Systematic bacterialization of yeast genes identifies a near-universally swappable pathway

Kachroo, Aashiq H.; Laurent, Jon M; Akhmetov, Azat; Szilagyi-Jones, Madelyn; McWhite, Claire D.; Zhao, Alice; Marcotte, Edward M.

doi:10.7554/elife.25093

Cited by 31 publications

(48 citation statements)

References 67 publications

(93 reference statements)

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“…72 expression profiles) rather than direct functional assays. However, recent systematic studies of 73 functional replacement of yeast genes by their orthologs from humans [18][19][20][21] and bacteria [22] 74 demonstrate the power of inter-species gene swaps to directly test functional divergence and 75 identify properties that determine functional conservation across vast evolutionary distances. Here, 76we sought to investigate the functional divergence across gene families by directly swapping 77 human genes belonging to extended families into yeast.…”

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Humanization of yeast genes with multiple human orthologs reveals principles of functional divergence between paralogs

Laurent

Garge

et al. 2019

Preprint

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1Despite over a billion years of evolutionary divergence, several thousand human genes possess 2 clearly identifiable orthologs in yeast, and many have undergone lineage-specific duplications in 3 one or both lineages. The ortholog conjecture postulates that orthologous genes between species 4 retain ancestral functions despite divergence over vast timescales, but duplicated genes will be free 5 to diverge in function. However, the retention of ancestral functions among co-orthologs between 6 species and within gene families has been difficult to test experimentally at scale. In order to 7 investigate how ancestral functions are retained or lost post-duplication, we systematically 8 replaced hundreds of essential yeast genes with their human orthologs from gene families that have 9 undergone lineage-specific duplications, including those with single duplications (one yeast gene 10 to two human genes, 1:2) or higher-order expansions (1:>2) in the human lineage. We observe a 11 variable pattern of replaceability across different ortholog classes, with an obvious trend towards 12 differential replaceability inside gene families, rarely observing replaceability by all members of 13 a family. We quantify the ability of various properties of the orthologs to predict replaceability, 14showing that in the case of 1:2 orthologs, replaceability is predicted largely by the divergence and 15 tissue-specific expression of the human co-orthologs, i.e. the human proteins that are less diverged 16 from their yeast counterpart and more ubiquitously expressed across human tissues more often 17 replace their single yeast ortholog. These trends were consistent with in silico simulations 18demonstrating that when only one ortholog is replaceable, it tends to be the least diverged of the 19 pair. Replaceability of yeast genes having more than two human co-orthologs was marked by 20 retention of orthologous interactions in functional or protein networks as well as by more ancestral 21 subcellular localization. Overall, we performed >400 human gene replaceability assays revealing 22 56 new human-yeast complementation pairs, thus opening up avenues to further functionally 23 characterize these human genes in a simplified organismal context. 24 25 and are distinguished from those related by speciation, termed orthologs [8,9]. Importantly, an 46 expanded family of paralogs in one lineage may be co-orthologs to a single gene in another lineage. 47How ancestral functions are partitioned, lost, or retained between paralogs and orthologs during 48 these duplication events is a major topic of study for evolutionary biology. To address these 49 questions, the ortholog conjecture was put forth, a major thesis stating that orthologs are more 50 likely to retain function between species than paralogs, which will tend to diverge in function after 51 duplication due to drift and relaxed selection [10,11]. This conjecture underlies common methods 52 by which functions are assigned to newly discovered or understudied genes in un-annotated 53 4 genomes ...

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Humanization of yeast genes with multiple human orthologs reveals principles of functional divergence between paralogs

Laurent

Garge

et al. 2019

Preprint

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“…S2A ) (ii) inactivated via a temperature-sensitive allele 22, 36 ( Fig. S3A ) or (iii) endogenously replaced by homologous recombination repair with the human ortholog(s) following CRISPR-Cas9 cleavage 8, 37 ( Fig. S4A ).…”

Section: Resultsmentioning

confidence: 99%

“…29 human genes were assayed using a CRISPR-Cas9 mediated yeast genome editing protocol as previously described 8, 37 . For every yeast gene to be replaced endogenously, a minimum of 2 synthetic guide RNAs (sgRNA) with high ON-target and low OFF-target scores were designed using the Geneious v10.2.6 CRISPR-Cas9 tools suite.…”

Section: Methodsmentioning

confidence: 99%

“…Transformants were selected on SC-URA medium to select for the knockout plasmid. While CRISPR-mediated double-stranded breaks are intrinsically lethal, targeted editing of an essential gene locus acts as an added layer of selection in replacing the human gene of interest, allowing cells to survive only if the human ortholog being assayed functionally replaces the yeast gene at the appropriate locus 8, 37 . Surviving colonies ( Fig.…”

Section: Methodsmentioning

confidence: 99%

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Systematic humanization of the yeast cytoskeleton discerns functionally replaceable from divergent human genes

Garge

Laurent

Kachroo³

et al. 2019

Preprint

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“…We assessed features that predict complementation of nonessential yeast gene deletion mutants. Previous screens that tested complementation of essential yeast genes determined that the strongest predictive feature of complementation is that genes in the same pathway, process, or complex tended to be similarly replaceable or nonreplaceable (Hamza et al 2015;Kachroo et al 2015Kachroo et al , 2017Sun et al 2016). Figure S1.…”

Section: Identification Of Human-yeast Complementation Pairs For the mentioning

confidence: 99%

Cross-Species Complementation of Nonessential Yeast Genes Establishes Platforms for Testing Inhibitors of Human Proteins

Hamza¹,

Driessen²,

Tammpere³

et al. 2020

Genetics

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Cross-species complementation can be used to generate humanized yeast, which is a valuable resource with which to model and study human biology. Humanized yeast can be used as an in vivo platform to screen for chemical inhibition of human protein drug targets. To this end, we report the systematic complementation of nonessential yeast genes implicated in chromosome instability (CIN) with their human homologs. We identified 20 human–yeast complementation pairs that are replaceable in 44 assays that test rescue of chemical sensitivity and/or CIN defects. We selected a human–yeast pair (hFEN1/yRAD27), which is frequently overexpressed in cancer and is an anticancer therapeutic target, to perform in vivo inhibitor assays using a humanized yeast cell-based platform. In agreement with published in vitro assays, we demonstrate that HU-based PTPD is a species-specific hFEN1 inhibitor. In contrast, another reported hFEN1 inhibitor, the arylstibonic acid derivative NSC-13755, was determined to have off-target effects resulting in a synthetic lethal phenotype with yRAD27-deficient strains. Our study expands the list of human–yeast complementation pairs to nonessential genes by defining novel cell-based assays that can be utilized as a broad resource to study human drug targets.

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Systematic bacterialization of yeast genes identifies a near-universally swappable pathway

Cited by 31 publications

References 67 publications

Humanization of yeast genes with multiple human orthologs reveals principles of functional divergence between paralogs

Humanization of yeast genes with multiple human orthologs reveals principles of functional divergence between paralogs

Systematic humanization of the yeast cytoskeleton discerns functionally replaceable from divergent human genes

Cross-Species Complementation of Nonessential Yeast Genes Establishes Platforms for Testing Inhibitors of Human Proteins

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