Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Summary

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

Abstract

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Introduction

The genetic tools and resources available in the model organism Saccharomyces cerevisiae have enabled large-scale functional genomics studies that collectively provide new insight into how genes function as networks to fulfill the requirements of biological systems. The cornerstone of these tools was the collaborative creation of a complete set of non-essential gene deletions of all open reading frames in yeast^1,2. A striking observation was that only ~20% of yeast genes are required for viability when grown as haploids under standard laboratory conditions. This highlights the ability of a cell to buffer against genomic perturbations through the utilization of alternative biological pathways. Genetic mutants that are viable individually, but lethal in combination, signal connected or convergent parallel biological pathways and form genetic interaction networks that describe biological function. With the development of conditional temperature-sensitive and hypomorphic alleles of essential genes the technology has not been limited to the study of non-essential genes^3,4. This concept has been applied at a genomic scale producing an unbiased genetic interaction map illustrating how genes involved in similar cellular processes cluster together⁵.

Chemical perturbations of genetic networks mimic gene deletions (Figure 1)⁶. Querying growth-inhibitory compounds against a high-density array of deletion strains for hypersensitivity identifies a chemical-genetic interaction profile, i.e. a list of genes that is required to tolerate chemical stress. Like genetic interactions, large-scale screens of chemical libraries have shown that compounds with a similar mode of action cluster together⁷. Therefore, by establishing the chemical-genetic interaction profile of a compound the mode of action may be inferred by comparing it with large-scale synthetic genetic and chemical genetic interaction datasets^8,9.

Large-scale chemical-genetic screens, where scores of compounds are interrogated, have been performed by barcode competition assays. In this approach, the pooled collection of deletion strains is grown en masse for several generations in a small volume of media containing a chemical. Since each deletion mutant harbors a unique genetic barcode, the viability/growth of individual mutants within the pool of deletion strains is tracked by microarray or high-throughput sequencing¹⁰.

Inferring fitness by monitoring colony size of physically arrayed mutants grown on solid agar containing a bioactive compound is also an effective method to identify chemical-genetic interactions^11,12. This approach provides a cost-effective alternative to competition-based screening and is well suited for assaying small libraries of chemicals. Outlined here is a simple methodology for producing a list of chemical-genetic interactions in S. cerevisiae that does not rely on molecular biology manipulations or infrastructure. It requires only a yeast deletion collection, a robotic or manual pinning apparatus, and freely available image analysis software.

Protocol

NOTE: The general workflow of this procedure is outlined in Figure 2.

1. Determination of Growth-inhibitory Dose

1. Preparation of yeast overnight culture
   NOTE: The yeast-extract-peptone-dextrose growth media (YEPD) used in this protocol is a standard recipe¹³.
   1. Streak out BY4741 (MATahis3Δ1 leu2Δ0 met15Δ0 ura3Δ0) cells on a YEPD agar plate and incubate for 48 hr at 30 °C or until visible colonies form.
   2. Prepare overnight cultures by inoculating 5 ml of YEPD liquid media in a sterile culture vessel with a single colony. Incubate overnight at 30 °C with continuous rotation or shaking. The culture will typically reach saturation by the morning (~2 × 10⁸ cells/ml).
2. Solid agar media preparation containing varying chemical doses
   1. Prepare several stocks of the chemical to be tested at 100x desired final concentration in an appropriate solvent.
      NOTE: Effective concentrations will depend on the chemical, and must be determined empirically. It is therefore advisable to initially test a broad final concentration range; i.e., from low µM to high mM.
   2. For each concentration of chemical to be tested, aliquot 3 ml of molten YEPD agar to a several sterile culture tube. Place in 55 °C water bath to keep from solidifying.
   3. For each concentration of chemical to be tested add 30 µl of the 100x compound to molten media, vortex 2-3 sec to mix, and pipette 1 ml into each pair of duplicate wells in a 12-well plate. Be sure to include a vehicle only control. Allow plates to set overnight at room temperature. Discard any extra media.
3. Spread plating cells
   1. Inoculate 10 ml of YEPD liquid media with 2 µl of a saturated yeast culture, grown overnight as in Step 1.1.2.
   2. Plate 25 µl of diluted yeast culture per well, spread evenly using sterile glass beads or rod, and allow the plate to dry under a flame. Incubate plate at 30°C for 48 hr.
   3. Evaluate the growth of yeast (both total number and size of colonies) on the plates. Select a sub-lethal concentration of compound that does not inhibit growth by greater than 10%-15% to perform the screen.

2. Systematic Chemical Genetic Screen

1. Maintenance of deletion-mutant-array (DMA) and Preparation of Source Plate
   1. For a detailed description on constructing deletion mutant arrays from glycerol stocks please refer to Baryshnikova et al.¹⁴. Once deletion mutants have been arrayed at a density of 384 colonies per plate, the DMA can be stored for several months at 4 °C. Replicate on YEPD agar containing 200 µg/ml of G418 as necessary to prevent colonies from growing into each other.
      NOTE: Multiple serial replications should be avoided to prevent loss of slow growing strains and minimize the appearance of suppressor mutants. This is particularly relevant if maintaining the collections as haploids.
   2. Perform all replica-plating steps using a microbial arraying robotic system. Alternatively, manipulate arrayed colonies using manual pinning tools.
      NOTE: The yeast deletion collection is available as haploids and diploids from several commercial sources and is typically shipped as glycerol stocks in 96-well plates.
2. Condensing the deletion mutant array
   1. Prepare 250 ml of YEPD agar media containing 200 µg/ml of G418. The effective concentration of G418 may vary and each lot should be tested empirically.
   2. Prepare five YEPD agar plates by pouring 50 ml of YEPD agar containing 200 µg/ml of G418 per plate. Do this one day before condensing the array and allow the plates to cool at room temperature overnight on a flat even surface.
      NOTE: Four plates will be required for the collection at 1,536-strain density, the fifth plate is poured as an extra.
   3. Remove the 16 Petri dishes containing the mutant deletion collection at a density of 384 colonies per plate and allow to come to room temperature (~1 hr).
   4. Wipe any condensation that has formed on the lid of each plate using a delicate task wipe. Failure to do so may result in water droplets being deposited on the array and cross contamination of arrayed mutants.
   5. Using a microbial array pinning robot, condense the DMA from a density of 384 colonies per plate (16 Petri dishes) to 1,536 colonies per plate (4 Petri dishes). Perform this so that the 1^st colony from plate 1 pins to row 1 column 1 of the condensed array, the 1^st colony of plate 2 to row 1 column 2, the 1^st colony of plate 3 to row 2 and column 1, and the 1^st colony of plate 4 to row 2 column 2.
   6. Incubate the consolidated array at 30°C overnight.
   7. Examine the array to ensure uniform transfer of colonies from source plates. There will be several blank positions, purposely incorporated into the array, which serve as a guide to ensure the DMA has been condensed correctly (Figure 4A).
      NOTE: These will be the source plates for replica plating onto media containing the test compound. A single source plate can be used for multiple pinnings. Also many robots will have an offsetting feature, which will ensure similar numbers of yeast are being transferred each time.
3. Replica plate deletion mutant array
   1. Prepare 700 ml of control (vehicle only) and 700 ml of experimental media (chemical-containing). This is enough for performing the assay in triplicate (12 plates per condition, plus two extras).
   2. Pour 50 ml of YEPD agar containing test chemical or control per plate. Allow the plates to cool at room temperature overnight on a flat even surface.
   3. Using the robot to replica plate, inoculate the DMA onto three sets of plates containing chemicals at the experimentally determined concentration, as well as three sets of plates containing the vehicle control. Incubate plates at room temperature for 24-48 hr.

3. Imaging Plates and Data Analysis

1. Remove plates from incubator and allow them to come to room temperature (~1 hr) prior to imaging. This will prevent condensation from forming while imaging the plates.
2. Image plates at 24 and 48 hr by removing the lid and placing face down on a flat bed scanner. Capture images at a resolution of at least 300 dpi. Alternatively, use a digital camera to capture images. If doing so, place plates face up on a dark background and remove the lids to image.
   NOTE: The file format and positioning of plates will depend on the quantification program used.
3. Perform quantification and comparison of array colony sizes using one of several open source programs, including Balony ¹⁵, SGAtools ¹⁶, and ScreenMill ¹⁷.

4. Validation of Screen

NOTE: At this stage several yeast deletions mutants will score as hypersensitive to the chemical of interest. These chemical-genetic interactions must next be validated in two ways. First the identity of sensitive strains should be confirmed by PCR. Second, chemical sensitivity must be independently scored. Outlined below is a brief protocol for performing spotting assays to validate strain hypersensitivity, a technique common in yeast biology.

1. Streak out desired (hypersensitive) mutants from the yeast deletion collection onto YEPD agar containing 200 µg/ml of G418. Incubate at 30 °C until colonies form. Verify the genotype of the strain by diagnostic PCR with primers according to the manufacturer’s protocol.
2. Inoculate 5 ml of the YEPD liquid for each strain and incubate overnight at 30 °C with rotation or shaking.
3. Measure OD₆₀₀ and dilute each strain to OD₆₀₀ = 1 in sterile dH₂O. These are now the normalized yeast cultures.
4. Dispense 100 µl of normalized wild-type yeast (BY4741) culture to well A1 of a 96-well plate. Dispense 100 µl of up to seven additional strains to wells B1-H1.
5. Use a multichannel pipettor to dispense 90 µl of sterile dH₂O to wells A2-H2 through A6-H6. Finally, prepare a series of 1:10 dilutions of normalized yeast cultures. Start by serially pipetting 10 µl of column 1 to column 2 with a multichannel pipettor, and continue across the 96-well plate until six dilutions are made (i.e., 10⁰ to 10^-5).
6. Transfer 2-5 µl of diluted yeast cultures in a grid using a multichannel pipettor onto YEPD solid media containing chemical and vehicle control. Incubate plates at the appropriate temperature for 24-48 hr.
7. Inspect plates and compare the sensitivity of select mutants to chemical (relative to BY4741 control). Image plates at 24 and 48 hr as in step 3.2.
   NOTE: While the identification of several hypersensitive strains with related gene ontologies or functions lends confidence to the validity of the screen, transformation of a hypersensitive deletion strain with a plasmid containing a wild-type copy of the gene is required to formally establish that a gene deletion of interest (and not hidden second site mutations) causes drug sensitivity.

Results

As a validation of this approach we performed a representative chemical genetic interaction screen of the chemotherapeutic agent 5-fluorouracil (5-FU) following the above outlined protocol. 5-FU is known to disrupt thymidylate synthase as well as DNA and RNA metabolism¹⁸. The chemical genetic interactions of 5-FU are well studied and have been investigated by yeast barcode microarray techniques using both heterozygous and homozygous deletion collections^8,19. Here we show that similar results can be ...

Discussion

Outlined here is an approach for generating chemical-genetic interaction profiles. The method is simple: by comparing colony sizes of each strain in comprehensive gene deletion collections in the presence and absence of a chemical, all genes needed to tolerate a chemical insult are identified. Annotation enrichment analysis of the resulting list of sensitive strains can then be used to provide insight into a chemicals mode of action. While this protocol has been optimized for budding yeast, it can also be adapted for use...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Yeast Extract	BioBasic	G0961	For YEPD liquid/solid media add to 1% final concentration (w/v)
Tyrptone Powder	BD Biosciences	211820	For YEPD liquid/solid media add to 2% final concentration (w/v)
Dextrose	Anachemia	31096-380	For YEPD liquid/solid media add to 2% final concentration (w/v) — do not autoclave. Prepare 20% stock solution, filter sterilize, and add to media after autoclaving.
Agar A	Bio Basic	FB0010	For YEPD solid media add to 2% final concentration (w/v)
G418	A.G. Scientific Inc.	G-1033	Prepare 1,000x stock at 200 mg/ml in dH2O and filter sterilize.
12-well plate	Greiner Bio One	655180
5 ml culture tubes	Evergreen Scientific	222-2376-080
10 cm Petri Dish	VWR	25384-302
ROTOR HDA	Singer Instruments	ROT-001	high-throughput microbial array pinning robot
PLUSPLATE© Petri Dish	Singer Instruments	PLU-001	Box of 200 dishes
384 Short-Pin RePad	Singer Instruments	RP-MP-384	Box of 1,000 pads
1536 Short-Pin RePad	Singer Instruments	RP-MP-1536	Box of 1,000 pads
Alternative Pinning Tools:
Fully Automated Robtic Systems	S&P robotics	http://www.sprobotics.com	Several automated colony handling robitic and imagining systems available.
Manual Pinning Tools	V&P Scientific	http://www.vp-scientific.com	Handheld replication tools and accessories.