Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Summary

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.

Abstract

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 chromosome^2,3. 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.

Protocol

1. BrdU Incorporation (Terminal Labeling)

1. Plate cells to about 70% confluence in a 150 mm tissue culture dish 24 hr prior to the addition of BrdU.
2. Replace media with fresh complete media containing 20 μg/ml BrdU (Sigma) at appropriate time points prior to harvest. The length of time cells are cultured in media with BrdU will vary with cell type and species, typically the G2 phase lasts between 2 and 5 hr (Figure 1).

2. Chromosome Harvest of Monolayer Cells Cultures

1. Remove culture media from the plate and collect 10 ml in a 15 ml conical centrifuge tube. Discard remaining media.

Results

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 .......

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 Formamide Solutions 70% Formamide/2x SSC 35 ml Formamide* (Sigma) 10 ml 10x SSC 5 ml d2H20 pH to 7.0 with HCl (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) 10 ml 10x SSC 15 ml dH2O pH to 7.0 with HCl (Sigma) 20x SSC, 4 L 702 g NaCl (Sigma) 358 g Na Citrate (Sigma) dH2O to volume 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): dH2O to volume 0.1 M NaH2P04 1 L 14.2 g NaH2P04 (Sigma) dH2O to volume. 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) 25 ml PN buffer (Recipe above) 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.