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1Department of Biochemistry and Molecular Biology, Knight Cancer Institute, Oregon Health & Science University
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A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.
Keywords: Genetics, Issue 70, Biochemistry, Molecular Biology, Cellular Biology, Chromosome replication timing, fluorescent in situ hybridization, FISH, BrdU, cytogenetics, chromosome rearrangements, fluorescence microscopy
Smith, L., Thayer, M. Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization. J. Vis. Exp. (70), e4400, doi:10.3791/4400 (2012).
Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of >3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.
1. BrdU Incorporation (Terminal Labeling)
2. Chromosome Harvest of Monolayer Cells Cultures
3. RNase Treatment and Ethanol Dehydration
4. Preparation of Probe Cocktails for BAC Plus Chromosome-specific Centromere Enumeration Probe (CEP) or for Whole Chromosome Paints In situ Hybridization
5. Slide and Probe Denaturation and In situ Hybridization
6. Post Hybridization Washes
7. BrdU Detection
8. Capturing Images and Quantifying BrdU Incorporation
9. Nick Translation of BAC DNA for Fluorescent Labeling (Vysis)
An example of the replication timing analysis for human chromosome 6 is shown in Figure 2. Cells containing a deletion of the ASAR6 gene4, located at 6q16.1, were exposed to BrdU for 5 hr, harvested for mitotic cells and processed for FISH with a chromosome 6 paint probe (Vysis) and for BrdU incorporation. Note that there is a significant difference in the BrdU banding pattern between the two 6's, which is consistent with a delay in replication timing of >2 hr for one of the chromosome 6's [see4 for the replication timing banding pattern of chromosome 6 prior to the deletion]. In addition, there is a significant difference in the total amount of BrdU incorporation (pixels) when compared to a similar analysis of the DAPI staining (Figure 2D).
Another example of the replication timing analysis for human chromosome 6 is shown in Figure 3. Cells containing the same deletion of the ASAR6 gene4 shown if Figure 2 were exposed to BrdU for 5 hr, harvested for mitotic cells and processed for FISH with a chromosome 6 CEP probe plus a BAC containing the ASAR6 gene and for BrdU incorporation. Note that the deleted chromosome 6 (Δ6) displays more BrdU incorporation and a more extended banding pattern of BrdU incorporation than the non-deleted chromosome 6. Mitotic spreads from 7 different cells were processed as in Figure 3 and the pixel profiles for both DAPI and BrdU are shown in Table 1.
Figure 1. BrdU terminal label scheme. A typical S phase in mammalian cells last for 8 to 10 hr, and G2 is typically 2 to 5 hr. BrdU is added for increasing periods of time (green arrows) to label the last portions of the chromosomes to replicate. The chromosomes in mammalian cells replicate according to a temporal program, with early and late replication occurring at the beginning and end of S phase respectively (black line). Inactive X chromosomes are delayed in replication timing with the majority of DNA synthesis occurring during the second half of S phase (blue line). Chromosomes with delayed replication timing are delayed in both initiation and completion of DNA synthesis, with active replication continuing through G2 (red line). Asynchronous cultures are harvested for mitotic cells and processed for BrdU incorporation and FISH.
Figure 2. Asynchronous replication of human chromosome 6. Cells containing an engineered deletion of the ASAR6 gene4 were treated with BrdU for 5 hr, harvested for mitotic cells and processed for BrdU incorporation and FISH using a chromosome 6 paint as probe. The DNA was stained with DAPI (blue). A) A mitotic spread containing a typical "banded" pattern of BrdU incorporation (green) is shown. The chromosome 6 paint probe hybridized to two chromosome 6's (red) in this cell. B) The two chromosome 6's (i and ii) were "cut out" and displayed with the three fluorescent labels separated into distinct images. C) The chromosome 6's were analyzed using the Cytovision software and the signal intensity profiles for both DAPI (blue) and BrdU (green) are shown. The red line indicates the path used, from short arm (p) to long arm (q) for the quantification of both BrdU and DAPI. The Distance refers to the length of each chromosome from short arm (p) to long arm (q) in pixels. D) Quantification of the total signal for both DAPI and BrdU fluorescence. The total values represent the average pixel intensity multiplied by the area represented by those pixels.
Figure 3. Delayed replication of human chromosome 6 containing a deletion of ASAR6. Cells containing an engineered deletion of the ASAR6 gene4 were treated with BrdU for 5 hr, harvested for mitotic cells and processed for BrdU incorporation and FISH using a chromosome 6 CEP plus a BAC containing the ASAR6 gene as probes. The DNA was stained with DAPI (blue). A) A mitotic spread containing a typical "banded" pattern of BrdU incorporation (green) is shown. The chromosome 6 CEP probe hybridized to two chromosome 6's (large red centromeric signal), and the BAC hybridized to a single chromosome 6 (small red signal on the long arm) in this cell. Note the difference in the BrdU banding pattern between the two 6's. B) The deleted chromosome 6 is represented by Δ6 and the non-deleted 6 by 6. The two 6's were "cut out" and displayed with the three fluorescent labels separated into distinct images. C) The chromosome 6's were analyzed using the Cytovision software and the signal intensity profiles for both DAPI (blue) and BrdU (green) are shown. The red line indicates the path used, from short arm (p) to long arm (q) for the quantification of both BrdU and DAPI. The Distance refers to the length of each chromosome from short arm (p) to long arm (q) in pixels. D) Quantification of the total signal for both DAPI and BrdU fluorescence. The total values represent the average pixel intensity multiplied by the area represented by those pixels.
Figure 4. Quantification of the replication timing difference between chromosome 6's. Cells containing an engineered deletion of the ASAR6 gene4 were treated with BrdU for 5 hr, harvested for mitotic cells and processed for BrdU incorporation and FISH using a chromosome 6 CEP probe plus a BAC containing the ASAR6 gene as probe. Mitotic spreads were processed as in Figure 3 and the values for 7 different cells are shown (see Table 1). A) The DAPI staining was quantified and the total number of pixels is displayed for each cell. Note that there is only ~10-20% difference between chromosomes within the same cell. B) The BrdU incorporation was quantified from the same chromosomes as panel B above. Note that there is >2 fold difference between the total pixel values for the deleted and non-deleted chromosome 6's.
The preparation of chromosome spreads is a critical step for the successful replication-timing assay described here. The inclusion of a colcemid pretreatment step prior to the hypotonic treatment may aid in the frequency and spreading of mitotic cells. We typically expose cells to colcemdi for 1-3 hr prior to harvest, and use colcemid at a final concentration of 10 ug/ml. However, the inclusion of a colcemid pretreatment step may alter the length of G2 and consequently may alter the apparent replication timing and state of condensation of the chromosomes3.
This procedure can be applied to many different cell types and species by varying the length of BrdU incubation, which depends on the cell cycle duration. For most human and mouse cell lines, the G2 phase is typically 3-6 hr; therefore, BrdU treatments are typically in this range. An alternative to BrdU is 5-ethynyl-2'deoxyuridine (EdU) and its subsequent detection using a fluorescent azide and "click chemistry" reaction10. The EdU detection scheme has several advantages over the BrdU detection scheme. For example, detection of EdU does not require sample fixation or DNA denaturation. Thus, the use of EdU to asses replication timing can be combined with simple G-banding techniques instead of FISH to identify the chromosomes of interest.
The replication timing protocol described here is specifically designed to assay late S phase plus any DNA synthesis extending into G2. In addition, DNA replication can be monitored throughout S phase using this procedure by using short (15-30 min) pulses of BrdU followed by relatively long chase periods of 6-10 hr. This allows for the visualization of BrdU incorporation in both early and middle S-phase. For example, certain tumor derived chromosome rearrangements are delayed in both the initiation and completion of DNA synthesis along the entire length of the chromosomes3.
One advantage of this replication timing procedure is that it assays replication timing in individual cells. Therefore, it has the ability to detect differences in replication timing between homologous chromosomes contained within the same cell. While there are other procedures, e.g. replication timing in situ hybridization (ReTiSH;11), that have the ability to detect differences in replication timing between alleles at specific loci on homologous chromosomes, the procedure described here can detect differences in the replication timing along the entire length of chromosomes. In addition, this procedure can assay differences in the replication timing of chromosomes that are present in only a fraction of cells of a population3. For example many cancer cell lines and primary tumor samples contain chromosome rearrangements that are present in less than 50% of cells. We are currently using this procedure to assay chromosomes in primary tumor samples, and have been able to detect asynchronous replication between chromosomes in multiple samples. However, given that primary tumor samples have a limited number of mitotic figures, about one third of the primary cultures failed to give sufficient numbers of mitotic spreads.
Another advantage that this procedure has over microarray or sequencing based assays is that individual chromosomes are assayed rather than immunoprecipitated DNA from pools of cells. In the immunoprecipitation-based assays polymorphisms must be identified and linked to specific alleles in order to distinguish the replication timing between alleles.
Furthermore, with the recognition that many cancer cells contain numerous chromosome rearrangements12, and the observation that DNA replication stress is associated with genomic instability in cancer cells13, we believe that this protocol is a useful and simple tool for the routine analysis of the replication timing of chromosomes in cancer cells.
No conflicts of interest declared.
This work was supported by a grant from the National Cancer Institute, CA131967.
|Anti-BrdU-FITC||Roche Millipore||11202593001 MAB326F||50 μg/μl|
|Nick Translation Kit||Abbott Molecular (Vysis)||07J00-0001|
|Spectrum Orange dUTP||Abbott Molecular (Vysis)||02N33-050|
|CEP||Abbott Molecular (Vysis)||Varies|
|LSI/WCP hybridization buffer||Abbott Molecular (Vysis)||06J67-011|
|CEP hybridization buffer||Abbott Molecular (Vysis)||07J36-001|
|Chromosome paints||MetaSystems Group||D-14NN-050-TR|
|Olympus BX61 Fluorescent Microscope||Olympus||BX61TRF-1-5|
|Microscope imaging software system||Applied Imaging||Cytovision 3.93.1|
IN SITU HYBRIDIZATION RECIPES
35 ml Formamide* (Sigma)
* It is important to use formamide that has been stored at -20 °C. Prolonged room temperature storage will generate formic acid and the pH will be too low.
50% Formamide/2x SSC
25 ml formamide (Sigma)
20x SSC, 4 L
702 g NaCl (Sigma)
PN Buffer [0.1 M NaP04 0.1% NP_40 (Sigma)]
Make a 0.1 M solution each of sodium phosphate (Filter sterilize and store in 500 ml aliquots).
0.1 M NaH2P04 , 1 L
13.8 g NaH2P04 (Sigma):
0.1 M NaH2P04 1 L
14.2 g NaH2P04 (Sigma)
PN: Adjust pH of 0.1 M Na2HP04 to pH 8.0 with .1 M NaH2P04. Filter sterilize and add 1 ml of NP-40.
PNM 50 ml
1.25 g Non-fat dry milk (Sigma)
Mix for 15-20 min with constant stirring. Spin 2 times at 400 x g for 10 min. Use supernatant, and make sure not to disturb precipitated milk proteins.
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