HIGHLIGHTED ARTICLE | INVESTIGATION Systematic Humanization of the Yeast Cytoskeleton Discerns Functionally Replaceable from Divergent Human Genes Riddhiman K. Garge,* Jon M. Laurent,*,† Aashiq H. Kachroo,‡,1 and Edward M. Marcotte*,1 *Center for Systems and Synthetic Biology, Department of Molecular Biosciences, The University of Texas at Austin, Texas 78712, †Institute for Systems Genetics, Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York 10016, and ‡The Department of Biology, Centre for Applied Synthetic Biology, Concordia University, Montreal, H4B 1R6 Quebec, Canada ORCID IDs: 0000-0002-6774-0172 (R.K.G.); 0000-0001-6583-4741 (J.M.L.); 0000-0001-9770-778X (A.H.K.); 0000-0001-8808-180X (E.M.M.) ABSTRACT Many gene families have been expanded by gene duplications along the human lineage, relative to ancestral opisthokonts, but the extent to which the duplicated genes function similarly is understudied. Here, we focused on structural cytoskeletal genes involved in critical cellular processes, including chromosome segregation, macromolecular transport, and cell shape maintenance. To determine functional redundancy and divergence of duplicated human genes, we systematically humanized the yeast actin, myosin, tubulin, and septin genes, testing 81% of human cytoskeletal genes across seven gene families for their ability to complement a growth defect induced by inactivation or deletion of the corresponding yeast ortholog. In five of seven families—all but a-tubulin and light myosin, we found at least one human gene capable of complementing loss of the yeast gene. Despite rescuing growth defects, we observed differential abilities of human genes to rescue cell morphology, meiosis, and mating defects. By comparing phenotypes of humanized strains with deletion phenotypes of their interaction partners, we identify instances of human genes in the actin and septin families capable of carrying out essential functions, but failing to fully complement the cytoskeletal roles of their yeast orthologs, thus leading to abnormal cell morphologies. Overall, we show that duplicated human cytoskeletal genes appear to have diverged such that only a few human genes within each family are capable of replacing the essential roles of their yeast orthologs. The resulting yeast strains with humanized cytoskeletal components now provide surrogate platforms to characterize human genes in simplified eukary- otic contexts. KEYWORDS humanized yeast; evolution; gene families; orthologs; cytoskeleton ENE duplication is regarded as one of the key drivers of how duplicated genes retain function or diverge are still un- Gevolution, contributing to the generation and accumu- clear. The processes governing the distribution of molecular lation of new genetic material within species (Soukup 1970; roles within gene families also have important consequences Koonin 2005). Duplication creates an initial multiplication of for annotating genes, which generally takes advantage of se- dosage and functional redundancy, but the trends dictating quence similarity and conservation over vast timescales of di- vergence to infer functions of homologous genes across Copyright © 2020 Garge et al. species. Many individual studies have directly tested the con- doi: https://doi.org/10.1534/genetics.120.303378 servation of function among orthologs from different species Manuscript received January 6, 2020; accepted for publication June 8, 2020; published Early Online June 10, 2020. by swapping them from one species into another (Heinicke Available freely online through the author-supported open access option. et al. 2007; Cherry et al. 2012). However, only recently have This is an open-access article distributed under the terms of the Creative Commons efforts been made to test such functional equivalence more Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, systematically, with several recent large-scale studies harness- provided the original work is properly cited. ing “the awesome power of yeast genetics” to systematically Supplemental material available at figshare: https://doi.org/10.25386/genetics. 12456206. replace yeast genes by their human, plant, or even bacterial 1Corresponding author: The University of Texas at Austin, 2500 Speedway, Austin, TX counterparts and assay for functional compatibility (Kachroo 78712. E-mail: [email protected]; and Centre for Applied Synthetic Biology, Department of Biology, 7141 Sherbrooke St W, Concordia University, Montreal, QC, et al. 2015, 2017; Hamza et al. 2015; Laurent et al. 2016; Sun Canada. H4B 1R6. E-mail: [email protected] et al. 2016; Yang et al. 2017). Although humans and yeast last Genetics, Vol. 215, 1153–1169 August 2020 1153 shared a common ancestor nearly a billion years ago, these by other paralogs complicating the determination of paralog- studies have demonstrated that substantial fractions (12%– specific roles within cytoskeletal gene families. The high de- 47%) of tested essential yeast genes could be replaced by their gree of sequence conservation among paralogs within each human equivalents (Kachroo et al. 2015, 2017; Hamza et al. cytoskeletal family make functional analysis of individual cy- 2015; Sun et al. 2016; Yang et al. 2017; Laurent et al. 2019). toskeletal genes directly in human cells both experimentally The ability of many human genes to functionally replace their and computationally cumbersome. However, cross-species yeast orthologs demonstrates the high degree of functional gene swaps have the potential to provide direct assays of conservation in eukaryotic systems over billion-year evolution- individual paralogs within these expanded gene families, ary timescales (Kachroo et al. 2015; Laurent et al. 2016). thereby revealing the extent to which present-day ortholo- Previous humanization efforts in yeast have focused pri- gous genes retain ancestral function. marily on ortholog pairs with no obvious duplications within To understand the extent to which human cytoskeletal yeast and human lineages (1:1 orthologs), only partially genes in expanded orthogroups retain cross-species functional testing the orthologs in expanded gene families (Kachroo equivalence, we systematically humanized major elements of et al. 2015; Hamza et al. 2015; Sun et al. 2016; Yang et al. the yeast cytoskeleton. We tested 81% (50/62) of all human 2017; Laurent et al. 2019) and seldom beyond assaying im- genes from actin, myosin, septin, and tubulin families, using a pact on growth rate. In this study, to better understand func- combination of classical yeast genetics and CRISPR-Cas9 me- tional conservation across expanded gene families in core diated genome editing to assay the replaceability of essential eukaryotic processes, we focused on the major structural cytoskeletal orthologs, as initially determined via simple components of the eukaryotic cytoskeleton, including actins, growth rescue complementation assays. Overall, we show that myosins, septins, and tubulin genes. Genes constituting the (13/50) members from five of seven tested gene families (ac- eukaryotic cytoskeleton play key roles in critical cellular tin, heavy myosin, septin, b-andg-tubulin) can indeed exe- processes, mainly organizing the contents of the cell by dy- cute essential roles of their yeast counterparts. Within each namically controlling cell shape, positioning organelles, and replaceable family, we show that several present-day human transporting macromolecules including chromosomes across orthologs still possess functional roles of their respective the cell through the generation of coordinated mechanical opisthokont ancestors compatible in a yeast cellular context. forces (Wickstead and Gull 2007, 2011; Wickstead et al. Additionally, we characterized cellular phenotypes beyond 2010). Importantly, cytoskeletal gene families have under- growth and observed differential abilities among complement- gone large expansions along the human lineage, while being ing human cytoskeletal genes to carry out nonessential cyto- restricted to only a few family members in yeast (Figure 1). skeletal roles, including those in cell morphology, sporulation, Advances in comparative genomics have shed light on the mating, meiosis, and cytokinesis. Besides revealing human cy- likely cytoskeletal components present in the last eukaryotic toskeletal orthologs capable of performing their core eukary- common ancestor (LECA) (Wickstead and Gull 2007, 2011; otic roles, yeast strains with human cytoskeleton components Wickstead et al. 2010). Additionally, phylogenetic profiling additionally serve as cellular reagents to study the specific studies of the cytoskeleton have been highly informative in roles of expanded cytoskeletal gene family members in a sim- inferring gene loss and retention events across eukaryotic plified unicellular eukaryotic context. clades (Wickstead and Gull 2007; Wickstead et al. 2010) (Figure 1). Such studies suggest that the origins of the Materials and Methods eukaryotic cytoskeleton predate eukaryogenesis and ances- Curating human orthologs to yeast genes trally trace back to primitive tubulin- and actin-like homologs in bacteria13. These components, critical in cell division, sub- Yeast genes to be tested for replaceability were curated from sequently evolved to incorporate families of accessory motors Saccharomyces Genome Database (Cherry et al. 1998, 2012), and regulatory proteins expanding toward