Analysis of cell cycle stage, replicated DNA, and chromatin-associated proteins using high-throughput flow cytometry

2.2.1 High-throughput cytometric analysis of DNA replication proteins on chromatin

Next, we employed the newly developed protocol to investigate the replicative helicase MCM2-7 on chromatin (Figure 3 and Supplementary Figures 4 and 5). Cells from asynchronous and G1-phase synchronised and released cultures (up to 120 min post-release) were withdrawn and analysed on all dimensions for their replication patterns. As before (Figure 2), DNA content and DNA synthesis recapitulated progress and completion of S-phase within 60 min of G1-phase release, cell division, and a second S-phase. When probing for MCM2-7, asynchronous cells presented four major populations: A low- and high-signal population corresponding to G1-phase, a low-signal population corresponding to G2-phase, and a population connecting the high G1- and low G2-phase populations (Figure 3A, top panel). This pattern matches the life cycle of the replicative helicase on chromatin along the cell cycle and is in line with previous studies in human cells (see below and (Frisa and Jacobberger 2010; Håland et al. 2015)). Within the G1-phase cell populations, the high-fluorescence population represents loaded MCM2-7 double hexamers on licensed origins (Evrin et al. 2009). Upon release into S-phase (>15 min), the high-fluorescence population shifts to higher DNA content levels (increased SYTOX Green intensity) while progressively losing signal intensity. This reflects the gradual unloading of MCM2-7 until DNA replication is completed (Figure 3A, panel 30–60 min). As MCM2-7 is completely unloaded after fully replicating the genomic DNA, the low-fluorescent populations in G1- and G2-phase most likely present cells harbouring only soluble, nuclear MCM2-7 complexes or subunits. When entering mitosis (Figure 3A, panel 75 min), the signal intensity of the MCM2-7 complex shifts to the G1-phase level (low, pre-licensing), followed by another round of MCM2-7 double-hexamer loading, and entry into the next S-phase (high G1-phase population; Figure 3A, panels 75–120 min). Controls without antibodies and secondary antibody only confirmed that the protein signal is specific for MCM2-7 (Supplementary Figure 5B). We also validated the feasibility and effectiveness of different dye combinations by testing different secondary antibodies coupled to various dyes (Supplementary Figure 5C). Hereby, only minor signal intensity differences could be observed, illustrating the feasibility and effectiveness of dye selection and the overall protocol.

Figure 3:

Analysis of DNA content, DNA synthesis, and MCM2-7 complex association with chromatin by flow cytometry in yeast cells. Cells were arrested in G1-phase with alpha factor. Upon release into fresh media, samples were withdrawn every 15 min for 120 min at 30 °C. DNA content (left panel), DNA synthesis (EdU incorporation, middle panel) and MCM2-7 complex association with chromatin (right panel) were visualised. Cells synchronously enter S-phase within 30 min and finish replication within 60 min. Cell division and another round of DNA synthesis are observable from 75 min post G1-phase release and onwards. A strong signal for MCM2-7 is observed in G1-phase, representing the inactive double-hexamer (G1 panel). Upon initiation of replication MCM2-7 complexes mature into CMGs, travel along the DNA, and are gradually unloaded until DNA replication has finished in G2-phase (60 min panel). A low signal is retained and cells divide before MCM2-7 is again loaded onto chromatin (75 min panel). A second replicate is shown in Supplementary Figure 4. (B) Bar chart representing the cell cycle phase distribution of cells in (A). G1-, S-, and G2-phase populations were calculated using the DNA content panel employing the Jean-Dean-Fox modelling algorithm (Fox 1980). (C) The appearance of EdU-positive cells coincides with S-phase progression. (D) MCM2-7 association with chromatin allows cell cycle status prediction. Dot plots from (A, right panels) were analysed for MCM2-7 binding and classified into high, intermediate, and low signal. This correlates with the cell cycle phase distribution depicted in panel (B) and reproduces the wave-like pattern of synchronised cell cycle progression. DNA synthesis (C, indicated by EdU incorporation) correlates with the moving helicase (CMG) in S-phase and allows prediction of DNA replication initiation timing. Gating strategy for MCM2-7 classification was used as described (Supplementary Figure 3). a.u. = arbitrary units. Presented are the averages and standard deviations derived from four biological replicates.

To gain qualitative information on the DNA replication status, we quantified DNA content, EdU incorporation, and MCM2-7 chromatin binding (Supplementary Figures 3, 4 and Figure 3B–D). Relative cell cycle distributions were calculated from the DNA content using the Dean-Jett-Fox modelling algorithm and showed a typical progression of synchronised cells through the cell cycle (Figure 3B). Similarly, a continuous increase of EdU-positive cells was observed upon entering S-phase (Figure 3C). Finally, when quantifying the different populations of MCM2-7 binding, we found that the relative MCM2-7 population distribution recapitulated the cell cycle distribution (Figure 3D). Furthermore, the increase in EdU incorporation was highest at the point, when MCM2-7 complex signals started to decrease (compare Figure 3A, panels 30, 90, and 105 min with Figure 3C and D).

Furthermore, to test our protocol with other chromatin-associated proteins we choose Hta2 (Histone H2A) and Orc2 (ORC complex) as targets. Both proteins showed a bimodal distribution matching the DNA content of the samples (Supplementary Figure 6A and B). Hta2 showed a gradual increase with DNA synthesis and Orc2 produced a similar but less pronounced phenotype. These phenotypes were expected, as histones are continuously incorporated into newly replicated DNA within nucleosomes while ORC binds to free origin DNA. These data show that various proteins binding permanently or transiently to genomic DNA can be studied with our approach.

2.2.2 High-throughput cytometric analysis of DNA replication mutants in vivo

To further benchmark the robustness of the presented method, we sought to analyse mutants of DNA replication and their effects on the distribution of MCM2-7 on chromatin. To this end, we used two well-characterised, temperature-sensitive (ts) mutants cdc6-1 and cdc46-1 (mcm5) that, when shifted to the non-permissive temperature, exhibit a strong growth defect and cell cycle arrest in G1-phase (Aparicio et al. 1997; Hennessy et al. 1991; Tanaka et al. 1997, and Supplementary Figure 7A). Indeed, when shifting cycling cells to the non-permissive temperature, both cdc6-1 (Figure 4A) and cdc46-1 (Supplementary Figure 7B) cells produced a gradual arrest in G1-phase within 2 h. The typical MCM2-7 chromatin distribution (Figure 3A and 4B and Supplementary Figure 7C) was abolished, coinciding with an accumulation of low protein signal fraction in G1-phase (Figure 4C and Supplementary Figure 7D). As both mutants are known for failing to produce mature MCM2-7 DHs on DNA, the accumulating and low MCM2-7 signal observed in G1- and G2-phase (Figure 3A, 4 and Supplementary Figures 3–7) presents soluble, nuclear MCM2-7 complexes or subunits in a pre-licensing (G1-phase) or post-replicative (G2-phase) state.

Figure 4:

Cdc6-1 cells arrest in a pre-licensing state in G1-phase of the cell cycle under non-permissive growth conditions. Cells were grown to exponential phase, split into two flasks, and continuously shifted to 37 °C, the non-permissive temperature of cdc6-1, (A) or kept at 25 °C (B) for indicated times. DNA content (left panels) and MCM2-7 complex association with chromatin (right panels) were visualised. a.u. = arbitrary units. (C) Quantification of the relative population distribution (A and B) of MCM2-7 under permissive and non-permissive growth conditions. Presented are the averages and standard deviations derived from three biological replicates.

Taken together, the optimised conditions and the development of new protocols for detection of DNA content, DNA synthesis, and protein chromatin binding in a 96-well format allow for a fast, cost-effective, and accurate description of DNA replication parameters in budding yeast.

4.8.1 DNA content

400 µL of culture (OD₆₀₀ >0.5) were fixed with 1 mL 100 % ethanol in a 96-well deepwell plate and kept at 4 °C sealed with a foil overnight or stored at 4 °C for up to a few months. Cells were washed twice with 500 µL citrate buffer pH 7.0 (2,500 g, 2 min), the plate was decanted and hit gently onto a piece of paper to dry. Cells were resuspended in 500 µL citrate buffer pH 7.0 supplemented with RNaseA (10 mg/mL, 1/400 dilution) and incubated at 50 °C for 1 h. Then, 4 µL Proteinase K was added and the plate was incubated at 50 °C for 1 h. Subsequently, samples were either stored at 4 °C if needed or 100 µL of the cell suspension was transferred into a new U-shaped-bottom 96-well plate. After sonication to separate mother and daughter cells (70 % output 2 s on, 2 s off for 20 s for 8-tip horn), SYTOX™ Green (final [c]= 2 µM) or propidium iodide (final [c]= 25 ug/mL) was added to each well of the plate and mixed. Samples were measured on a flow cytometer equipped to measure 96-well plates (see Note 1).

4.8.2 DNA content and newly synthesised DNA

400 µL of culture (OD₆₀₀ >0.5) were fixed with 1 mL 100 % ethanol in a 96-well deepwell plate and kept at 4 °C sealed with a foil overnight or stored at 4 °C for up to a few months. Cells were washed twice with 500 µL citrate buffer pH 7.0 (2,500 g, 2 min), the plate was decanted and hit gently onto a piece of paper to dry. Cells were resuspended in 500 µL citrate buffer pH 7.0 supplemented with RNaseA (10 mg/mL, 1/400 dilution) and incubated at 50 °C for 1 h. Then, 4 µL Proteinase K was added and the plate was incubated at 50 °C for 1 h. Cells were spun for 2 min at 2,500 g and the cell pellet was resuspended 500 μL of 1✕ PBS+3 % BSA and incubated for 15 min (see Note 2). For the click reaction, samples were spun (2,500 g, 2 min), the supernatant was discarded, and the cell pellet was resuspended in 40 μL of freshly prepared dye azide mix. Per well, 27.8 μL of ultrapure water, 8 μL 5 ✕ PBS, 2 μL of CuSO₄ (100 mM), 0.2 μL of AlexaFluor 647 picolyl-azide (500 μM), and 2 μL of sodium ascorbate (200 mg/mL) (see Note 3) were mixed. The click mix was distributed and the reaction incubated in the dark for 30–60 min. Cells were washed once with 500 μL of 1✕ PBS+3 % BSA, then with 500 μL of 1✕ PBS. Finally, cells were resuspended in 150 μL (cycling cells) to 300 μL (remaining samples) of 1✕ PBS and 100 µL of the cell suspension was transferred into a new U-shaped-bottom 96-well plate. Sample sonication separated mother and daughter cells (70 % output 2 s on, 2 s off for 20 s for 8-tip horn). SYTOX™ Green (final [c]= 2 µM) or propidium iodide (final [c]= 25 μg/mL) was added to each well of the plate and mixed. Samples were protected from light and measured on a flow cytometer equipped to measure 96-well plates (see Note 1).

4.8.3 DNA content, newly synthesised DNA, and protein association with chromatin

10 µL of 10 % sodium azide (final [c]= 0.1 % w/v) were added to 1 mL of culture (OD₆₀₀ >0.5) in a 96-well deepwell plate to block mitochondrial respiration and inhibit cell growth. Cells were stored at 4 °C overnight or for up to few weeks until use. The plate was then removed from 4 °C and warmed up to RT. Cells were fixed with formaldehyde (final [c]= 1 % w/v) and incubated 30 min. The plate was centrifuged at 2,500 g for 2 min, the supernatant discarded and cells were washed with 1 mL of ultrapure water. After that, cells were resuspended in 500 µL spheroplasting buffer supplemented with DTT (final [c]= 10 mM) and Zymolyase 20T (final [c]= 10 U/mL). Next, the plate was incubated at 30 °C for 1 h without shaking and cell wall digestion was monitored (see Notes 4 and 5). The plate was spun (3,200 g, 2 min), the supernatant discarded, and cells were washed with 500 µL of spheroplasting buffer. Samples were centrifuged (3,200 g, 2 min), the supernatant discarded, and cells were resuspended in 50 µL permeabilisation buffer supplemented with the primary antibody (1/250, 4 µg; see Note 6). After incubation for at least 1 h, the plate was spun (3,200 g, 2 min), the supernatant discarded, and cells were washed with 250 µL permeabilisation buffer. Subsequently, cells were mixed with 50 μL permeabilisation buffer containing secondary antibody (1/200, 5 µg, fluorescently labelled; see Notes 6–8) and cells were incubated for at least 1 h in the dark. The plate was centrifuged (3,200 g, 2 min), the supernatant discarded, and cells were washed with 250 µL permeabilisation buffer. If EdU staining was not needed, the following steps (until SYTOX™ Green addition) were skipped, otherwise included. Having spun the plate and removed the supernatant, cells were resuspended in 250 μL permeabilisation buffer +3 % BSA and incubated for >15 min (see Note 2). For the click reaction, samples were spun (3,200 g, 2 min), the supernatant was discarded, and the cell pellet was resuspended in 40 μL of freshly prepared click mix. Per well, 27.8 μL of ultrapure water, 8 μL 5✕ PBS, 2 μL of CuSO₄ (100 mM), 0.2 μL of AlexaFluor 647 picolyl-azide (500 μM), and 2 μL of sodium ascorbate (200 mg/mL) (see Note 3) were mixed. The click mix was distributed and the reaction incubated in the dark for 30 min. Cells were washed once with 250 μL of 1✕ PBS +3 % BSA, then with 250 μL of permeabilisation buffer. Finally, cells were resuspended in 100 μL of permeabilisation buffer supplemented with RNaseA (10 mg/mL, 1/400 dilution), SYTOX™ Green (final [c]= 2 µM) or propidium iodide (final [c]= 25 μg/mL), and 100 µL of the cell suspension was transferred into a new U-shaped-bottom 96-well plate. Samples were protected from light and measured on a flow cytometer equipped to measure 96-well plates (see Note 1).

4.9.1 DNA content

The appropriate number of cells was grown in the respective medium. Confluency was avoided to ensure that all cell cycle stages were represented in the final analysis. Cells were washed with 1x DPBS, trypsinised, washed with 1x PBS, and diluted to 1–1.5✕10⁶ cells/mL in 1x PBS. Cells were then transferred to a 96-well deepwell plate, spun at 400 g for 3 min at 4 °C and resuspended in 50 μL 1✕ PBS before fixation by adding 1 mL of ice-cold 70 % ethanol dropwise while vortexing the plate on a low setting. Cells were covered with a foil incubated at 4 °C overnight or for at least 1 h before storage at −20 °C if cells were not used within the next days. After fixation, cells were centrifuged at 1,000 g for 10 min at 4 °C, washed with 1✕ PBS and resuspended in 100 μL of 1✕ PBS supplemented with RNaseA (10 mg/mL, 1/400 dilution) and SYTOX™ Green (final [c]= 2 µM) or propidium iodide (final [c]= 25 μg/mL). The cell suspension was transferred into a new U-shaped-bottom 96-well plate. Samples were incubated for 20 min while protected from light and finally analysed on a flow cytometer, where DNA content was measured.

4.9.2 DNA content and newly synthesised DNA

The appropriate number of cells was grown in the respective medium. Confluency was avoided to ensure that all cell cycle stages were represented in the final analysis. Cells were washed with 1✕ DPBS, trypsinised, washed with 1x PBS, and diluted to 1–1.5✕10⁶ cells/mL in 1✕ PBS. 1–1.5✕10⁶ cells were then transferred to a 96-well deepwell plate and spun at 400 g for 3 min at 4 °C and resuspended in 50 μL 1✕ PBS before fixation by adding 1 mL of ice-cold 70 % ethanol dropwise while vortexing the plate on a low setting. Cells were covered with a foil and incubated at 4 °C overnight or for at least 1 h before storing at −20 °C if cells were not used within the next days. After fixation, cells were centrifuged at 1,000 g for 10 min at 4 °C, washed once with 1 mL 1✕ PBS and 1 mL 1 % BSA in 1✕ PBS. Permeabilisation of cells was achieved by resuspending cells in 100 μL 1x permeabilisation buffer (eBioscience) and pulse-vortexing followed by 30 min incubation. The plate was centrifuged (400 g, 3 min) cells were washed once with 200 μL 1✕ permeabilisation buffer, before adding 50 μL of the click mix. To prepare the click mix, 33.75 μL of ultrapure water, 10 μL 5✕ PBS, 1 μL of CuSO₄ (100 mM), 0.25 μL of AlexaFluor 647 picolyl-azide (500 μM), and 5 μL of sodium ascorbate (200 mg/mL) (see Note 3) were mixed per well. Cells were treated in 50 μL of click mix and the reaction was incubated >30min in the dark. 500 μL 1✕ permeabilisation buffer was added and the plate was centrifuged (400 g, 3 min). The supernatant was discarded and cells were resuspended in 100 μL of 1✕ permeabilisation buffer supplemented with RNaseA (10 mg/ml, 1/400 dilution) and SYTOX™ Green (final [c]= 2 µM) or propidium iodide (final [c]= 25 μg/mL). The cell suspension was transferred into a new U-shaped-bottom 96-well plate. Samples were incubated for 20 min while protected from light and finally analysed on a flow cytometer, where. DNA content and newly synthesised DNA were measured.

4.9.3 DNA content, newly synthesised DNA, and protein association with chromatin

The appropriate number of cells was grown in the respective medium. Confluency was avoided to ensure that all cell cycle stages were represented in the final analysis. Cells were washed with 1✕ DPBS, trypsinised, washed with 1✕ PBS, and diluted to 1–1.5✕10⁶ cells/mL in 1✕ PBS. 1–1.5✕10⁶ cells were then transferred to a 96-well deepwell plate, spun at 400 g for 3 min at 4 °C and resuspended in 100 μL cold extraction buffer to pre-extract cytoplasmic proteins. After 7 min of incubation on ice, 1 mL PBS-B buffer was added on ice and the plate was centrifuged for 3 min, 400 g, at 4 °C. The supernatant was discarded and the cell pellet fixed in 100 μL fixation buffer for 20 min. The protocol was either continued directly, or cells were spun, the supernatant removed and cells resuspended in 1✕ PBS +3 % BSA and stored at 4 °C overnight, covered with a foil. If the plate was stored overnight, cells were washed once with 250 µL flow buffer before continuing. To stain chromatin-bound proteins, cells were washed with 500 μL flow buffer and resuspended in 50 µL flow buffer + 4 % milk powder with first antibody (1/250, 4 µg, see Note 9). After incubation for at least 1h, the plate was spun (400 g, 3 min), the supernatant discarded, and cells were washed with 250 µL flow buffer. Subsequently, cells were mixed with 50 μL flow buffer + 4 % milk powder containing diluted secondary antibody (1/200, 5 μg, fluorescently labelled) and the cells were incubated for at least 1 h in the dark at RT. The plate was centrifuged (400 g, 3 min, RT), the supernatant discarded, and cells were washed with 250 µL flow buffer + 4 % milk powder. If EdU staining was not needed the following steps (until SYTOX™ Green addition) were skipped, otherwise included. To prepare the click mix, 33.75 μL of ultrapure water, 10 μL 5✕ PBS, 1 μL of CuSO₄ (100 mM), 0.25 μL of AlexaFluor 647 picolyl-azide (500 μM), and 5 μL of sodium ascorbate (200 mg/mL) (see Note 3) per well were mixed. Cells were treated in 50 μL of click mix and the reaction was incubated >30 min in the dark. 500 μL flow buffer + 4 % milk powder was added thereafter and the plate was centrifuged (400 g, 3 min, RT). The supernatant was discarded and cells were washed with flow buffer (no milk powder) twice. Cells were then resuspended in 100 μL of flow buffer supplemented with RNaseA (10 mg/mL, 1/400 dilution) and SYTOX™ Green (final [c]= 2 µM) or propidium (final [c]= 25 μg/mL). The cell suspension was transferred into a new U-shaped-bottom 96-well plate. Samples were incubated for 20 min while protected from light and finally analysed on a flow cytometer, where DNA content, newly synthesised DNA, and chromatin-bound proteins were measured with appropriate settings.