding and inhibition functions of
      p57KIP2.
    • fig. S7. DNA combing analysis of freshly explanted fetal livers from wild-type and p57KIP2+/−m embryos associated with the experiment in Fig. 6.
    • fig. S8. DNA combing experiments.
    • fig. S9. DNA combing: Example of fluorescence image file used for scoring data.

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • data file S1 (Microsoft Excel format). Spreadsheet containing the data set for the
      DNA combing experiment in Fig. 6.
    • Image files (.pdf format)

    Files in this Data Supplement:

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