Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

Summary

This work describes a protocol for a multicopy suppressor genetic screen in Schizosaccharomyces pombe. This screen uses a genome-wide plasmid library to identify suppressor clone(s) of a loss-of-function phenotype associated with a query mutant strain. Novel genetic suppressors of the ell1 null mutant were identified using this screen.

Abstract

Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships with other genes and organization into biological pathways and processes. Although the majority of the genetic screens were initially developed in Saccharomyces cerevisiae, a complementary platform for carrying out these genetic screens has been provided by Schizosaccharomyces pombe. One of the common approaches used to identify genetic interactions is by overexpression of clones from a genome-wide, high-copy-number plasmid library in a loss-of-function mutant, followed by selection of clones that suppress the mutant phenotype.

This paper describes a protocol for carrying out this 'multicopy suppression'-based genetic screen in S. pombe. This screen has helped identify multicopy suppressor(s) of the genotoxic stress-sensitive phenotype associated with the absence of the Ell1 transcription elongation factor in S. pombe. The screen was initiated by transformation of the query ell1 null mutant strain with a high-copy-number S. pombe cDNA plasmid library and selecting the suppressors on EMM2 plates containing 4-nitroquinoline 1-oxide (4-NQO), a genotoxic stress-inducing compound. Subsequently, plasmid was isolated from two shortlisted suppressor colonies and digested by restriction enzymes to release the insert DNA. Plasmids releasing an insert DNA fragment were retransformed into the ell1 deletion strain to confirm the ability of these suppressor plasmid clones to restore growth of the ell1 deletion mutant in the presence of 4-NQO and other genotoxic compounds. Those plasmids showing a rescue of the deletion phenotype were sequenced to identify the gene(s) responsible for suppression of the ell1 deletion-associated genotoxic stress-sensitive phenotype.

Introduction

Networks of genetic interactions provide functional information about genes and delineate pathways and biological processes that these genes may be involved in in vivo. In addition, they may also provide insights into how different genes interact with one another, resulting in a specific phenotype^1,2,3. Over the years, a variety of genetic screens have been designed by researchers to answer fundamental biological questions and study human diseases. Screens for the identification of genetic interactions can be performed in multiple ways. Genetic interactions identified in different genetic screens can represent distinct mechanistic relationships between genes. Furthermore, studies have revealed that a common set of genetic interactions are shared by genes that encode proteins belonging to the same pathway or complex^4,5. Thus, genetic interaction networks can be used to establish the functional organization in a cell, wherein genes sharing the most similar profiles belong to the same complex or pathway, those genes sharing somewhat less similar profiles belong to the same biological process, and those genes exhibiting overlapping but more diverse profiles reflect members belonging to the same cellular compartment⁶.

Genetic interaction screens based on dosage suppression ('high-copy or multicopy suppression') are one of the commonly used approaches. These screens can be performed by transforming a query mutant strain with a high-copy-number genomic or cDNA library, followed by suitable assays/selection techniques to identify suppressing or enhancing genetic interactions^7,8,9. To ensure a comprehensive genome-wide coverage, these screens have also been carried out by overexpressing a specific gene of interest in a collection of genome-wide loss-of-function mutant or by overexpressing a high-copy-number plasmid-encoded genomic or cDNA library in a loss-of-function query mutant^{9,10,11,12,13,14,15}. The multicopy strategy could also work using a dominant/overexpression approach using a regulatable promoter.

The main advantages of using suppressor-based screens are that suppression of a preexisting phenotype in a mutant strain by another gene establishes a genetic relationship among these two gene products that may not have been demonstrated using other approaches. Second, it has been observed that the presence of a preexisting mutation sensitizes a particular pathway, allowing additional components of that pathway to be identified by the isolation of suppressors, which may have not been identified by more direct genetic selections. Moreover, this screen can be used to identify suppressors that have different mechanisms of suppression¹⁶. Suppressor interactions usually occur between genes that are functionally related and can be used to elucidate hierarchies in pathways. The exact underlying mechanism of suppression may differ based on several factors, including the type of query mutant used in the screen, experimental conditions, and the level of gene expression. One of the common dosage suppression mechanisms involves genes encoding products that function together in the same complex or in parallel in the same cellular/biological process. The results of such screens in simpler model organisms such as yeast can be extended to higher eukaryotic organisms since most fundamental biological pathways and processes are conserved across evolution.

These genetic screens can also be modified in several ways to answer different biological questions. For example, orthologous genes from different organisms that can suppress the phenotype of the query mutant strain can be identified. It has also been used to delineate potential resistance mechanisms and determine protein targets of novel antibacterial^17,18, antifungal^19,20, antiparasitic²¹, and anticancer²² compounds. This screen has also been exploited to identify suppressors of the activity of pharmaceutical drugs whose mechanism of action is not known. Thus, in principle, these multicopy suppressor screens can be optimized and used in a variety of applications in different organisms. Although most of the genetic screens employed by yeast researchers have been initially developed in S. cerevisiae, S. pombe has emerged as a complementary model system for carrying out various genetic screens and assays²³. Moreover, genomic organization and biological processes in S. pombe, such as occurrence of introns in more genes, complexity of origins of DNA replication, centromere structure, organization of the cell cycle and presence of the RNAi machinery, show greater resemblance between S. pombe and higher eukaryotes^23,24, underscoring the importance of designing and using genetic tools in S. pombe.

This paper describes a protocol for identifying genetic interactors based on 'dosage suppression' of a loss-of-function mutant phenotype in S. pombe. The basis of this protocol is that it is a rapid and efficient method to screen a cDNA library overexpressing wild-type genes either on a multicopy plasmid and/or from a strong promoter. This protocol has four main steps: transformation of the library into a query mutant strain, selection of plasmid clones that suppress the desired phenotype of the query mutant strain, retrieving the plasmid(s) from these suppressor clones, and identification of the gene responsible for the suppression of the phenotype. As is true for any method based on the selection and identification of cDNAs from a library, the success of the screen is dependent on using a high-quality and high-complexity library as the screen can retrieve only those cDNA clones that are present in the library.

Using this protocol, we have successfully identified two novel suppressors of the genotoxic stress-sensitive phenotype of the query S. pombe ell1 null mutant. The ELL (Eleven Nineteen Lysine Rich Leukemia) family of transcription elongation factors suppress transient pausing of RNA polymerase II on DNA templates in in vitro biochemical assays and are conserved across various organisms, from fission yeast to humans²⁵. Earlier work has provided evidence that an S. pombe ell1 null mutant shows genotoxic stress sensitivity in the presence of 4-nitroquinoline 1-oxide (4-NQO) and methyl methanesulfonate (MMS)²⁶. Therefore, we transformed a S. pombe plasmid-encoded multicopy cDNA library into the query S. pombe ell1 null mutant and identified two putative clones that exhibited the ability to suppress the genotoxic stress sensitivity of the S. pombe ell1 null mutant in the presence of 4-NQO, a compound that induces DNA lesions. Subsequent sequencing of the insert present in the plasmid clones identified that the genes encoding rax2⁺ and osh6⁺ were responsible for suppressing the genotoxic stress sensitivity of ell1 null mutant when overexpressed in the ell1 null mutant.

Protocol

1. Transformation of the cDNA library into the query S. pombe mutant strain to screen for multicopy suppressors

NOTE: The Standard Lithium-Acetate method²⁷ was followed to transform the S. pombe cDNA library into the query S. pombe ell1Δ strain with a few modifications:

1. Grow the S. pombe ell1Δ strain at 32 °C on a YE medium (Table 1) plate supplemented with 225 µg/mL each of adenine, leucine, and uracil. Inoculate a loop full of inoculum of ell1Δ strain from the above plate in 15-20 mL of YE medium supplemented with 225 µg/mL each of adenine, leucine, and uracil. Incubate it overnight at 32 °C with shaking (200-250 rpm).
2. Next day, dilute the overnight culture in 100 mL of fresh YE medium with the desired supplements to an OD_{600 nm} (optical density at 600 nm) of 0.3 and incubate it at 32 °C with shaking at 200-250 rpm until the OD_{600 nm} reaches mid-log phase.
3. Divide the 100 mL culture into two 50 mL centrifuge tubes, followed by centrifugation at room temperature for 10 min at 4,000 × g.
4. Discard the supernatant and wash the cell pellet with 1 mL of sterile water, i.e., resuspend the cells in 1 mL of sterile water and centrifuge at 4,000 × g for 5 min at room temperature.
5. Discard the supernatant and wash each cell pellet with 1 mL of 1x LiAc-TE (100 mM lithium acetate, 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) solution as described in step 1.4.
6. Remove the supernatant and resuspend each cell pellet in 250 µL of 1x LiAc-TE. Use these S. pombe competent cells (500 µL) for transformation with the DNA of interest. Transfer 125 µL of the competent cells into four different sterile microcentrifuge tubes for subsequent transformation steps.
7. Boil carrier DNA from Salmon testes or herring sperm for 1 min and immediately place on ice. For each transformation, add 10 µL of 10 mg/mL denatured, single-stranded carrier DNA to the microcentrifuge tube containing 125 µL of the competent cells, followed by gentle mixing using a micropipette tip. Subsequently, add 50 µg of the S. pombe cDNA library to the competent cells-carrier DNA mixture-containing microcentrifuge tube.
8. Incubate the microcentrifuge tubes at room temperature for 10 min, and then add 260 µL of polyethylene glycol-lithium acetate solution (40% [w/v] PEG 4,000, 100 mM lithium acetate, 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) to the tubes and mix gently with the help of a micropipette tip.
9. Incubate the microcentrifuge tubes containing the transformation mixture at 32 °C for 2 h without shaking. Add 43 µL of prewarmed dimethyl sulfoxide (DMSO) to the microcentrifuge tube and mix gently. Subsequently, expose the tubes to heat shock at 42 °C for 5 min.
10. Pellet the cells at 4,000 × g for 5 min at room temperature. Discard the supernatant and remove the residual PEG- LiAc-TE solution with the help of a micropipette tip.
11. Resuspend the cells in 100 µL of sterile water and plate on one EMM2 (Table 1) plate (150 mm) with required supplements and 0.2 µg/mL of 4-NQO.
12. Incubate the plates at 32 °C for 5-6 days to allow colonies to appear on the plates. Streak the obtained colonies on the same medium plate in the presence of 0.2 µg/mL of 4-NQO and use for further screening.
13. For one library transformation experiment, use 500 µL of competent cells (125 µL of competent cells x 4 microcentrifuge tubes) for transformation as described in steps 1.8 to 1.14 above.
14. As a control, plate 1/10^th of the transformation mixture on one EMM2 plate (150 mm) with required supplements, but lacking 4-NQO, to calculate the total number of library clones obtained after transformation of the library, and screen for isolation of the suppressor clones.

2. Test and validate the rescue/suppression of the phenotype associated with the query mutant strain by the putative suppressor

NOTE: Stress spot assays were carried out as described below to test and validate the rescue/suppression of the ell1 deletion-associated 4-NQO stress sensitivity by the putative suppressor(s).

1. Add 100-200 µL sterile water in each of the different wells of a sterile 96-well microtiter plate.
2. Pick a small amount of inoculum from each of the different colonies obtained after cDNA library transformation on the plate containing 4-NQO using a sterile toothpick or a 20-200 μL micropipette tip. Add the inoculum from each of the different colonies into separate independent wells of the microtiter plate containing 100-200 µL of sterile water. Mix thoroughly.
3. Spot 3 µL of the cell suspension from each well onto EMM2 agar plates containing adenine (225 µg/mL) and uracil (225 µg/mL) but lacking leucine (the selectable auxotrophic marker present on the plasmid vector used for construction of the library used in this work). Add appropriate concentrations of 4-NQO to the plates as required.
4. Incubate the plates at 32 °C for 3-4 days to allow the cells to grow. Identify the colonies that show growth in the presence of different concentrations of 4-NQO as putative suppressors.
5. To further validate the suppressors, grow the selected suppressors along with appropriate control strains overnight at 32 °C with shaking (200-250 rpm) in EMM2 medium containing 225 µg/mL each of adenine and uracil but lacking leucine.
6. Next day, dilute the cells to an OD_{600 nm} of 0.3 in fresh EMM2 medium and grow them at 32 °C with shaking until mid-log phase (approximately OD_{600 nm} of 0.6-0.8).
7. Spot appropriate serial dilutions (1:10 or 1:5) of cultures on EMM2 plates with required supplements containing either 0.4 µM 4-NQO or 0.01% MMS. For control, spot the strains on an EMM2 plate with required supplements but lacking any DNA-damaging agent.
8. Incubate the plates at 32 °C for 3-5 days to monitor growth.
   NOTE: Suitable assays based on the phenotype associated with the query mutant strain should be used to test the rescue or suppression of the phenotype. For example, if suppressors of a cold-sensitive phenotype of a query mutant strain need to be identified, the assay for testing and validation of the suppressor clones would involve growing transformants at low temperature.
9. Spot the mutant strains transformed with full-length gene of interest (Spell1 in this case) or empty vector as positive and negative controls, respectively, along with the library transformants.

3. Isolation of the plasmid from the S. pombe suppressor clones

NOTE: Plasmid isolation from S. pombe was carried out by following the protocol described in Fission yeast: a laboratory manual²⁸ with a few modifications.

1. Inoculate a single yeast colony in EMM2 medium containing 225 µg/mL of adenine and uracil and grow the cells overnight at 32 °C with shaking at 200-250 rpm. Next day, harvest 5 O.D. cells (i.e., 10 mL culture of O.D. 0.5) by centrifuging for 2 min at 4,000 × g at room temperature.
2. Remove the supernatant and resuspend the cell pellet in 0.2 mL of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris HCl (pH 8.0), and 1 mM Na₂EDTA).
3. Add 0.2 mL of phenol:chloroform:isoamyl alcohol (25:24:1) and 0.3 g of acid-washed glass beads to the microcentrifuge tube. Vortex the microcentrifuge tube for 2 min at 4 °C and incubate on ice for 1 min. Repeat this step 6x.
4. Centrifuge the tube at 10,000 × g for 15 min at room temperature. Transfer the upper aqueous layer to a fresh microcentrifuge tube and add 200 µL of phenol:chloroform:isoamyl alcohol (25:24:1).
5. Centrifuge the tube at 10,000 × g for 10 min. Transfer the upper aqueous layer to a fresh tube and add two volumes of 100% ethanol (~400 µL) and 1/10 volume of sodium acetate (3 M, pH 5.8) (~20 µL) to the tube. Incubate the microcentrifuge tube -70 °C for 1 h.
6. Precipitate the DNA by centrifugation at 10,000 × g for 15 min at 4 °C and remove the supernatant. Wash the DNA pellet with 70% ethanol (~500 µL) and keep it at room temperature to air-dry.
7. Resuspend the DNA in 20 µL of sterile water. Use 2-5 µL of plasmid DNA for transformation of competent E. coli cells.
   NOTE: Plasmid can also be isolated from yeast cells using any of the commercially available yeast plasmid isolation kits.

4. Identification of the gene encoded by the suppressor clone

1. Transform the isolated yeast plasmids into E. coli Top10 strain [F-mcrA (mrr-hsdRMS-mcrBC) 80LacZM15 lacX74 recA1 ara139 (ara-leu)7697 galUgalKrpsL (StrR) endA1 nupG] using the standard protocol²⁹ and spread the cells on LB (Luria Broth) plates with required antibiotics.
2. Isolate the plasmid(s) from E. coli transformants using the standard alkaline lysis protocol²⁹, and follow the appropriate combinations of restriction enzymes to check for the release of the insert DNA fragment by restriction digestion.
   NOTE: Suppressor plasmid 84 was digested with BamHI restriction enzyme, and Suppressor plasmid 104 was digested with PstI/BamHI restriction enzymes.
3. Retransform the plasmids showing insert release after restriction digestion in the ell1Δ strain to check for their ability to rescue the genotoxic stress-sensitive phenotype of the ell1Δ strain.
4. Select the plasmid clones showing suppression of the genotoxic stress sensitivity and sequence the insert DNA fragment present in these plasmid clones using vector-specific forward and reverse primers.
   NOTE: In this study, the adh1 promoter-specific universal forward primer (5'CATTGGTCTTCCGCTCCG 3') was used³⁰.
5. To identify the gene responsible for the suppression, align the sequence obtained using NCBI nucleotide blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&BLAST_SPEC=GeoBlast&PAGE_TYPE=BlastSearch) or Pombase sequence alignment tool (https://fungi.ensembl.org/Schizosaccharomyces_pombe/Tools/Blast?db=core). Select the sequence showing maximum alignment and identify it as the gene for the multicopy suppressor plasmid.

Results

Screening for multicopy suppressor(s) of ell1 deletion-associated genotoxic stress sensitivity in S. pombe
We performed the genetic screen using the protocol described above to identify multicopy suppressors of the loss-of-function phenotype of the query ell1 deletion mutant strain. The growth-related sensitivity of the ell1 deletion strain observed in the presence of the 4-NQO genotoxic agent was adopted as the ...

Discussion

Yeasts have been widely used to investigate the basic biological processes and pathways that are evolutionarily conserved across eukaryotic organisms. The availability of genetic and genomic tools along with their amenability to various biochemical, genetic, and molecular procedures make yeasts an excellent model organism for genetic research^34,35,36. Over the years, various genetic screens have been designed by yeast researcher...

Materials

Name	Company	Catalog Number	Comments
4-NQO	Sigma	N8141
Acetic Acid, glacial	Sigma	1371301000
Adenine Sulphate	Himedia	GRM033
Agar	Himedia	GRM026
Agarose	Lonza	50004L
Ammonium Chloride	Himedia	MB054
BamHI	Fermentas	ER0051
Biotin	Himedia	RM095
Boric Acid	Himedia	MB007
Calcium Chloride	Sigma	C4901
Chloroform:Isoamyl alcohol 24:1	Sigma	C0549
Citric Acid	Himedia	RM1023
Disodium hydrogen phospahte anhydrous	Himedia	GRM3960
single stranded DNA from Salmon testes	Sigma	D7656
EDTA disodium	Sigma	324503
Ferric Chloride Hexahydrate	Himedia	RM6353
Glucose	Amresco	188
Ionositol	Himedia	GRM102
Isopropanol	Qualigen	Q26897
Leucine	Himedia	GRM054
Lithium Acetatae	Sigma	517992
Magnesium Chloride Hexahydrate	Himedia	MB040
Molybdic Acid	Himedia	RM690
Nicotinic Acid	Himedia	CMS177
PEG, MW 4000	Sigma	81240
Pentothinic Acid	Himedia	TC159
Phenol	Himedia	MB082
Plasmid Extraction Kit	Qiagen	27104
Potassium Chloride	Sigma	P9541
Potassium hydrogen Pthallate	Merc	DDD7D670815
Potassium iodide	Himedia	RM1086
RNAse	Fermentas	EN0531
SDS	Himedia	GRM205
Sodium Hydroxide	Himedia	GRM1183
Sodium Sulphate	Himedia	RM1037
Tris free Base	Himedia	MB209
Uracil	Himedia	GRM264
Yeast Extract Powder	Himedia	RM668
Zinc Sulphate Heptahydrate	Merc	DJ9D692580