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.
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.
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 …
The authors have nothing to disclose.
This work was supported by a grant from the National Cancer Institute, CA131967.
Name of Reagent | Company | Catalogue Number | Comments (optional) |
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 | |
Digital Camera | Olympus | UCMAD3 | |
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. |