Measurement of spatial proximity and accessibility of chromosomal loci in yeast using Cre/loxP site-specific recombination

Abstract

Several methods have been developed to measure interactions between homologous chromosomes during meiosis in budding yeast. These include cytological analysis of fixed, spread nuclei using fluorescence in situ Hybridization (FISH) (1, 2), visualization of GFP-labeled chromosomal loci in living cells (3), and Chromosome-Conformation Capture (3C) (4). Here we describe a quantitative genetic assay that uses exogenous site-specific recombination to monitor the level of homolog associations between two defined loci in living cells of budding yeast (5). We have used the Cre/loxP assay to genetically dissect nuclear architecture and meiotic homolog pairing in budding yeast. Data obtained from this assay report on the relative spatial proximity or accessibility of two chromosomal loci located within the same strain and can be compared to measurements from different mutated strains.

1.Introduction

The frequency of Cre-mediated recombination events (referred to here as “collisions”) between pairs of loxP sites engineered into the yeast genome provides a measure of the relative spatial proximity of those sites to one another in a living cell. A 13-fold level of induction of meiosis specific collision events in wild-type cells indicates a state of close, stable homolog juxtaposition (CSHJ) (6). From mutant analysis, we have shown that CSHJ is achieved primarily through the function of trans-acting factors required for early and intermediate stages of meiotic recombination (6–8).

Cre recombinase is supplied under the control of galactose-inducible promoter (pGAL1). Cre-mediated crossing over between loxP sites engineered into specific chromosomal loci gives a genetically detectable product (Fig. 1) (6). One loxP insert bears an upstream constitutive promoter (pGPD) and the other loxP insert bears a downstream open reading frame from a reporter gene (e.g. ADE2, URA3 or GFP). Collisions between pairs of loxP sites can be measured at allelic positions on homologous chromosomes and at ectopic positions on nonhomologous chromosomes in the same strain by using two different reporters. While ectopic collisions are consistently low, allelic collisions increase as cells progress through meiosis (6). Depending on the type of reporter used for the assay, the output is either the frequency of prototroph formation upon return-to-growth (RTG) on media supporting vegetative growth or the frequency of fluorescent spores. For the protocol described here, pGPD1-loxP inserted in Chromosome V recombines with loxP:ura3 inserted at an allelic site, or with loxP:ade2 on Chromosome VIII at an ectopic position to generate Ura+ or Ade+ prototrophs, respectively. Strains listed in Table 1 are available upon request.

Figure 1.

Table1.

Yeast Strains

Name	Genotype1
SBY 1438	MATα lys2::GAL1-Cre-LYS2   flo8::LEU2-pGPD1-loxP-lacZ ndt80::LEU2
SBY 1160	MATa lys2 flo8::LEU2-loxP-ura3
SBY 1338	MATa lys2 flo8::LEU2-loxP-ura3 ndt80::LEU2-loxP-ade2
SBY 2877	MATa lys2 arg4::LEU2-loxP-ade2 ndt80::LEU2
SBY 2879	MATα lys2::GAL1-Cre-LYS2  arg4::LEU2-pGPD-loxP ndt80::LEU2
SBY 2865	MATa lys2  flo8::LEU2-loxP-ade ndt80::LEU2
SBY 2867	MATa lys2  flo8::LEU2-loxP-ade
SBY 2021	MATα lys2::GAL1-Cre-LYS2  flo8::LEU2-pGPD1-loxP-lacZ
SBY 2061	MATα lys2  arg4::LEU2-loxP-ade2
SBY 2063	MATa lys2::GAL1-Cre-LYS2  arg4::LEU2-pGPD-loxP
SBY 1060	MATa lys2 arg4::LEU2-loxP-ura3
SBY 1780	MATa lys2::GAL1-Cre-LYS2  arg4::LEU2-loxP-ura3 ndt80::LEU-loxP-ade2
SBY 1755	MATa lys2  arg4::LEU2-loxP-ura3 ndt80::LEU2-loxP-ade2
SBY 1192	MATα lys2  ndt80::LEU2-pGPD1-loxP-lacZ
SBY 1693	MATalys2::GAL1-Cre-LYS2 arg4::LEU2-act1-loxP-GFP       ste7-1  cyh2-z MATα lys2       arg4::LEU2-GPD-act1-loxP-GFP ste7-1  CYH2
SBY 1476	MATa lys2::GAL1-Cre-LYS2 flo8::LEU2-pGPD1-loxP-lacZ ndt80::LEU2 MATα lys2         flo8::LEU2-loxP-ura3    ndt80::LEU2-loxP-ade2

¹All strains are SK1 and contain the following genotype: ho::hisG ura3D::hisG leu2::hisG ade2D::hisG trp1::hisG GAL3. Wild type SK1 is gal3 (15). FLO8 is on Chromosome V and NDT80 is on Chromosome VIII.

2.Materials

2.1 Synchronization of cells for entry into meiosis

1. All media and solutions are made with deionized water.

2. Solid growth media: YPG (1% yeast extract, 2% peptone, 3% glycerol, 0.004% tryptophan, 0.01% adenine sulfate, 2% agar). YPD is the same as YPG except in place of glycerol, dextrose is added from a filter-sterilized 40% (w/v) stock to a final concentration of 2% (w/v) after autoclaving. For all media, tryptophan and adenine sulfate are added from filter-sterilized 1% (w/v) stock solutions to autoclaved media cooled to 65°C. Keep 1% tryptophan stock at 4°C and shield from light. Stored 1% adenine sulfate will form crystals that can be melted by heating the solution.

3. Liquid growth medium: Liquid YPD is prepared as above except agar is omitted. YPA is 1% yeast extract, 2% peptone, 1% potassium acetate, 0.004% tryptophan, 0.01% adenine sulfate.

4. Synthetic complete (SC) powder: mix together 0.8 g adenine sulfate, 0.8 g arginine, 2 g aspartic acid, 0.8 g histidine, 2.4 g leucine, 1.2 g lysine, 0.8 g methionine, 2 g phenylalanine, 8 g threonine, 0.8 g tryptophan, 1.2 g tyrosine, 0.8 g uracil and grind to a fine powder with a mortar and pestle. SC-URA powder is made similarly except that uracil is omitted.

5. Liquid sporulation medium (SPM): 1% potassium acetate, 0.02% raffinose, 0.009% SC powder.

6. 18-cm and 25-cm test tubes

2.2 Cre induction and return-to-growth

1. SC-URA medium: 0.67% yeast nitrogen base without amino acids, 0.09 % SC-URA drop-out powder, 2% dextrose, 0.004% tryptophan, 0.01% adenine sulfate, 2% agar. Supplement with dextrose, tryptophan and adenine sulfate as described above.

2. YPD-ADE solid medium: same as YPD but without supplementation with adenine sulfate.

3. 2% (w/v) galactose is made from a 20% (w/v) filter-sterilized stock solution, 2% (w/v) glucose from a 40% (w/v) filter-sterilized stock solution.

4. 550 Sonic ZD-dismembrator (Fisher Scientific)

5. Glass pipets (1-mL and 0.2-mL)

6. Fixed volume or calibrated liquid dispenser

7. 16-cm test tubes

3. Methods

Cells from three independent colonies are cultured and synchronized to enter meiosis upon transfer to sporulation medium (SPM). Transfer of cells to SPM culture marks t = 0 h of the meiotic time course. At t = 1 h (about the time of DNA replication), Cre recombinase expression is induced by the addition of galactose. Sample aliquots are removed from the culture at t = 1 (before induction), 2, 4, 6, 8, and 10 h after transfer to SPM and diluted appropriately for plating on selective and nonselective media for RTG. The ndt80Δ mutant arrests in pachytene with full-length synaptonemal complexes and unresolved double-Holliday junction intermediates (9, 10). The use of the ndt80Δ mutation allows for recovery of cells in most mutants via RTG that would otherwise be inviable if allowed to complete meiotic divisions and sporulate (11–13). Random spore analysis can be performed with strains that are NDT80. Expression of the GFP reporter can be visualized using fluorescence microscopy in individual spores contained in an ascus due to the spore-autonomous expression of the GPD promoter (14).The number of prototrophs per colony forming unit (C.F.U.) or the number of fluorescent tetrads per total tetrads for each time point indicates the frequency of collisions. Comparison of C.F.U. at each time point relative to t = 1 h reports on survivability of cells by RTG.

3.1 Synchronization of cells for entry into meiosis

1. From frozen stocks stored in 15% (v/v) glycerol at −80°C, patch cells onto YPG plates. Incubate less than 15 h at 30°C (see Note 1).

2. From YPG plate, streak for single well-spaced colonies on YPD plates. Incubate 2 d at 30°C.

3. Inoculate 5 mL YPD (18-cm test tube) with single colony from YPD plate. In a typical experiment, analysis of each strain is carried out in triplicate and is performed in conjunction with a wild-type control strain, also in triplicate (see Note 2). Incubate at 30°C on roller drum at 56 rpm for at least 30 h.

4. In a 25-cm test tube, inoculate 10 mL of YPA to a final OD₆₀₀ of 0.23 (see Note 3). Incubate on roller drum at 30°C at 56 rpm for 14 h.

5. Examine cultures microscopically to determine whether vegetative growth has been arrested at G0/G1. Quantify the fraction of mitotic dividing cells, which are cells with small buds, for one culture of each strain. If more than 5% of cells are small-budded in any culture, consider incubating all of the cultures another 30 min. If more than 10% of cells have small buds, abort the experiment.

6. Pellet cells at 2,400g for 3 min.

7. Resuspend cells in 10 mL of sterile deionized water, pellet as described in Subheading 3.1, step 6.

8. Resuspend cells in 10 mL of SPM pre-warmed to 30°C and transfer to fresh 25-cm test tubes. Incubate on roller drum at 30°C at 56 rpm. The time at which the SPM cultures are placed back in the roller drum is designated as t = 0 h.

3.2 Cre induction and return-to-growth

1. Prepare one 16-cm test tube that contains 5 mL sterile deionized water and two 16-cm test tubes that contain 4.5 mL for each timepoint for each culture. These tubes will be used to perform the dilution series in Subheading 3.2, step 7. They can be prepared in advance using a fixed-volume water dispenser. We typically carry out analysis on four strains in triplicate so 36 test tubes are required for each time point.

2. At t = 1 h, remove 1 mL of uninduced cells from each culture and transfer to a 1.5 mL microcentrifuge tube.

3. After removing the first aliquot, induce Cre recombinase expression by adding 135 µL of 2% galactose into 9 mL of remaining culture (final concentration of 0.03% - see Note 4).

4. Place cultures back on rollerdrum to incubate at 30°C at 56 rpm. For GFP analysis, see Note 5.

5. Pellet the 1mL aliquot of cells for 1 min at 13,000g in a microcentrifuge and resuspend pellet in 1 mL of 2% glucose. For random spore analysis, see Note 6.

6. Briefly vortex resuspended cells.

7. To disperse cells, sonicate for 3 sec at setting 1.5 (15% maximum power) using the microtip of a 550 Sonic ZD-dismembrator.

8. Dilution series and plating: Add 25 µL of the sonicated cell suspension to a 16-cm test tube containing 5 mL of sterile deionized water. Vortex briefly. Transfer 0. 5 mL using a 1-mL pipet to a 16-cm test tube containing 4.5 mL water. This is the 10⁻³ dilution to be used for counting Ade⁺ prototrophs and Ura⁺ prototrophs. Transfer 0.5 mL from 10⁻³ dilution to 4.5 ml water. This is the 10⁻⁴ dilution to be used for determining C.F.U.s.

9. Briefly vortex the test tube before plating. Spread 0.2 mL from the 10⁻³ dilution onto YPD-ADE and SC-URA plates (see Note 7). From the 10⁻⁴ dilution, spread 0.2 mL onto YPD-ADE.

10. Incubate plates at 30°C.

11. Repeat Subheading 3.2, steps 5–10 at t = 2, 4, 6, 8, and 10 h.

12. Count the total number of colonies on 10⁻⁴ dilution plate after 2 d of growth (see Note 8).

13. Count white colonies (Ade⁺ prototrophs) on 10⁻³ dilution plate after 2 d of growth (see Note 9).

14. Count colonies (Ura⁺ prototrophs) on SC-URA plates after 4–5 d of growth.

3.3 Analysis

1. To calculate the frequency of ectopic collisions for each culture at each time point, divide the number of white colonies (Ade⁺ prototrophs) on the 10⁻³ plate by 10 times the total number of colonies on the 10⁻⁴ plate. To calculate the frequency of allelic collisions for each culture at each time point, divide the number of colonies (Ura⁺ prototrophs) on the 10⁻³ plate by 10 times the total number of colonies on the 10⁻⁴ plate. Viability of a strain is monitored by comparing colony-forming units (C.F.U.s) at each time point to C.F.U.s at the first time point. These calculations give the frequency of events per meiosis. If performing random spore analysis, the frequency is per chromatid.