Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

Summary

We describe here a relatively fast and simple approach for mapping genome-wide mammalian replication timing, from cell isolation to the basic analysis of the sequencing results. A genomic map of a representative replication program will be provided following the protocol.

Abstract

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic region is replicated at a distinct time during S phase through the simultaneous activation of multiple origins of replication. Time of replication (ToR) correlates with many genomic and epigenetic features and is linked to mutation rates and cancer. Comprehending the full genomic view of the replication program, in health and disease is a major future goal and challenge.

This article describes in detail the "Copy Number Ratio of S/G1 for mapping genomic Time of Replication" method (herein called: CNR-ToR), a simple approach to map the genome wide ToR of mammalian cells. The method is based on the copy number differences between S phase cells and G1 phase cells. The CNR-ToR method is performed in 6 steps: 1. Preparation of cells and staining with propidium iodide (PI); 2. Sorting G1 and S phase cells using fluorescence-activated cell sorting (FACS); 3. DNA purification; 4. Sonication; 5. Library preparation and sequencing; and 6. Bioinformatic analysis. The CNR-ToR method is a fast and easy approach that results in detailed replication maps.

Introduction

Mammalian DNA replication is tightly regulated to ensure the precise replication of each chromosome exactly once during the cell cycle. Replication occurs according to a highly regulated order — multiple large genomic regions (~Mb) replicate at the beginning of S phase (early replicating domains) whereas other genomic regions replicate later at middle or late S phase (middle and late replicating domains)¹. Most of the genome replicates at the same time in all tissues (constitutive ToR domains), whereas 30% - 50% of the genome, changes its ToR between tissues², during differentiation^3,4 and to a lesser extent also during cancer transformation⁵. Moreover, certain genomic regions replicate asynchronously^6,7,8, namely there is a difference in the ToR between the two alleles.

ToR correlates with many genomic and epigenomic features including transcription levels, GC content, chromatin state, gene density, etc.^1,9. ToR is also associated with mutation rates and types^10,11 and therefore unsurprisingly, perturbations of the replication program are linked to cancer^12,13. The causal relationship between ToR and chromatin structure is not yet understood. It is possible that open chromatin facilitates early replication. However, an alternative model suggests that the chromatin is assembled during replication and the different chromatin regulators present at the beginning and end of S phase lead to differential packaging of early and late replicating regions^1,14. We have recently shown that the ToR shapes the GC content by affecting the type of mutations that occur in different genomic regions¹¹.

Fluorescence in situ hybridization (FISH) is the main method for measuring ToR at individual loci. It is performed simply by counting the percentage of S phase cells that exhibit single FISH signals vs. the percentage of doublets for a given allele^15,16. An alternative method, consists of pulse labeling the DNA with BrdU, sorting cells according to their DNA content to multiple time points along S, immunoprecipitating DNA containing BrdU, and checking the abundance of precipitated DNA with qPCR¹⁷.

Genomic ToR mapping can be achieved by two methods. The first method is a genomic version of the BrdU-IP based method described above, in which the quantification of the amount of precipitated DNA in each fraction is done simultaneously for the entire genome through hybridization to microarrays or by deep sequencing. The second method, CNR-ToR, is based on measuring the copy number of each genomic region of S phase cells and normalizing by the DNA content in G1 cells. In this method, cells are sorted by FACS into non-replicating (G1 phase) and replicating (S phase) groups (Figure 1). Cells in G1 have the same copy number in all genomic regions and thus their DNA content should be the same. On the other hand, the DNA copy number in S depends on the ToR, since early replicating regions underwent replication in most cells and therefore their DNA content is doubled, whereas late replicating regions have not replicated yet in most cells and therefore their DNA content will be similar to that of G1 cells. Hence the S to G1 ratio of DNA content is indicative of the ToR. The amount of DNA for each genomic region is measured either by hybridization to microarrays or by deep sequencing^2,8. The advantages of the CNR-ToR method will be further discussed.

This paper describes the CNR-ToR method for genomic ToR mapping as described in Figure 2. The paper discusses the fine details of the entire process from collecting cells until the basic analysis of the results and the creation of genomic ToR maps. The protocol described in this paper has been successfully performed on various cell types grown in culture. Future improvements of this protocol can lead to the mapping of the ToR in vivo and in rare cell types.

Protocol

Note: ToR can be measured only on growing, unsynchronized cells. The procedure should begin with at least 1 - 2 x 10⁶ fast growing cells, which will usually result in ~1 x 10⁵ cells in S phase (the rate limiting step). It is recommended to conduct each experiment using two or three replicates. The entire process of CNR-ToR can be completed within one week — two days should be dedicated to all steps up to library preparation, one to two days are needed for sequencing and an additional day is necessary for the initial data analysis.

1. Collection of Cells from Culture

NOTE: The protocol is written for cells growing in culture in 10 cm plates (containing approximately 2 - 5 x 10⁶ cells), but can be easily adjusted to other platforms.

1. For cells that were grown in suspension, proceed to fixation (section 2).
2. For adherent cells, aspirate and wash the plate with 3 mL PBS without Ca²⁺ and Mg²⁺.
3. Discard the PBS and incubate the cells for 5 min at 37 °C with 1 mL commercial trypsin-EDTA until cells detach.
   NOTE: The duration of the trypsin treatment should be adjusted to each cell type.
4. Add 3 mL culture media to neutralize the trypsin and collect cells in a 15 mL conical tube or a 5 mL polystyrene tube. Keep on ice.

2. Fixation

NOTE: For this part, all steps should be done at 4 °C.

1. Centrifuge the cells at 300 x g for 5 min at 4 °C.
2. Aspirate and wash cells twice with 1 mL cold PBS.
3. Resuspend cells in 250 µL (total) cold PBS.
4. While gently vortexing the tube, slowly add dropwise 800 µL of -20 °C 100% ethanol. This leads to a final ethanol concentration of 70 - 80%.
   NOTE: High purity ethanol is recommended at this step.
5. Incubate cells on ice for 30 min.
   NOTE: At this stage cells can be kept for a few days at 4 °C or for a few months at -20 °C.

3. PI Staining

1. Centrifuge the cells at 500 x g for 10 min at 4 °C.
2. Aspirate the supernatant carefully and wash cells twice with 1 mL cold PBS.
3. Aspirate and resuspend each sample with the following mixture: 1 mL PBS, 5 µL 10 mg/mL RNaseA, 50 µL 1 mg/mL propidium iodide (PI; mix bottle before use). Adjust concentration up to ~2 x 10⁶ cells/mL).
   NOTE: Keep out of light — PI is light sensitive.
4. Filter through 35 µm mesh to a 5 mL polystyrene tube, and close with Parafilm.
5. Incubate at RT in the dark, for 15 - 30 min.
   NOTE: Cells are now ready for FACS analysis. If necessary, the stained cells can be stored for at least 24 h at 4 °C in the dark.

4. Sort

1. Sort cells using a FACS machine. Use the 561 nm laser to differentiate cells based on their PI intensity. Other lasers near the 535 nm excitation maximum like 488 nm or 532 can be used depending on the FACS machine configuration.
2. For optimal results, use the smallest nozzle recommended for the specific cell size (for most cells 85 µm). Prioritize purity modes over yield. Use a steady slow flow, usually up to 300-500 events/s, with a sheath pressure of 45 psi.
3. Using gating, discriminate dead cells and subcellular debris (low FCS and high SSC) by Plotting FCS vs SSC. From the viable cells discriminate doublets by plotting SSC-Width (W) vs SSC-Height (H) followed by a FSC-W vs FSC-H plot and by PI- W vs PI-H (doublets will have the same H-value but bigger W-value). For the viable single cells draw a histogram of the PI-Area (A) intensity which represents the DNA content of the cells.
4. Sort cells into G1 and S phases, as shown in Figure 1. Gating for S should be wide and intrude into the G1 and G2 phases, while G1 gating should be narrow and as far from S as possible.

Figure 1. Cell cycle phase determination based on PI intensity. Histogram showing the distribution of the cellular DNA content (measured by PI-Area) of mouse embryonic fibroblast (MEF) population. The DNA content is used to sort the population into two sub-populations i) G1 cells (2N DNA content) and ii) S phase cells (2N - 4N DNA content), using the marked regions. Please click here to view a larger version of this figure.

NOTE: The purpose of the collection of G1 cells is to account for biases in the sequencing efficiency between different genomic regions. An alternative approach is to use G1 arrested cells from the same cell type. This approach gives cleaner results (since it minimizes S phase contamination) but it may introduce biases stemming from genetic differences between the arrested cells and the measured cells.

5. Sort in cold conditions and collect sorted cells into 1.5/2 mL tubes. Keep tubes on ice following the sort.
   NOTE: In order to improve DNA recovery, it is better to use low binding tubes or to use tubes coated with 4 - 5% BSA for 1 - 3 h at 4 °C ¹⁸.

5. DNA Purification

1. For each sample (G1 and S) purify DNA using a DNA purification kit.
   NOTE: When using the commercial kit, elute with 400 µL elution buffer into a 2 mL tube for high yield, as recommended by the manufacturer.
2. Check DNA concentration using fluorometer.
   NOTE: From 100,000 mammalian cells collected by the FACS, one should get ~ 1 µg of DNA. At this stage DNA can be kept for a few days at 4 °C or at -20 °C for long storage.

6. Sonication

1. Transfer DNA into a 1.7 mL tube that is compatible with the magnetic stand used.
2. Concentrate DNA using 2x SPRI beads according to the manufacturer's instructions, using a magnetic stand, and elute in 50 µL elution buffer.
3. Shear DNA with a focused ultrasonicator to an average target peak size of 250 bp. Use the following settings for a 50 µL DNA sample: 50 W, 20% Duty factor, 200 Cycles per burst, 20 °C, 120 s.
4. Verify the size of the sheared DNA by electrophoresis. The recommended size distribution is 200 - 700 bp with a peak at ~ 250 bp.
   NOTE: At this stage DNA can be kept for a few days at 4 °C or at -20 °C for long storage.

7. Library Preparation, and Sequencing

NOTE: Many library preparation kits and different sequencing platforms should work similarly to the ones used by us and mentioned in the materials section. Actually in the past, ToR maps were generated using a very similar method with microarray platforms².

1. Prepare libraries using any commercial library preparation kit.
2. At the end of library preparation, select size using magnetic beads for 300 - 800 bp.
3. After preparing libraries, measure DNA concentration using fluorometer.
4. Measure DNA size using electrophoresis.
5. Perform sequencing on any platform.
   NOTE: Sequencing at least 10 M reads per sample is recommended. This depth is equivalent to a read approximately every 300 bp (for ~3 Gb size genomes), and is sufficient for ToR measurements at a resolution of 50 - 100 kb. Increasing the depth will result in a decrease in the size of the windows and thus will enable increasing resolution with higher certainty. Paired end sequencing is not necessary in this protocol since only coverage information is collected. It, however, may help in resolving the location of reads that contain repetitive sequences.

8. Analysis

NOTE: Data analysis is based on the method used by A. Koren et al.¹⁹.

1. Map sequencing data to the corresponding genome using bowtie2 or any reliable short read aligner. Define varying-size, equal-coverage chromosomal windows as segments covered by 200 reads in the G1 fraction and count S phase reads in the same windows.
2. Calculate the S/G1 ratio for each window. This should generate a map with large fluctuations in the S/G1 ratio along the genome (Figure 3). A good control for the reliability of the ToR measurements is to compare this map to the G1/G1 ratio (from two separate measurements of G1) which should be much flatter.
3. Normalize data to 0 mean and 1 SD by subtracting from each value the average value of all windows (excluding the X chromosome) and dividing the results by the standard deviation of the S/G1 of all windows. This is done in order to convert to z scores and enable comparison between different experiments.
4. Remove all gap regions listed by the UCSC genome browser as well as each remaining inter gap fragment containing fewer than 15 data windows.
5. Smooth the remaining fragments with a cubic smoothing spline via the Matlab function csaps with a parameter of 10⁻¹⁶ and interpolate at set points every 100 kb.
   NOTE: Parameters of smoothing and interpolation should be adjusted based on the depth of the data. Other suitable smoothing methods and functions exist and can be used.
6. After visually confirming the reliability of each replicate, merge all the reads and calculate a deeper resolution profile by performing the same process described above on this data.

Results

A typical ToR map is shown in Figure 3 for mouse embryonic fibroblasts (MEFs). This figure demonstrates the analysis process since it shows both the dots, which are the normalized S/G1 ratio for individual windows (step 8.3), as well as the line which results from the cubic smoothing and interpolation (step 8.5).

Such maps capture the organization of the replication program, which is a patchwork of two types of ...

Discussion

CNR-ToR can be performed in principle on any eukaryotic proliferating cell population that can be divided by FACS to S and G1 phases (reviewed by Rhind N. and Gilbert DM²⁰). The method described here has been adjusted to mammalian cells with a genome size of ~3 Gb such as human and mouse. Small changes in the CNR-ToR protocol (in cell preparation and sequencing depth) are needed, in order to adjust it to other eukaryotes. Attention must be paid to the collection of sufficient amounts of S phase ce...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PBS	BI (Biological Industries)	02-023-1A
Trypsin-EDTA	BI (Biological Industries)	03-052-1B
15 mL conical tube	Corning	430790
5 mL Polystyrene round Bottom tube with cell strainer cap	BD-Falcon	352235
Ethanol	Gadot	64-17-5
RNAse-A 10 mg/mL	Sigma	R4875
Propidiom iodide 1 mg/mL	Sigma	P4170
Parafilm	Parafilm	PM-996
1.5 mL DNA LoBind Eppendorf tubes	Eppendorf	22431021
BSA	Sigma	A7906
1.7 mL MaxyClear tube	Axygen	MCT-175-C
magnetic beads - Agencourt AMPure XP	Beckman Coulter	A63881
Ultrasonicator	Covaris	M-series  -530092
50 µL microTUBE AFA Fiber Screw-Cap 6 x 16 mm	Covaris	520096
Qubit fluorometer	Invitrogen
Qubit dsDNA High Sensitivity (HS) Assay Kit	Invitrogen	Q32854
Electrophoresis 2200 Tape station system	Agilent	D1000 ScreenTape
Seqeuncing - Illumina NextSeq system	Illumina	SY-415-1001
Dneasy kit for DNA purification	Qiagen	69504
PureProteom Magnetic Stand	Millipore	LSKMAGS08
Anti-BrdU/FITC	DAKO	F7210
FACS sorter	BD	FACSARIA III
FACS software	BD	FACSDiva v 8.0.1