Name: genome-wide determination of mammalian replication timing by dna content measurement
Uploaded: 2017-01-19T14:00:00
Description: Watch this Scientific Journal Video about Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement at JoVE.com

We thank Oriya Vardi for assistance in generating figures. Work in the IS group was supported by the Israel Science Foundation (grant No. 567/10) and the European Research Council Starting Grant (#281306).

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 &#8212; 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 ne...

<ol>
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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...

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 &#8212; 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 differentiation3,4 and to a lesser extent also during cancer transformation5. Moreover, certain genomic regions replicate asynchronously6,7,8, 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 types10,11 and therefore unsurprisingly, perturbations of the replication program are linked to cancer12,13. 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 regions1,14. 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 allele15,16. 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 sequencing2,8. 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.

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genome-wide determination of mammalian replication timing by dna content measurement

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 &#34;Copy Number Ratio of S/G1 for mapping genomic Time of Replication&#34; 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.

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 ...

Watch this Scientific Journal Video about Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement at JoVE.com

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

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.

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 ...

The overall goal of this procedure, is to map the genome-wide replication timing program of a cell of choice. This method can help answer key questions in the replication field. Such as, how the genomic replication program changes in health and disease.The main advantage of this technique is that it is a fast and simple way to map replication timing in various cells, and conditions. The man writing the fax procedure would be Dan Lehmann, a fax expert in The Faculty of Medicine. This procedure usually starts with at least one to two times ten to the sixth fast growing unsynchronized cells.For adherent cells, aspirate and wash each plate with three milliliters of PBS without calcium of magnesium. Discard the PBS and add one milliliter of commercial Trypsin-EDTA to each plate. Incubate the cells for five minutes, at 37 degrees Celsius, until they detach.Add three milliliters of culture media to neutralize the Trypsin and collect the cells in a 15 milliliter conical tube or a five milliliter polystyrene tube. Keep the cells on ice. To begin theisprocedure, centrifuge the cells at 300 x G for five minutes at four degrees Celsius.Aspirate and wash the cells with one milliliter of cold PBS. Then, spin down the cells. After the second wash, remove the PBS and resuspend the cells in 250 microliters of cold PBS.While gently vortexing the tube, slowly add drop wise 800 microliters of minus 20 degree Celsius, 100 percent high purity ethanol. This leads to our final ethanol concentration of 70 to 80 percent. Incubate the cells on ice for 30 minutes.Begin this procedure by centrifuging the fixed cells at 500 x G for ten minutes at 4 degrees Celsius. Aspirate the supernatant carefully and wash the cells twice with one milliliter of cold PBS. Spin down after each wash.After the second wash, aspirate the supernatant and resuspend each cell with the following PI mixture. One milliliter of PBS containing five microliters of ten milligram per milliliter RNAas, and 50 microliters of one milligram per milliliter propidium iodide. Filter the sample through a 35 micrometer mesh into a 5 milliliter polystyrene tube.Seal the tube with parafilm. Incubate at room temperature for 15 to 30 minutes. Keep the samples in the dark as propidium iodide is light sensitive.The cells will be sorted using a fax machine. Use the 561 nanometer laser to differentiate cells based on their propidium iodide intensity. For optimal results, use the smallest nozzle recommended for the specific cell size.For most cells, it is 85 micrometers. Use a steady, slow flow. Usually up to 300 to 500 events per second.Using Gating, discriminate dead cells and subcellular debris by plotting FCS versus SSC. From the viable cells, discriminate doublets by plotting SSC width versus SSC height, followed by an FFC width versus FFC height plot, and a propidium iodide width versus propidium iodide height plot. For the viable signal cells, draw a histogram of the propidium iodide area intensity, which represents the DNA content of the cells.Sort cells in cold conditions into G1 and S phases. 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.For come cell types, it may be hard to get a nice cell cycle distribution. It is most critical to take the G1 phase without S phase cells. Remove tubes with sorted cells from the fax machine.Keep tubes on ice following the sort. Following the cell sorting, the DNA for each sample is purified using a commercial DNA purification kit following the manufacturer's directions. Next, sheer the DNA with a focused ultrasonicator to an average target peak size of 250 base pairs.Verify the size of the sheer DNA by electroforesis. The recommended size distribution is 200 to 700 base pairs with a peak at about 250 base pairs. Subsequently, the library is prepared, sequenced, and analyzed as described in the text protocol.A time of replication map for the entire chromosome one from mouse embryonic fibroblasts is shown. With a closer look at a region of approximately 50 megabases. The dots represent the normalized S to G1 ratio for individual windows, and the solid line represents the results from the cubic smoothly and interpolation.High S to G1 values corresponds to early replication, whereas low values correspond to late replication. The red arrows point to examples of constant time of replication regions, and the green arrows point to temporal transition regions. This figure shows the reproducibility of time of replication maps between triplicates of mouse embryonic fibroblasts, compared to triplicates of mouse pre-B cells in the same 18 megabase region on mouse chromosome one.The orange arrows, point to examples of differential regions between the cell lines. A heat map of the spearman correlations between the six samples further confirm the results. Once mastered, this technique from the collection of cells to the generation of maps, can be completed in one week if performed properly.After watching this video, you should have a good understanding of how to generate time of replication genomic maps for growing cells.

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