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PubMed2005

Genetic interactions between Nhp6 and Gcn5 with Mot1 and the Ccr4-Not complex that regulate binding of TATA-binding protein in Saccharomyces cerevisiae.

Our previous work suggests that the Nhp6 HMGB protein stimulates RNA polymerase II transcription via the TATA-binding protein TBP and that Nhp6 functions in the same functional pathway as the Gcn5 histone acetyltransferase. In this report we examine the genetic relationship between Nhp6 and Gcn5 with the Mot1 and Ccr4-Not complexes, both of which have been implicated in regulating DNA binding by TBP. We find that combining either a nhp6ab or a gcn5 mutation with mot1, ccr4, not4, or not5 mutations results in lethality. Combining spt15 point mutations (in TBP) with either mot1 or ccr4 also results in either a growth defect or lethality. Several of these synthetic lethalities can be suppressed by overexpression of TFIIA, TBP, or Nhp6, suggesting that these genes facilitate formation of the TBP-TFIIA-DNA complex. The growth defect of a not5 mutant can be suppressed by a mot1 mutant. HO gene expression is reduced by nhp6ab, gcn5, or mot1 mutations, and the additive decreases in HO mRNA levels in nhp6ab mot1 and gcn5 mot1 strains suggest different modes of action. Chromatin immunoprecipitation experiments show decreased binding of TBP to promoters in mot1 mutants and a further decrease when combined with either nhp6ab or gcn5 mutations.

Adenosine Triphosphatases↗
PubMed2005

Commensurate distances and similar motifs in genetic congruence and protein interaction networks in yeast.

BACKGROUND: In a genetic interaction, the phenotype of a double mutant differs from the combined phenotypes of the underlying single mutants. When the single mutants have no growth defect, but the double mutant is lethal or exhibits slow growth, the interaction is termed synthetic lethality or synthetic fitness. These genetic interactions reveal gene redundancy and compensating pathways. Recently available large-scale data sets of genetic interactions and protein interactions in Saccharomyces cerevisiae provide a unique opportunity to elucidate the topological structure of biological pathways and how genes function in these pathways. RESULTS: We have defined congruent genes as pairs of genes with similar sets of genetic interaction partners and constructed a genetic congruence network by linking congruent genes. By comparing path lengths in three types of networks (genetic interaction, genetic congruence, and protein interaction), we discovered that high genetic congruence not only exhibits correlation with direct protein interaction linkage but also exhibits commensurate distance with the protein interaction network. However, consistent distances were not observed between genetic and protein interaction networks. We also demonstrated that congruence and protein networks are enriched with motifs that indicate network transitivity, while the genetic network has both transitive (triangle) and intransitive (square) types of motifs. These results suggest that robustness of yeast cells to gene deletions is due in part to two complementary pathways (square motif) or three complementary pathways, any two of which are required for viability (triangle motif). CONCLUSION: Genetic congruence is superior to genetic interaction in prediction of protein interactions and function associations. Genetically interacting pairs usually belong to parallel compensatory pathways, which can generate transitive motifs (any two of three pathways needed) or intransitive motifs (either of two pathways needed).

Protein Interaction Mapping↗
PubMed2004

The coordinate regulation of pharyngeal development in C. elegans by lin-35/Rb, pha-1, and ubc-18.

Organ development is a complex process involving the coordination of cell proliferation, differentiation, and morphogenetic events. Using a screen to identify genes that function coordinately with lin-35/Rb during animal development, we have isolated a weak loss-of-function (LOF) mutation in pha-1. lin-35; pha-1 double mutants are defective at an early step in pharyngeal morphogenesis leading to an abnormal pharyngeal architecture. pha-1 is also synthetically lethal with other class B synthetic multivulval (SynMuv) genes including the C. elegans E2F homolog, efl-1. Reporter analyses indicate that pha-1 is broadly expressed during embryonic development and that its functions reside in the cytoplasm. We also provide genetic and phenotypic evidence to support the model that PHA-1, a novel protein, and UBC-18, a ubiquitin-conjugating enzyme that we have previously shown to function with lin-35 during pharyngeal development, act in parallel pathways to regulate the activity of a common cellular target.

Animals↗
PubMed2026

The role of CHAMP1 in chromatin-mediated DNA damage repair.

Defects in the replication stress response are major drivers of cancer development and present key targetable vulnerabilities that can be exploited for anti-cancer therapy. Recent studies have identified CHAMP1 as a novel DNA damage repair factor with roles in double-strand break repair and the replication stress response. Mutations in CHAMP1 are associated with the neurodevelopmental disorder CHAMP1 Syndrome. More recently, children with CHAMP1 Syndrome have developed leukemia, suggesting that CHAMP1 mutations are a potential cancer risk factor. CHAMP1 is part of two DNA damage repair complexes: CHAMP1-POGZ-REV7 (Complex I) and CHAMP1-POGZ-HP1α (Complex II). Complex I promotes homologous recombination by removing the Shieldin complex from the ends of double strand breaks and allowing DSB end resection to occur. Complex II enriches heterochromatin content through the recruitment of the methyltransferase SETDB1 to DNA damage sites. Increased heterochromatin at stalled forks is associated with proper fork stability and restart, demonstrating the importance of CHAMP1 in maintaining genomic integrity. Loss of CHAMP1 leads to increased sensitivity to DNA damaging agents and increased dependence on other DNA damage repair pathways, such as the DNA damage checkpoint and the Fanconi Anemia pathway. CHAMP1 is overexpressed in breast and ovarian cancer cells with high levels of replication stress, providing a molecular mechanism for the tolerance of replication stress. These new findings on the relationship of CHAMP1 with well-established DNA damage repair pathways, suggest that targeting CHAMP1 could present a new synthetic lethality opportunity for cancer cells with high levels of replication stress.

CHAMP1↗
PubMed2000

Disruption of cytoplasmic and mitochondrial folylpolyglutamate synthetase activity in Saccharomyces cerevisiae.

Similar to other eukaryotes, yeasts have parallel pathways of one-carbon metabolism in the cytoplasm and mitochondria and have folylpolyglutamate synthetase activity in both compartments. The gene encoding folylpolyglutamate synthetase is MET7 (also referred to as MET23) on chromosome XV and appears to encode both the cytoplasmic and mitochondrial forms of the enzyme. In order to determine the metabolic roles of both forms of folylpolyglutamate synthetase, we disrupted the met7 gene and determined that the strain is a methionine auxotroph and an adenine and thymidine auxotroph when grown in the presence of sulfanilamide. The met7 mutant becomes petite under normal growth conditions but can be maintained with a grande phenotype if the strain is tup and all media are supplemented with dTMP. A met7 gly1 strain is auxotrophic for glycine when grown on glucose but prototrophic when grown on glycerol. A met7 ser1 strain cannot use glycine to suppress the serine auxotrophy of the ser1 phenotype. A met7 shm2 strain is nonviable. In order to disrupt just the mitochondrial folylpolyglutamate synthetase activity, we constructed mutants with an inactivated chromosomal MET7 gene complemented by genes that express only cytoplasmic folylpolyglutamate synthetase, including the Lactobacillus casei folC gene and the yeast MET7 gene with its mitochondrial leader sequence deleted (MET7Deltam). All the genes providing cytoplasmic folylpolyglutamate synthetase complemented the methionine auxotrophy as well as the synthetic lethality of the shm2 strain and the synthetic glycine auxotrophy of the gly1 strain. The strains lacking the mitochondrial folylpolyglutamate synthetase had longer doubling times than the isogenic wild-type strains but retained the function of the mitochondrial folate-dependent enzymes to produce formate, serine, and glycine. Mutants complemented by the bacterial folC gene or by the MET7Deltam gene on a 2mu plasmid remained grande without the tup mutation and supplementation and dTMP. Mutants complemented by the MET7Deltam gene integrated in single copy became petites under those conditions, indicating a deficiency in dTMP production but this is likely due to lower expression of cytoplasmic folylpolyglutamate synthetase by the MET7Deltam gene.

Amino Acids↗
PubMed2025

De novo pyrimidine synthesis is a collateral metabolic vulnerability in NF2-deficient mesothelioma.

Pleural mesothelioma (PM) is one of the deadliest cancers, with limited therapeutic options due to its therapeutically intractable genome, which is characterized by the functional inactivation of tumor suppressor genes (TSGs) and high tumor heterogeneity, including diverse metabolic adaptations. However, the molecular mechanisms underlying these metabolic alterations remain poorly understood, particularly how TSG inactivation rewires tumor metabolism to drive tumorigenesis and create metabolic dependencies. Through integrated multi-omics analysis, we identify for the first time that NF2 loss of function defines a distinct PM subtype characterized by enhanced de novo pyrimidine synthesis, which NF2-deficient PM cells are critically dependent on for sustained proliferation in vitro and in vivo. Mechanistically, NF2 loss activates YAP, a downstream proto-oncogenic transcriptional coactivator in the Hippo signalling pathway, which in turn upregulates CAD and DHODH, key enzymes in the de novo pyrimidine biosynthesis pathway. Our findings provide novel insights into metabolic reprogramming in PM, revealing de novo pyrimidine synthesis as a synthetic lethal vulnerability in NF2-deficient tumors. This work highlights a potential therapeutic strategy for targeting NF2-deficient mesothelioma through metabolic intervention.

Pyrimidines↗
PubMed1999

Genetic approaches to the study of protein-protein interactions.

This article describes genetic approaches to the study of heterologous protein-protein interactions, focusing on the yeast Saccharomyces cerevisiae as a useful eukaryotic model system. Several methods are described that can be used to search for new interactions, including extragenic suppression, multicopy suppression, synthetic lethality, and transdominant inhibition. Strategies for screening, genetic characterization, and clone identification are described, along with recent examples from the literature. In addition, genetic methods are discussed that can be used to further characterize a newly discovered protein-protein interaction. These include the creation of mutant libraries of a given protein by chemical mutagenesis or polymerase chain reaction, the production of dominant-negative mutants, and strategies for introducing these mutant alleles back into yeast for analysis. Although these genetic methods are quite powerful, they are often just a starting point for further biochemical or cell biological experiments.

Cloning, Molecular↗
PubMed1992

A gene encoding a putative tyrosine phosphatase suppresses lethality of an N-end rule-dependent mutant.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. In the yeast Saccharomyces cerevisiae, mutational inactivation of the N-end rule pathway is neither lethal nor phenotypically conspicuous. We have used a "synthetic lethal" screen to isolate a mutant that requires the N-end rule pathway for viability. An extragenic suppressor of this mutation was cloned and found to encode a 750-residue protein with strong sequence similarities to protein phosphotyrosine phosphatases. This heat-inducible gene was named PTP2. Null ptp2 mutants grow slowly, are hypersensitive to heat, and are viable in either the presence or absence of the N-end rule pathway. We discuss possible connections between dephosphorylation of phosphotyrosine in proteins and the N-end rule pathway of protein degradation.

Amino Acid Sequence↗
PubMed2002

fzr-1 and lin-35/Rb function redundantly to control cell proliferation in C. elegans as revealed by a nonbiased synthetic screen.

We report here a synthetic-lethal screen in Caenorhabditis elegans that overcomes a number of obstacles associated with the analysis of functionally redundant genes. Using this approach, we have identified mutations that synthetically interact with lin-35/Rb, a SynMuv gene and the sole member of the Rb/pocket protein family in C. elegans. Unlike the original SynMuv screens, our approach is completely nonbiased and can theoretically be applied to any situation in which a mutation fails to produce a detectable phenotype. From this screen we have identified fzr-1, a gene that synthetically interacts with lin-35 to produce global defects in cell proliferation control. fzr-1 encodes the C. elegans homolog of Cdh1/Hct1/FZR, a gene product shown in other systems to regulate the APC cyclosome. We have also uncovered genetic interactions between fzr-1 and a subset of class B SynMuv genes, and between lin-35 and the putative SCF regulator lin-23. We propose that lin-35, fzr-1, and lin-23 function redundantly to control cell cycle progression through the regulation of cyclin levels.

Amino Acid Sequence↗
PubMed1997

Mso1p: a yeast protein that functions in secretion and interacts physically and genetically with Sec1p.

The yeast Sec1p protein functions in the docking of secretory transport vesicles to the plasma membrane. We previously have cloned two yeast genes encoding syntaxins, SSO1 and SSO2, as suppressors of the temperature-sensitive sec1-1 mutation. We now describe a third suppressor of sec1-1, which we call MSO1. Unlike SSO1 and SSO2, MSO1 is specific for sec1 and does not suppress mutations in any other SEC genes. MSO1 encodes a small hydrophilic protein that is enriched in a microsomal membrane fraction. Cells that lack MSO1 are viable, but they accumulate secretory vesicles in the bud, indicating that the terminal step in secretion is partially impaired. Moreover, loss of MSO1 shows synthetic lethality with mutations in SEC1, SEC2, and SEC4, and other synthetic phenotypes with mutations in several other late-acting SEC genes. We further found that Mso1p interacts with Sec1p both in vitro and in the two-hybrid system. These findings suggest that Mso1p is a component of the secretory vesicle docking complex whose function is closely associated with that of Sec1p.

Biological Transport↗
PubMed2001

Gamma-tubulin and the C-terminal motor domain kinesin-like protein, KLPA, function in the establishment of spindle bipolarity in Aspergillus nidulans.

Previous research has found that a gamma-tubulin mutation in Schizosaccharomyces pombe is synthetically lethal with a deletion of the C-terminal motor domain kinesin-like protein gene pkl1, but the lethality of the double mutant prevents a phenotypic analysis of the synthetic interaction. We have investigated interactions between klpA1, a deletion of an Aspergillus nidulans homolog of pkl1, and mutations in the mipA, gamma-tubulin gene. We find that klpA1 dramatically increases the cold sensitivity and slightly reduces the growth rate at all temperatures, of three mipA alleles. In synchronized cells we find that klpA1 causes a substantial but transient inhibition of the establishment of spindle bipolarity. At a restrictive temperature, mipAD123 causes a slight, transient inhibition of spindle bipolarity and a more significant inhibition of anaphase A. In the mipAD123/klpA1 strain, formation of bipolar spindles is more strongly inhibited than in the klpA1 single mutant and many spindles apparently never become bipolar. These results indicate, surprisingly, that gamma-tubulin and the klpA kinesin have overlapping roles in the establishment of spindle bipolarity. We propose a model to account for these data.

Alleles↗
PubMed2002

Saccharomyces cerevisiae MPT5 and SSD1 function in parallel pathways to promote cell wall integrity.

Yeast MPT5 (UTH4) is a limiting component for longevity. We show here that MPT5 also functions to promote cell wall integrity. Loss of Mpt5p results in phenotypes associated with a weakened cell wall, including sorbitol-remedial temperature sensitivity and sensitivities to calcofluor white and sodium dodecyl sulfate. Additionally, we find that mutation of MPT5, in the absence of SSD1-V, is lethal in combination with loss of either Ccr4p or Swi4p. These synthetic lethal interactions are suppressed by the SSD1-V allele. Furthermore, we have provided evidence that the short life span caused by loss of Mpt5p is due to a weakened cell wall. This cell wall defect may be the result of abnormal chitin biosynthesis or accumulation. These analyses have defined three genetic pathways that function in parallel to promote cell integrity: an Mpt5p-containing pathway, an Ssd1p-containing pathway, and a Pkc1p-dependent pathway. This work also provides evidence that post-transcriptional regulation is likely to be important both for maintaining cell integrity and for promoting longevity.

Cell Cycle Proteins↗
PubMed2006

ASPP2 is a haploinsufficient tumor suppressor that cooperates with p53 to suppress tumor growth.

ASPP2 stimulates the apoptotic function of the p53 family in vivo. We show here that ASPP2-/- pups died before weaning. This postnatal lethality was significantly enhanced in p53+/- background and both deletions are synthetic lethal. ASPP2+/- mice developed spontaneous tumors. The tumor onset was accelerated by gamma-irradiation or in p53+/- background. Tumors derived from ASPP2+/- mice retained wild-type ASPP2 allele even though some of them lost p53. These provide the first genetic evidence that ASPP2 is a haploinsufficient tumor suppressor that shares overlapping function(s) with p53 in mouse development and tumor suppression.

Alleles↗
PubMed2025

KIF18A promotes chromosome congression in cooperation with CENP-E downstream of CENP-C.

Chromosome congression is a key process that acts to align chromosomes at the spindle equator via kinetochore-microtubule interactions, with defects in chromosome alignment leading to chromosomal instability. However, defining the mechanisms that underlie chromosome congression is limited due to the multiple factors that act in parallel to regulate chromosome movement. Here, we conducted a genome-wide Cas9-based functional genetics screen using a hypomorphic CENP-C mutant that affects its kinetochore interactions. Our analysis identified KIF18A, whose knockout resulted in synthetic lethality with the CENP-C mutant. Further analysis revealed that the synthetic defect was due to a reduction in CENP-E function in the CENP-C mutant. Our work suggests that KIF18A promotes chromosome alignment in cooperation with CENP-E downstream of CENP-C during early prometaphase. Thus, our analysis enables us to dissect parallel molecular mechanisms for chromosome congression and identify sensitivities and biomarkers that might guide anti-KIF18A chemotherapeutics.

Kinesins↗
PubMed1999

LST1 is a SEC24 homologue used for selective export of the plasma membrane ATPase from the endoplasmic reticulum.

In Saccharomyces cerevisiae, vesicles that carry proteins from the ER to the Golgi compartment are encapsulated by COPII coat proteins. We identified mutations in ten genes, designated LST (lethal with sec-thirteen), that were lethal in combination with the COPII mutation sec13-1. LST1 showed synthetic-lethal interactions with the complete set of COPII genes, indicating that LST1 encodes a new COPII function. LST1 codes for a protein similar in sequence to the COPII subunit Sec24p. Like Sec24p, Lst1p is a peripheral ER membrane protein that binds to the COPII subunit Sec23p. Chromosomal deletion of LST1 is not lethal, but inhibits transport of the plasma membrane proton-ATPase (Pma1p) to the cell surface, causing poor growth on media of low pH. Localization by both immunofluorescence microscopy and cell fractionation shows that the export of Pma1p from the ER is impaired in lst1Delta mutants. Transport of other proteins from the ER was not affected by lst1Delta, nor was Pma1p transport found to be particularly sensitive to other COPII defects. Together, these findings suggest that a specialized form of the COPII coat subunit, with Lst1p in place of Sec24p, is used for the efficient packaging of Pma1p into vesicles derived from the ER.

Amino Acid Sequence↗
PubMed2001

Rpm2, the protein subunit of mitochondrial RNase P in Saccharomyces cerevisiae, also has a role in the translation of mitochondrially encoded subunits of cytochrome c oxidase.

RPM2 is a Saccharomyces cerevisiae nuclear gene that encodes the protein subunit of mitochondrial RNase P and has an unknown function essential for fermentative growth. Cells lacking mitochondrial RNase P cannot respire and accumulate lesions in their mitochondrial DNA. The effects of a new RPM2 allele, rpm2-100, reveal a novel function of RPM2 in mitochondrial biogenesis. Cells with rpm2-100 as their only source of Rpm2p have correctly processed mitochondrial tRNAs but are still respiratory deficient. Mitochondrial mRNA and rRNA levels are reduced in rpm2-100 cells compared to wild type. The general reduction in mRNA is not reflected in a similar reduction in mitochondrial protein synthesis. Incorporation of labeled precursors into mitochondrially encoded Atp6, Atp8, Atp9, and Cytb protein was enhanced in the mutant relative to wild type, while incorporation into Cox1p, Cox2p, Cox3p, and Var1p was reduced. Pulse-chase analysis of mitochondrial translation revealed decreased rates of translation of COX1, COX2, and COX3 mRNAs. This decrease leads to low steady-state levels of Cox1p, Cox2p, and Cox3p, loss of visible spectra of aa(3) cytochromes, and low cytochrome c oxidase activity in mutant mitochondria. Thus, RPM2 has a previously unrecognized role in mitochondrial biogenesis, in addition to its role as a subunit of mitochondrial RNase P. Moreover, there is a synthetic lethal interaction between the disruption of this novel respiratory function and the loss of wild-type mtDNA. This synthetic interaction explains why a complete deletion of RPM2 is lethal.

Alleles↗
PubMed1992

A new subclass of nucleoporins that functionally interact with nuclear pore protein NSP1.

NSP1 is a nuclear pore protein (nucleoporin) essential for cell growth. To identify the components that functionally interact with NSP1 in the living cell, we developed a genetic screen for mutants that are lethal in a genetic background of mutated, but not wild type NSP1. Fourteen synthetic lethal mutants were obtained, belonging to at least four different complementation groups. The genes of two complementation groups, NSP116 and NSP49, were cloned. Like the previously described nucleoporins, these genes encode proteins with many repeat sequences. NSP116 and NSP49, however, contain a new repetitive sequence motif 'GLFG', which classifies them as a subclass of nucleoporins. NSP116 and NSP49, tagged with the IgG binding domain of protein A and expressed in yeast, are located at the nuclear envelope. These data provide in vivo evidence that distinct subclasses of nucleoporins physically interact or share overlapping function in nuclear pore complexes.

Amino Acid Sequence↗
PubMed1990

Sec2 protein contains a coiled-coil domain essential for vesicular transport and a dispensable carboxy terminal domain.

SEC2 function is required at the post-Golgi apparatus stage of the yeast secretory pathway. The SEC2 sequence encodes a protein product of 759 amino acids containing an amino terminal region that is predicted to be in an alpha-helical, coiled-coil conformation. Two temperature-sensitive alleles, sec2-41 and sec2-59, encode proteins truncated by opal stop codons and are suppressible by an opal tRNA suppressor. Deletion analysis indicates that removal of the carboxyl terminal 251 amino acids has no apparent phenotype, while truncation of 368 amino acids causes temperature sensitivity. The amino terminal half of the protein, containing the putative coiled-coil domain, is essential at all temperatures. Sec2 protein is found predominantly in the soluble fraction and displays a native molecular mass of greater than 500 kD. All phenotypes of the temperature-sensitive sec2 alleles are partially suppressed by duplication of the SEC4 gene, but the lethality of a sec2 disruption is not suppressed. The sec2-41 mutation exhibits synthetic lethality with the same subset of the late acting sec mutants as does sec4-8 and sec15-1. The Sec2 protein may function in conjunction with the Sec4 and Sec15 proteins to control vesicular traffic.

Amino Acid Sequence↗