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.
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.
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 phenotype1,2,3. 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 complex4,5. 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 interactions7,8,9. 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 mutant9,10,11,12,13,14,15. 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 antibacterial17,18, antifungal19,20, 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 eukaryotes23,24, 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.
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:
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).
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.
4. Identification of the gene encoded by the suppressor clone
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 loss-of-function phenotype to select for multicopy suppressors. Figure 1 shows a schematic representation of the genetic screen. To begin the screen, the genotoxic stress sensitivity of the ell1 deletion mutant (h- ura4-D18 leu1-32 ade6-M210 can1-1 ell1Δ :: kanMX6) was first confirmed with respect to its isogenic wild-type strain (h- ura4-D18 leu1-32 ade6-M210 can1-1) in the presence of 4-NQO (Figure 2A). Subsequently, the query S. pombe ell1 null mutant was transformed with a high-copy-number S. pombe cDNA library to select for those plasmid clones that could suppress/rescue the 4-NQO growth-related sensitivity of the ell1 null mutant. This library contains ~6 × 105 S. pombe cDNA fragments cloned in the pLEV3 S. pombe overexpression vector under the control of the adh1 promoter30. As a control, 1/10th of the transformation mixture was also plated on EMM2 plates lacking 4-NQO to calculate the total number of recombinant clones obtained after transformation of the library, an indication of the coverage of the library, and screened for isolation of the suppressor clones. This is critical, as screening fewer clones will reduce the possibility of identifying the suppressor clones.
Figure 2B shows representative images of the control and 4-NQO-containing plates. As shown in the figure, a large number of colonies are obtained on the control plate lacking 4-NQO showing a good coverage of the library. As expected, fewer transformants were obtained on the 4-NQO plate as these transformants likely represent the putative suppressors of the 4-NQO sensitivity of the ell1 null mutant. Next, the different transformant colonies obtained on EMM2 plates containing 0.2 µM 4-NQO were further streaked or spotted on EMM2 plates containing 0.2 µM 4-NQO to confirm the ability of these transformants to grow in the presence of 4-NQO. It was observed that 620 transformant colonies were able to grow on 0.2 µM 4-NQO-containing EMM2 plates.
Validation of the ability of the putative suppressor clones to restore the growth of ell1 deletion mutant under genotoxic stress conditions
To confirm the putative suppressors, it is important to test the rescue of the desired phenotype under higher stringency conditions as shown in the schematic representation (Figure 3A). Therefore, the 620 shortlisted transformants were spotted on EMM2 plates with 0.4 µM 4-NQO. As can be seen in Figure 3B, only 74 transformants showed growth at 0.4 µM 4-NQO. Subsequently, these 74 transformants were spotted on EMM2 plates containing 0.8 µM 4-NQO, and it was observed that only 16 showed growth in the presence of 0.8 µM 4-NQO (Figure 3C). To further confirm these observations, these 16 transformants were spotted on EMM2 plates with required supplements, containing either 0.8 µM 4-NQO or no 4-NQO (control), along with the ell1 deletion mutant transformed with empty vector. Results of these spotting assays revealed that, as expected, the ell1 deletion strain transformed with empty vector, as well as all the 16 transformants, showed growth on the plate lacking 4-NQO. In comparison, while the ell1 deletion mutant containing only the empty vector showed no growth at 0.8 µM 4-NQO, six transformants exhibited growth at 0.8 µM 4-NQO (Figure 3D), suggesting that the library clone(s) present in them had the ability to suppress the genotoxic stress phenotype of the ell1 null mutant.
Identification of the gene encoded by the suppressor clone
We next selected 02 genetic suppressors of the ell1 deletion strain, sup 84 and sup 104, showing complete suppression of the ell1-associated growth sensitivity in the presence of 4-NQO. The library plasmid clone(s) was isolated from these two yeast suppressors and transformed into competent E. coli cells. Subsequently, plasmid was isolated from E. coli cells using standard protocols31. The two isolated plasmids, labeled as 84 and 104, were subjected to restriction digestion, which revealed the presence of approximately 1,000 bp and 800 bp insert DNA fragments, respectively (Figure 4A,B). To further validate the results of this suppressor screen, these plasmids were retransformed into the ell1 deletion mutant and checked if they could restore the growth of the ell1 null mutant in the presence of 4-NQO. The stress spot assays revealed that the suppressor plasmid clones resulted in the suppression of the 4-NQO-associated growth sensitivity when reintroduced into the ell1 null mutant, suggesting that the suppression was plasmid-dependent (Figure 4C). Furthermore, we decided to determine whether these suppressor clones could also suppress the growth-related sensitivity of the ell1 deletion strain in the presence of another DNA damage-inducing compound, MMS. The spot assays demonstrated that transformation of the suppressor plasmid clones into the ell1 null mutant resulted in more growth on MMS-containing medium than transformation of the ell1 deletion mutant with empty vector, indicating that these plasmid clones could suppress the genotoxic stress sensitivity of the ell1 deletion mutant in the presence of both genotoxic agents, i.e., 4-NQO and MMS (Figure 4D).
Next, to identify the gene present in these suppressor plasmid clones, sequencing of the insert DNA fragment was carried out using the adh1 promoter-specific universal forward primer30. The nucleotide sequence obtained was searched by using 'BLAST at Ensembl' tool available on the PomBase website (https://www.pombase.org), which revealed that the two plasmid clones 84 and 104 contained rax2+ and osh6+ genes, respectively, providing evidence that Rax2 and Osh6 were responsible for suppressing the genotoxic stress-sensitive phenotype associated with absence of ell1+ de S. pombe. Knowledge about the functions of both these proteins in S. pombe is limited. Rax2 has been shown to regulate the localization of For3p to control the cell polarity during vegetative growth in S. pombe32. Osh6 is a lipid transport protein that belongs to the oxysterol-binding protein-related protein family. It is involved in the transport of phosphatidylserine from the endoplasmic reticulum to the plasma membrane33.
Figure 1: Schematic representation of the genetic multicopy suppressor screen. 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. Abbreviation: 4-NQO = 4-nitroquinoline 1-oxide. Please click here to view a larger version of this figure.
Figure 2: Screening for multicopy suppressor(s) of ell1 deletion-associated genotoxic stress sensitivity in S. pombe. (A) Stress spot assay showing 4-NQO sensitivity of ell1-deleted S. pombe strain as compared to its isogenic wild-type strain. Serial dilutions (1:10) of appropriate strains were spotted on EMM2 medium containing 225 µg/mL each of adenine, leucine, and uracil with or without (control) 0.4 µM 4-NQO. Plates were then incubated for 3-5 days at 32 °C. (B) A high-copy-number cDNA library was transformed in ell1-deleted S. pombe strain, and transformed colonies were plated on EMM2 medium lacking leucine but containing 0.2 µM 4-NQO. A small aliquot of the transformation mixture was also plated on EMM2 plates lacking 4-NQO, as a control. Plates were incubated at 32 °C for 5-6 days and photographed. Panel B shows a representative image of these plates. Abbreviation: 4-NQO = 4-nitroquinoline 1-oxide. Please click here to view a larger version of this figure.
Figure 3: Validation of the ability of putative suppressor clones to suppress the genotoxic stress sensitivity of the ell1 deletion mutant. (A) A schematic representation of the screening of the library transformants by testing suppression of the desired phenotype. Growth of 620 transformants was tested on increasing concentrations of 4-NQO by spotting different transformants on EMM2 plates lacking leucine and containing either (B) 0.4 µM 4-NQO or (C) 0.8 µM 4-NQO. Plates were incubated at 32 °C for 5-6 days and photographed. Representative images of plates have been shown. (D) Sixteen transformants that were showing growth on 0.8 µM 4-NQO were spotted on EMM2 plates lacking leucine but containing either 0.8 µM 4-NQO or not containing 4-NQO (control), along with the ell1 deletion strain transformed with empty vector. Plates were incubated at 32 °C for 5-6 days and photographed. Only six colonies exhibited the ability to rescue ell1 deletion associated-genotoxic stress sensitivity. Abbreviation: 4-NQO = 4-nitroquinoline 1-oxide. Please click here to view a larger version of this figure.
Figure 4: Identification of the suppressor plasmid clones. Agarose gel images of (A) restriction digestion of suppressor plasmid 84 with BamHI restriction enzyme released an insert of ~1 kb. (B) Suppressor plasmid 104 digested with PstI/BamHI restriction enzymes released an insert of ~800 bp. (C) 1:10 serially diluted ell1 null mutant transformed with plasmids, sup 84 or sup 104, were spotted on EMM2 plates lacking leucine and containing either 0.4 µM 4-NQO or (D) 0.01% MMS. In control plates, no DNA-damaging agent was added. Plates were incubated at 32 °C for 4-5 days and photographed. Please click here to view a larger version of this figure.
Table 1: Composition of media for growth of S. pombe. Please click here to download this Table.
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 research34,35,36. Over the years, various genetic screens have been designed by yeast researchers to identify genetic interactions that would shed light on the functions of individual gene(s), as well as the pathways and biological processes in which they may be involved10,11,17,37. One of the common and useful screens to identify genetic interactions is known as 'multicopy or high copy number suppression'.
This screen requires a query mutant strain(s) exhibiting a loss-of-function phenotype, followed by transformation of this mutant query strain(s) with a high-copy-number genomic or cDNA library. Subsequently, the transformants obtained are screened to identify those genes, which when overexpressed, suppress the phenotype associated with the query mutant strain(s). This type of genetic interaction between the specific mutant strain and gene(s) present in the plasmid library clones result in the isolation and identification of gene products that are likely to act in the same pathway or affect the same biological process4. Thus, this screen provides insights into the functions of the gene(s) of interest and their functional organization into pathways and biological processes in vivo. Traditionally, this screen has been extensively used in S. cerevisiae38,39,40,41,42.
The goal of the present work is to describe a simple and straightforward protocol for carrying out a multicopy suppression genetic screen in S. pombe. We carried out the screen using an S. pombe strain carrying a deletion of the ell1 transcription elongation factor. ELL is a family of transcription elongation factors, which are present in a wide range of organisms, from fission yeast to humans. The functions of Ell1 are only beginning to be understood in S. pombe. Earlier work had highlighted the role of Ell1 in optimal growth during normal growth conditions and in survival during genotoxic stress conditions26. We have employed the multicopy suppressor screening as one of the approaches to uncover the molecular mechanism underlying the role of Ell1 in genotoxic stress sensitivity in S. pombe26. A multicopy plasmid-encoded S. pombe cDNA library was transformed into the S. pombe ell1 null mutant that exhibits sensitivity to 4-NQO. Subsequently, those transformants that could grow in the presence of 4-NQO were selected.
Isolation of plasmids from these transformants and sequencing of inserts present in these plasmids led to the identification of two novel genetic interactors of ell1+ de S. pombe, rax2+ and osh6+. A previous study using a similar genetic screen in S. pombe has also identified the anti-silencing protein, epe1+, as a multicopy suppressor of the ell1 deletion-associated phenotypes43. We have also used this method in identifying the multicopy suppressors of the non-essential Rpb9 subunit of S. pombe RNA polymerase II (unpublished observations). Collectively, these results validate the utility of the genetic multicopy suppressor screening approach as a method to identify putative genes/proteins that exhibit genetic interaction with a desired gene of interest, elucidating the function, pathways, and processes in which the gene of interest may be involved.
To use the screen described in this work, it is important to first have a query mutant strain that exhibits a clear phenotype associated with the mutation, which can be used to isolate the multicopy suppressor(s). Moreover, the success of this screen will depend upon the availability of a high-quality and good representative library containing clones representing the maximum number of wild-type genes or their corresponding cDNAs. Traditionally, both genomic and cDNA libraries have been used in these screens, but cDNA libraries have the advantage of being made from mRNAs expressed in the cell/organism. Furthermore, a high efficiency of its transformation into the yeast cells is imperative for the success of the screen because it is important to screen a large number of transformants, increasing the probability of identifying suppressors. If fewer transformants are obtained on the control plate after transformation of the library into the query mutant strain using the lithium acetate protocol, then electroporation can be used to transform the library.
It is also advisable to first standardize the transformation protocol with another plasmid before transforming the library. Otherwise, it will unnecessarily result in loss of the library. The protocol described here uses 4-NQO sensitivity as the mutant phenotype, but suitable screening methods/assays should be designed for selection of suppressors based on the specific mutant phenotype of interest. If no or very few transformants are obtained on the plate to select for suppressors after transformation of the library into the query mutant strain, then if possible, less stringent conditions/assays should be used when plating the library to select for suppressors so as to detect as many genetic suppressors as possible before confirming those under high-stringency conditions. In addition to using restriction digestion to test for the presence of the insert in the identified suppressor plasmid clones, PCR using vector-specific primers can be used to amplify the desired insert DNA fragment. Alternatively, the suppressor plasmid clones can be directly given for sequencing, avoiding the need for restriction digestion or PCR.
This screen may also be adopted for yeasts other than S. cerevisiae and S. pombe if a genetic tool kit with respect to appropriate mutant strains, plasmids, and promoter constructs is available that would facilitate overexpression of genes, allowing for the rescue of mutant phenotype present in the host yeast cell. Furthermore, with the availability of genome-wide resources, including collection of genome-wide deletion mutants44 and overexpression libraries37, this screen can also be performed by transformation of one or a few query plasmids into a systematic array of yeast mutant strain collection or by transformation of a systematic array of overexpression plasmid collection into one or a few mutant query strains45. In conclusion, this approach can be clearly generalized to a wide variety of experimental questions in different organisms.
The authors have nothing to disclose.
This work was funded by a research grant from the Department of Biotechnology, Government of India (Grant No. BT/PR12568/BRB/10/1369/2015) to Nimisha Sharma. The authors thank Prof. Charles Hoffman (Boston College, USA) for the gift of the S. pombe cDNA library and Prof. Susan Forsburg for the yeast plasmids.
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 |