Complex modifier landscape underlying genetic background effects Jing Houa,1, Guihong Tana, Gerald R. Finkb, Brenda J. Andrewsa,1, and Charles Boonea,1 aDonnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON M5S 3E1, Canada; and bWhitehead Institute for Biomedical Research, Cambridge, MA 02142 Edited by Jasper Rine, University of California, Berkeley, CA, and approved February 4, 2019 (received for review December 7, 2018) The phenotypic consequence of a given mutation can be influ- Recent large-scale comparative screens in many model sys- enced by the genetic background. For example, conditional gene tems, including yeasts, nematodes, Drosophila, mice, and human essentiality occurs when the loss of function of a gene causes cell lines, have revealed an extensive catalog of background ef- lethality in one genetic background but not another. Between two fects, mainly related to differential fitness consequences of well- individual Saccharomyces cerevisiae strains, S288c and Σ1278b, defined loss-of-function mutations across genetically distinct in- ∼1% of yeast genes were previously identified as “conditional dividuals (20–30). In the budding yeast, Saccharomyces cerevisiae, essential.” Here, in addition to confirming that some conditional we carried out a comparative study of systematic gene deletions essential genes are modified by a nonchromosomal element, we in two closely related individuals, S288c and Σ1278b, and showed show that most cases involve a complex set of genomic modifiers. that in this context, ∼1% (57 genes) of all yeast genes are con- From tetrad analysis of S288C/Σ1278b hybrid strains and whole- ditional essential, where the deletion of a given gene is lethal in genome sequencing of viable hybrid spore progeny, we identified one background but not another (23). These data provide an complex sets of multiple genomic regions underlying conditional es- opportunity to systematically dissect the modifiers involved in sentiality. For a smaller subset of genes, including CYS3 and CYS4, background-specific phenotypes related to gene deletion vari- each of which encodes components of the cysteine biosynthesis path- ants. In addition, an important advantage of the yeast model is way, we observed a segregation pattern consistent with a single the availability of a large number of genetically diverse natural modifier associated with conditional essentiality. In natural yeast iso- isolates. So far, the genomes of over 1,000 wild yeast isolates GENETICS lates, we found that the CYS3/CYS4 conditional essentiality can be originating from various ecological and geographical locations caused by variation in two independent modifiers, MET1 and OPT1, have been completely sequenced (31). Combining these re- each with roles associated with cellular cysteine physiology. Interest- sources, the yeast model offers a unique opportunity to explore ingly, the OPT1 allelic variation appears to have arisen independently specific cases of conditional essentiality in two defined genetic from separate lineages, with rare allele frequencies below 0.5%. backgrounds, and then to expand the analysis to the population Thus, while conditional gene essentiality is usually driven by genetic level to discover variant frequency, type, and trait predictability. interactions associated with complex modifier architectures, our anal- Here, we characterized the modifier complexity involved in ysis also highlights the role of functionally related, genetically inde- previously identified conditional essentiality cases between S288c pendent, and rare variants. Significance conditional gene essentiality | background effect | complex modifier interactions | rare variants Genetic background impacts the phenotypic outcome of a mutation in different individuals; however, the underlying enetic backgrounds can impact the phenotypic conse- molecular mechanisms are often unclear. We characterized Gquences of a specific mutation and might constitute an in- genes exhibiting conditional essentiality when mutated in two herent feature of biological traits, complicating our ability to genetically distinct yeast strains. Hybrid crosses and whole- predict individual phenotypes from genomic information (1–9). genome sequencing revealed that conditional essentiality can In many human Mendelian disorders, including cystic fibrosis, be associated with nonchromosomal elements or a single- sickle cell anemia, and neurofibromatosis, even though the modifier locus, but most involve a complex set of modifier causal variant is well established (1, 10–13), individuals carrying loci. Detailed analysis of the cysteine biosynthesis pathway the same causal mutation either do not always develop the dis- showed that independent, rare, single-gene modifiers, related ease, a phenomenon called incomplete penetrance, or do not to both up- and downstream pathway functions, can arise in display the same clinical symptoms, an effect known as variable multiple allelic forms from separate lineages. For several genes, expressivity. Variable penetrance or expressivity of a trait typically we also resolved complex sets of modifying loci underlying conditional essentiality, revealing specific genetic interactions reflects environmental or genetic background influences and sig- that drive an individual strain’s background effect. nificantly impacts the ability to connect genotype to phenotype in natural populations (14). Author contributions: J.H. and C.B. designed research; J.H. performed research; G.T. and While genetic background effects are commonly observed, the G.R.F. contributed new reagents/analytic tools; J.H., G.R.F., B.J.A., and C.B. analyzed data; underlying molecular mechanisms remain mostly unknown. In and J.H., B.J.A., and C.B. wrote the paper. general, genetic variants contributing to background effects are The authors declare no conflict of interest. termed “modifiers.” Identification of critical modifiers is diffi- This article is a PNAS Direct Submission. cult, likely due to the low population frequencies of the modified This open access article is distributed under Creative Commons Attribution-NonCommercial- trait and the heterogenic nature of the modifiers involved (7, 15). NoDerivatives License 4.0 (CC BY-NC-ND). In addition, recent evidence suggests that many genetic back- Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. PRJNA493856). ground effects are caused by highly complex modifier interac- 1To whom correspondence may be addressed. Email: [email protected], brenda. tions, which themselves can be confounded by other genetic and [email protected], or [email protected]. – environmental factors (5, 16 19). As a result, only a handful of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. examples of modifiers involved in human Mendelian diseases, 1073/pnas.1820915116/-/DCSupplemental. notably in cystic fibrosis, have been identified (4, 10, 12, 13). www.pnas.org/cgi/doi/10.1073/pnas.1820915116 PNAS Latest Articles | 1of10 Downloaded by guest on September 26, 2021 and Σ1278b. We mapped the genomic regions involved in a a 2:2 segregation pattern of viable progeny, with the deletion subset of cases and focused on a pair of genes, CYS3 and CYS4, marker cosegregating with the lethal phenotype. Previous work which are involved in the cysteine biosynthesis pathway and are showed that this segregation pattern reflects conditional essen- essential in Σ1278b but not S288c. We characterized and func- tiality associated with cytosolic factors related to the mitochon- tionally validated the modifier underlying the Σ1278b-specific drial genomes and/or the presence of killer viruses (19). For essentiality and expanded our analysis to other natural isolates. 6 other cases, including CYS3 and CYS4, we observed a segre- By surveying a large number of strains, we showed that cysteine gation pattern that was consistent with a single modifier associ- biosynthesis pathway essentiality can be caused by variation in ated with the conditional essential gene. In the single-modifier two independent modifiers, OPT1 and MET1, that are linked to cases, either a 2:2 segregation pattern in the homozygous de- upstream or downstream pathway functions. Sequence analyses letion hybrid or a predominance of tetrads containing four, revealed that allelic variants of the identified modifiers in- three, or two viable spores in the heterozygous deletion hybrid dependently arose from separate lineages and were extremely was observed, indicative of a single-modifier origin (Dataset S1). rare across the population. For the other 20 cases listed in Dataset S1, the segregation patterns in either heterozygous S288c/Σ1278b hybrid alone or Results both heterozygous and homozygous S288c/Σ1278b hybrids to- The Modifier Complexity of S288c/Σ1278b Conditional Gene Essentiality. gether indicate complex modifier origins, which is consistent with We previously compared growth phenotypes of gene deletion mu- the general conclusion of our previous preliminary findings tant collections constructed in two laboratory strains, S288c and basedontetradanalysis(23). Σ1278b, and identified a total of 57 genes as conditional essential, Depending on the interaction patterns among the modifiers, among which 13 were specific to S288c and 44 were specific to the number of modifiers involved in some of the complex cases Σ1278b (23). To select cases for modifier analysis, we first rean- could be inferred